METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER
Some embodiments of the present invention relate to methods and compositions for treating cancer. More embodiments include methods and compositions for modulating the activity of the Hedgehog pathway.
This application is a continuation of U.S. Ser. No. 14/919,448 filed Oct. 21, 2015, entitled “METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER” which is a continuation of U.S. Ser. No. 13/635,893 filed May 8, 2013 entitled “METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER” now U.S. Pat. No. 9,186,370 issued Nov. 17, 2015 which is the U.S. National Phase Application No. PCT/US2011/029093 entitled “METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER” filed Mar. 18, 2011 and published in English on Sep. 22, 2011 as WO2011/116351 which claims the benefit of U.S. Provisional Application No. 61/315,615 filed Mar. 19, 2010, the contents of which are incorporated by reference in their entireties.
STATEMENT REGARDING FEDERALLY SPONSORED R&DThis invention was made with government support under Department of Defense IDEA Award 07-1-0400, and National Institute of Health 1R01CA140472-01A1. The government has certain rights in the invention.
REFERENCE TO SEQUENCE LISTINGThe present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled USA002C2SEQLIST.TXT, created Nov. 29, 2016, which is approximately 34 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTIONSome embodiments of the present invention relate to methods and compositions for treating cancer. More embodiments include methods and compositions for modulating the activity of the Hedgehog pathway.
BACKGROUNDMembers of the hedgehog family of signaling molecules mediate many important short- and long-range patterning processes during invertebrate and vertebrate embryonic, fetal, and adult development. In Drosophila melanogaster, a single hedgehog gene regulates segmental and imaginal disc patterning. In contrast, in vertebrates, a hedgehog gene family (e.g., in mammals, SHH, DHH, IHH) is involved in the control of proliferation, differentiation, migration, and survival of cells and tissues derived from all three germ layers, including, e.g., left-right asymmetry, CNS development, somites and limb patterning, chondrogenesis, skeletogenesis and spermatogenesis.
Hedgehog signaling occurs through the Hedgehog pathway which includes interactions between hedgehog ligand with the hedgehog receptor, Patched (Ptch), and the co-receptor Smoothened (Smo). There are two mammalian homologs of Ptch, Ptch-1 and Ptch-2 (“collectively “Ptch”), both of which are 12 transmembrane proteins containing a sterol sensing domain (Motoyama et al., Nature Genetics 18: 104-106 (1998), Carpenter et al., P.N.A.S. (U.S.A.) 95: 13630-40 (1998)). The interaction of Hedgehog with Ptch triggers a signaling cascade that results in the regulation of transcription by zinc-finger transcriptions factors of the Gli family.
Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin. 43:7 (1993)). Cancer features can include an the increase in the number of neoplastic cells which proliferate to form a tumor mass; invasion of adjacent tissues by these neoplastic tumor cells; and generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
Reactivation of the Hedgehog pathway has been implicated in a wide variety of cancers and carcinogenesis. The earliest examples of Hedgehog signaling in cancers came from the discovery that Gorlin's syndrome, in which patients frequently suffer basal cell carcinomas and are also predisposed to medulloblastomas and rhabdomysocarcomas, is due to an inactivating mutation in Ptch, resulting in Hedgehog pathway activation (Hahn et al 1998 Cell 85:841; Johnson et al. 1996, Science 272:1668). Subsequently inactivating mutations in Ptch and/or activating mutations in Smo were found to be responsible for sporadic basal cell carcinomas (Xie et al. 1998, Nature 391: 90).
Hedgehog pathway proteins and genes have also been implicated in esophageal cancer (Ma, X., et al. Int J Cancer, 118: 139-148, 2006; Berman, D. et al. Nature, 425: 846-851, 2003) and are highly expressed in the majority of chemotherapy-resistant esophageal cancer specimens (Sims-Mourtada, J. et al. Clin Cancer Res, 12: 6565-6572, 2006). More cancers where the Hedgehog pathway are involved include biliary tract cancers (Berman, D. et al. Nature, 425: 846-851, 2003), melanoma (Stecca, B., et al. Proc Natl Acad Sci USA, 104: 5895-5900, 2007), and stomach cancer (Berman, D. et al. Nature, 425: 846-851, 2003; Ma, X., et al. Carcinogenesis, 26: 1698-1705, 2005). Tumors that contain highly proliferative “tumor stem cells” and which represent areas of therapy include glial cell cancers (Clement, V., et al. Curr Biol, 17: 165-172, 2007), prostate cancers (Li, C., Heidt, et al. Cancer Res, 67: 1030-1037, 2007), breast cancers (Liu, S., et al. Cancer Res, 66: 6063-6071, 2006), multiple myelomas (Peacock, C. D., et al. PNAS, 104: 4048-4053, 2007), and colon cancers (Ricci-Vitiani, L., et al. Nature, 445: 111-115, 2007).
In addition, the Hedgehog pathway plays a role in regulating cancer stem cells, which are discrete tumor cell populations that display highly enhanced survival, self-renewal, and tumorigenicity properties (Beachy, P. A., et al. Nature, 432: 324-331, 2004). Activation of the Hedgehog pathway has been shown to play a role in cancer stem cells of the breast (Liu, S., et al. Cancer Res, 66: 6063-6071, 2006), central nervous system (Clement, V., Curr Biol, 17: 165-172, 2007) as well as in hematological malignancies (Peacock, C. D., PNAS, 104: 4048-4053, 2007).
Modulators of the Hedgehog pathway are described herein.
SUMMARYSome embodiments of the present invention relate to methods and compositions for treating cancer. More embodiments include methods and compositions for modulating the activity of the Hedgehog pathway.
Some embodiments include methods for killing or retarding the growth of at least one neoplastic cell, comprising reducing the expression level of a nucleic acid encoding GLI1, such as the expression level of a nucleic acid encoding GLI1-130, or the expression level of GLI1 protein such as the expression level of the GLI1-130 protein in the cell.
In some aspects of such embodiments, the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding GLI1-130, or the level of GLI1 protein such as the level of the GLI1-130 protein is reduced by contacting the cell with an isolated nucleic acid selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1 or a fragment thereof, a sequence encoding antisense GLI1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the at least one neoplastic cell is selected from a breast cancer cell, melanoma cell, prostate cancer cell, colorectal cancer cell, head and neck cancer cell, lung cancer cell, colon cancer cell, oesophageal cancer cell, gastric cancer cell, testicular cancer cell, and ovarian cancer cell.
In some aspects of such embodiments, the cell is a mammalian cell.
In some aspects of such embodiments, the mammalian cell is a human cell.
In some aspects of such embodiments, the cell is in vivo.
In some aspects of such embodiments, the cell is in vitro.
In some aspects of such embodiments, the nucleic acid encoding GLI1 comprises a nucleic acid encoding GLI1-130, or the GLI1 protein comprises GLI1-130 isoform.
Some embodiments include methods for treating or ameliorating cancer in a subject comprising reducing the level of a nucleic acid encoding GLI1 or the level of GLI1 protein in a cell of the subject.
In some aspects of such embodiments, the expression level of a nucleic acid encoding GLI1 such as the expression level of a nucleic acid encoding GLI1-130, or the expression level of GLI1 protein such as the expression level of the GLI1-130 protein is reduced by administering an isolated nucleic acid to the subject, wherein the nucleic acid is selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1, such as GLI-130, or a fragment thereof, a sequence encoding antisense GLI1, a sequence encoding antisense GLI1-130, or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1-130 or a fragment thereof.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the cancer is selected from breast cancer, melanoma, prostate cancer, colorectal cancer, head and neck cancer, lung cancer, colon cancer, oesophageal cancer, gastric cancer, testicular cancer, and ovarian cancer.
In some aspects of such embodiments, the subject is a mammal.
In some aspects of such embodiments, the mammal is a human.
In some aspects of such embodiments, the nucleic acid encoding GLI1 comprises a nucleic acid encoding GLI1-130, or the GLI1 protein comprises GLI1-130 isoform.
Some embodiments include isolated nucleic acids comprising a sequence encoding GLI1, such as a sequence encoding GLI1-130, or a fragment thereof, or a sequence encoding antisense GLI1, such as a sequence encoding antisense GLI1-130, or a fragment thereof, an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof, such as, an antisense nucleic acid complementary to a sequence encoding GLI1-130 or a fragment thereof, wherein the nucleic acid reduces the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as the level of GLI1-130 protein in a cell.
In some aspects of such embodiments, the nucleic acid is selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the isolated nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the isolated nucleic acid comprises SEQ ID NO:01.
Some embodiments include isolated nucleic acids comprising a sequence encoding GLI1-130 or a fragment thereof, a sequence encoding antisense GLI1-130 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1-130 or a fragment thereof, wherein the nucleic acid reduces the level of a nucleic acid encoding GLI1-130 or the level of GLI1-130 protein in a cell.
In some aspects of such embodiments, the nucleic acid is selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the isolated nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the isolated nucleic acid comprises SEQ ID NO:01.
Some embodiments include vectors comprising the isolated nucleic acid of any one of the nucleic acids provided herein.
Some embodiments include cells comprising the isolated nucleic acid of any one of the nucleic acids provided herein.
Some embodiments include pharmaceutical compositions comprising the nucleic acid of any one of the nucleic acids provided herein, and a pharmaceutically acceptable carrier.
Some embodiments include methods for killing or retarding the growth of at least one cell comprising: reducing the level of a nucleic acid encoding GLI1, such as GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein, in the cell; and contacting the cell with an effective amount of the therapeutic compound, wherein the effective amount is reduced compared to a cell wherein the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein, is not reduced.
In some aspects of such embodiments, the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein, is reduced by contacting the cell with an isolated nucleic acid selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1, such as a sequence encoding GLI1-130, or a fragment thereof, a sequence encoding antisense GLI1, such as a sequence encoding antisense GLI1-130, or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof, such as an antisense nucleic acid complementary to a sequence encoding GLI1-130 or a fragment thereof.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the therapeutic compound comprises a chemotherapeutic agent.
In some aspects of such embodiments, the chemotherapeutic agent is selected from a platinum-based compound such as cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, and triplatin tetranitrate, a nitrogen mustard such as cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, and ifosfamide, a nitrosourea such as carmustine, lomustine, and streptozocin, an alkyl sulfonate such as busulfan, thiotepa, procarbazine, and altretamine.
In some aspects of such embodiments, the chemotherapeutic agent is selected from taxol and doxirubicin.
In some aspects of such embodiments, the therapeutic compound is an agent for which increased expression of ERRC1, XPD, or XRCC1 results in increased cellular resistance.
In some aspects of such embodiments, increased activity of the nucleotide excision repair pathway results in increased cellular resistance to the therapeutic compound.
In some aspects of such embodiments, increased activity of the base excision repair pathway results in increased cellular resistance to the therapeutic compound.
In some aspects of such embodiments, the cellular resistance further comprises clinical resistance to the therapeutic compound.
In some aspects of such embodiments, the at least one cell comprises at least one neoplastic cell.
In some aspects of such embodiments, the at least one neoplastic cell is selected from a breast cancer cell, melanoma cell, prostate cancer cell, colorectal cancer cell, head and neck cancer cell, lung cancer cell, colon cancer cell, oesophageal cancer cell, gastric cancer cell, testicular cancer cell, and ovarian cancer cell.
In some aspects of such embodiments, the cell is a mammalian cell.
In some aspects of such embodiments, the mammalian cell is a human cell.
In some aspects of such embodiments, the cell is in vivo.
In some aspects of such embodiments, the cell is in vitro.
In some aspects of such embodiments, the nucleic acid encoding GLI1 comprises a nucleic acid encoding GLI1-130, or the GLI1 protein comprises GLI1-130 isoform.
Some embodiments include methods for reducing the dosage of a therapeutic agent needed to treat a disorder in a subject comprising reducing the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein, in a cell of the subject.
In some aspects of such embodiments, the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein is reduced by administering to the subject an isolated nucleic acid selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1, such as a sequence encoding GLI1-130, or a fragment thereof, a sequence encoding antisense GLI1, such as a sequence encoding antisense GLI1, or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the therapeutic compound comprises a chemotherapeutic agent.
In some aspects of such embodiments, the chemotherapeutic agent is selected from a platinum-based compound such as cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, and triplatin tetranitrate, a nitrogen mustard such as cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, and ifosfamide, a nitrosoureas such as carmustine, lomustine, and streptozocin, an alkyl sulfonate such as busulfan, thiotepa, procarbazine, and altretamine.
In some aspects of such embodiments, the chemotherapeutic agent comprises a platinum-based compound.
In some aspects of such embodiments, the platinum-based compound comprises cisplatin.
In some aspects of such embodiments, the chemotherapeutic agent is selected from taxol and doxirubicin.
In some aspects of such embodiments, the therapeutic compound is an agent for which increased expression of ERRC1, XPD, or XRCC1 results in increased cellular resistance.
In some aspects of such embodiments, increased activity of the nucleotide excision repair pathway results in increased cellular resistance to the therapeutic compound.
In some aspects of such embodiments, increased activity of the base excision repair pathway results in increased cellular resistance to the therapeutic compound.
In some aspects of such embodiments, the cellular resistance further comprises clinical resistance to the therapeutic compound.
In some aspects of such embodiments, the disorder comprises cancer.
In some aspects of such embodiments, the cancer is selected from breast cancer, melanoma, prostate cancer, colorectal cancer, head and neck cancer, lung cancer, colon cancer, oesophageal cancer, gastric cancer, testicular cancer, and ovarian cancer.
In some aspects of such embodiments, the subject is a mammal.
In some aspects of such embodiments, the mammal is a human.
In some aspects of such embodiments, the nucleic acid encoding GLI1 comprises a nucleic acid encoding GLI1-130, or the GLI1 protein comprises GLI1-130 isoform.
Some embodiments include methods for identifying a therapeutic compound comprising: contacting a target cell with a test compound; and determining whether the test compound reduces the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein, in the target cell.
Some aspects of such embodiments also include comparing the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein, in a target cell which has not been contacted with the test compound to the level of a nucleic acid encoding, such as a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein, in a target cell contacted with the test compound.
Some aspects of such embodiments also include determining whether the test compound reduces the level of c-jun (Ser 63) protein in the target cell.
Some aspects of such embodiments also include comparing the level of c-jun (Ser 63) protein in a target cell which has not been contacted with the test compound to the level of c-jun (Ser 63) protein in a target cell contacted with the test compound.
Some aspects of such embodiments also include determining whether the test compound does not substantially decrease the level of GLI2 protein or a nucleic acid encoding GLI1 in the target cell.
Some aspects of such embodiments also include comparing the level of GLI2 protein in a target cell which has not been contacted with the test compound to the level of GLI2 protein in a target cell contacted with the test compound.
Some aspects of such embodiments also include determining whether the test compound reduces the level of OPN protein in the target cell.
Some aspects of such embodiments also include comparing the level of OPN protein in a target cell which has not been contacted with the test compound to the level of OPN protein in a target cell contacted with the test compound.
In some aspects of such embodiments, the target cell comprises a neoplastic cell.
In some aspects of such embodiments, the neoplastic cell is selected from a breast cancer cell, melanoma cell, prostate cancer cell, colorectal cancer cell, head and neck cancer cell, lung cancer cell, colon cancer cell, oesophageal cancer cell, gastric cancer cell, testicular cancer cell, and ovarian cancer cell.
In some aspects of such embodiments, the target cell is a mammalian cell.
In some aspects of such embodiments, the mammalian cell is a human cell.
In some aspects of such embodiments, the nucleic acid encoding GLI1 comprises a nucleic acid encoding GLI1-130, or the GLI1 protein comprises GLI1-130 isoform.
Some embodiments include methods for assessing the effectiveness of a compound or agent in treating a disorder comprising measuring the level of a nucleic acid encoding OPN or the level of OPN protein in a subject who has been contacted with the compound or agent.
Some aspects of such embodiments, also include comparing the level of a nucleic acid encoding OPN or the level of OPN protein in a subject having the disorder who has been contacted with the compound or agent to the level of a nucleic acid encoding OPN or the level of OPN protein in a subject who does not have the disorder.
Some aspects of such embodiments, also include comparing the level of a nucleic acid encoding OPN or the level of OPN protein in a subject having the disorder who has been contacted with the compound or agent to the level of a nucleic acid encoding OPN or the level of OPN protein in a subject who has not been contacted with the compound or agent.
In some aspects of such embodiments, a decrease in the level of a nucleic acid encoding OPN or the level of OPN protein is indicative of a favorable prognosis.
In some aspects of such embodiments, the agent is a nucleic acid is selected from the group consisting of a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1, such a sequence encoding GLI1130, or a fragment thereof, a sequence encoding antisense GLI1, such as a sequence encoding antisense GLI1-130, or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof, such as an antisense nucleic acid complementary to a sequence encoding GLI1-130 or a fragment thereof.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the disorder comprises cancer.
In some aspects of such embodiments, the cancer is selected from breast cancer, melanoma, prostate cancer, colorectal cancer, head and neck cancer, lung cancer, colon cancer, oesophageal cancer, gastric cancer, testicular cancer, and ovarian cancer
In some aspects of such embodiments, the subject is a mammal.
In some aspects of such embodiments, the mammal is a human.
Some embodiments include methods for assessing the potential effectiveness of a nucleic acid as a therapeutic agent comprising determining whether the nucleic acid reduces the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein in a cell, wherein the nucleic acid is identified as having potential effectiveness as a therapeutic agent if the nucleic acid reduces the level of the nucleic acid encoding GLI1, such as a nucleic acid encoding GLI1-130, or the level of GLI1 protein, such as GLI1-130 protein in said cell.
Some aspects of such embodiments, also include determining whether the nucleic acid has no substantial effect on the level of a nucleic acid encoding GLI2 or the level of GLI2 protein in a cell, wherein the nucleic acid is identified as having potential effectiveness a therapeutic agent if the nucleic acid has no substantial effect on the level of the nucleic acid encoding GLI2 or the level of the GLI2 protein in said cell.
In some aspects of such embodiments, the nucleic acid encoding GLI1 comprises a nucleic acid encoding GLI1-130, or the GLI1 protein comprises GLI1-130 isoform.
Some embodiments include nucleic acids identified as having potential effectiveness as a therapeutic agent by any one of the methods provided herein.
Some embodiments include methods of regulating transcription from the OPN promoter in a cell comprising regulating the activity of Hedgehog (Hh).
In some aspects of such embodiments, the activity of Hedgehog (Hh) is regulated by regulating the amount or activity of a nucleic acid encoding GLI1 or the amount or activity of GLI1 protein.
In some aspects of such embodiments, regulating the amount or activity of a nucleic acid encoding GLI1, such as GLI1-130, or the amount or activity of GLI1 protein, such as GLI1-130 protein comprises administering to said cell an isolated nucleic acid selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1, such as GLI1-130, or a fragment thereof, a sequence encoding antisense GLI1, such as GLI1-130 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof, such as an antisense nucleic acid complementary to a sequence encoding GLI1-130 or a fragment thereof.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the Hedgehog (Hh) comprises Sonic Hedgehog (SHh).
In some aspects of such embodiments, the cell comprises a neoplastic cell.
In some aspects of such embodiments, the neoplastic cell is selected from a breast cancer cell, melanoma cell, prostate cancer cell, colorectal cancer cell, head and neck cancer cell, lung cancer cell, colon cancer cell, oesophageal cancer cell, gastric cancer cell, testicular cancer cell, and ovarian cancer cell.
In some aspects of such embodiments, the cell is a mammalian cell.
In some aspects of such embodiments, the mammalian cell is a human cell.
In some aspects of such embodiments, the cell is in vivo.
In some aspects of such embodiments, the cell is in vitro.
Some embodiments include methods of regulating osteoclast differentiation in a cell comprising regulating the activity of Hedgehog (Hh) in said cell.
In some aspects of such embodiments, the activity or effects of Hedgehog (Hh) are regulated by regulating the levels or activity of OPN.
In some aspects of such embodiments, the activity of Hedgehog (Hh) is regulated by regulating the amount or activity of a nucleic acid encoding GLI1, such as GLI1-130, or the amount or activity of GLI1 protein, such as GLI-130 protein.
In some aspects of such embodiments, regulating the amount or activity of a nucleic acid encoding GLI1, such as GLI1-130, or the amount or activity of GLI1 protein, such as GLI1-130 protein, comprises administering to said cell an isolated nucleic acid selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1, such as GLI1-130, or a fragment thereof, a sequence encoding antisense GLI1, such as GLI1-130, or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1, such as GLI-130, or a fragment thereof.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the Hedgehog (Hh) comprises Sonic Hedgehog (SHh).
In some aspects of such embodiments, the cell comprises a neoplastic cell.
In some aspects of such embodiments, the neoplastic cell comprises a breast cancer cell.
In some aspects of such embodiments, the cell is a mammalian cell.
In some aspects of such embodiments, the mammalian cell is a human cell.
In some aspects of such embodiments, the cell is in vivo.
In some aspects of such embodiments, the cell is in vitro.
Some embodiments include methods of diminishing the levels of osteolysis in an individual having breast cancer comprising regulating the activity of Hedgehog (Hh).
In some aspects of such embodiments, the activity or effects of Hedgehog (Hh) are regulated by regulating the levels or activity of OPN.
In some aspects of such embodiments, the activity of Hedgehog (Hh) is regulated by regulating the amount or activity of a nucleic acid encoding GLI1 or the amount or activity of GLI1 protein.
In some aspects of such embodiments, regulating the amount or activity of a nucleic acid encoding GLI1, such as GLI1-130, or the amount or activity of GLI1 protein, such as GLI1-130 protein, comprises administering to said individual an isolated nucleic acid selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1, such as GLI1-130, or a fragment thereof, a sequence encoding antisense GLI1, such as GLI1-130, or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1, such as GLI-130, or a fragment thereof.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the Hedgehog (Hh) comprises Sonic Hedgehog (SHh).
Some embodiments include methods for assessing the extent to which breast cancer has metastasized to the bone comprising determining the level of expression of at least one protein selected from the group consisting of OPN, CTSK, and MMP9 or a nucleic acid encoding at least one of said OPN, CTSK, or MMP9 proteins in a bone sample.
Some embodiments include uses of an isolated nucleic acid comprising a sequence encoding GLI1 or a fragment thereof, a sequence encoding antisense GLI1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof for treatment of a disorder.
In some aspects of such embodiments, the isolated nucleic acid is selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the disorder is selected from breast cancer, melanoma, prostate cancer, colorectal cancer, head and neck cancer, lung cancer, colon cancer, oesophageal cancer, gastric cancer, testicular cancer, and ovarian cancer.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1-130 or a fragment thereof, a sequence encoding antisense GLI1-130 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1-130 or a fragment thereof for treatment of a disorder.
Some embodiments include isolated nucleic acids comprising a sequence encoding GLI1 or a fragment thereof, a sequence encoding antisense GLI1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof for use as a medicament.
In some aspects of such embodiments, the isolated nucleic acid is selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1-130 or a fragment thereof, a sequence encoding antisense GLI1-130 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1-130 or a fragment thereof.
Some embodiments include uses of an isolated nucleic acid comprising a sequence encoding GLI1 or a fragment thereof, a sequence encoding antisense GLI1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof for the preparation of a medicament for treating a disorder.
In some aspects of such embodiments, the isolated nucleic acid is selected from a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
In some aspects of such embodiments, the nucleic acid comprises a sequence selected from SEQ ID NO.s:01-10.
In some aspects of such embodiments, the nucleic acid comprises SEQ ID NO:01.
In some aspects of such embodiments, the disorder is selected from breast cancer, melanoma, prostate cancer, colorectal cancer, head and neck cancer, lung cancer, colon cancer, oesophageal cancer, gastric cancer, testicular cancer, and ovarian cancer.
In some aspects of such embodiments, the nucleic acid comprises a sequence encoding GLI1-130 or a fragment thereof, a sequence encoding antisense GLI1-130 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1-130 or a fragment thereof for treatment of a disorder.
Some embodiments of the present invention relate to methods and compositions for treating disorders relating to increased activity of the Hedgehog pathway. Some embodiments include methods and compositions for modulating the activity of the Hedgehog pathway. Additional methods and compositions relate to treating neoplastic cells and cancer.
The Hedgehog pathway has a central role in developmental patterning (ontogeny), in the maintenance of stem or progenitor cells in many adult tissues, and has been demonstrated to be active in multiple cancer types. Active Hedgehog signaling is also reported to influence the tumor stromal microenvironment and support stem cells in the tumor in an undifferentiated, proliferative state.
Hedgehog signaling in mammalian cells is mediated by the GLI family of zinc finger transcription factors comprising GLI1, GLI2, and GLI3. GLI1 is a strong transcriptional activator; GLI2 has both activator and repressor functions; and GLI3 is mostly a repressor. In the Hedgehog ligand-dependent pathway, in the absence of the ligand, Desert hedgehog (DHH), Indian hedgehog (IHH), or Sonic hedgehog (SHH), the Hedgehog signaling pathway is inactive, GLI1 is sequestered in the cytoplasm and repressed for its transcription activity. Binding the Hedgehog ligands to the receptor patched-1 or patched-2 (PTCH1 or -2) changes the GLI code: the transmembrane protein, Smoothened (Smo) is activated, and GLI1 is activated by release from a large protein complex and translocates to the nucleus to function as a transcriptional activator (Ingham, P. W., et al. (2001) Genes Dev. 15, 3059-3087; FIG. 1).
GLI1 is encoded by two alternatively spliced transcripts which give rise to at least five different protein isoforms, some of which exist in more than one form. As used herein the term “GLI1 protein” includes all of these isoforms, including the isoforms listed in Table 4 below. As used herein the term “nucleic acid encoding GLI1” includes nucleic acids encoding all of the GLI1 isoforms, including the isoforms listed in Table 4 below.
Signaling via the Hedgehog pathway plays a determinative role in the development of the dorsal brain, near the sites of origin of melanogenic precursors. The Hedgehog pathway is required for normal proliferation of human melanocytes in vitro and for proliferation and survival of human melanoma in vivo. Activation of Hedgehog signaling results in transcriptional activation of the expression of several genes including insulin-like growth factor-binding protein, cyclin D2, and osteopontin (OPN).
Expression of GLI1 and OPN increase progressively with the progression of melanoma from primary cutaneous cancer to metastatic melanoma in clinically derived specimens. OPN is a direct transcriptional target of GLI1. OPN expression is stimulated in the presence of Hedgehog ligands and inhibited in the presence of the Smo inhibitor, cyclopamine. Transcriptional silencing of GLI1 negatively impacts OPN expression and compromises the ability of cancer cells to proliferate, migrate, and invade in vitro and interferes with their ability to grow as xenografts and spontaneously metastasize in nude mice. These altered attributes can be rescued by re-expressing OPN in the GLI1-silenced cells, suggesting that OPN is a critical downstream effector of active GLI1 signaling. These findings suggest that the GLI1-mediated upregulation of OPN promotes malignant behavior of cancer cells. Expression levels of GLI1 and OPN are significantly elevated in surgically excised metastatic melanoma specimens compared with surgically obtained basal and squamous cell carcinomas and primary melanoma samples. The Hedgehog pathway acts via OPN to regulate malignant behavior of cancer cells. Thus, there exists a clinically relevant relationship between OPN and Hedgehog signaling.
Embodiments of the present invention relate to the finding that reducing the expression of GLI1 reduces the metastatic potential of particular cells. Some embodiments of the present invention relate to methods and compositions for reducing the expression level of GLI1 protein or the expression level of a nucleic acid encoding GLI1 in a cell or a subject. Some such methods and compositions can be useful to kill or retard the growth of neoplastic cells. More such methods can be useful to treat a disorder in a subject in which the disorder is related to an increase in the activity of the Hedgehog pathway. Such disorders can include cancer, for example, ovarian cancer and breast cancer.
More embodiments include methods and compositions for increasing the sensitivity of cells to therapeutic compounds. Such methods can be useful to reduce the dosage of a therapeutic agent to treat a subject. For example, some methods and compositions provided herein can be used to increase the sensitivity of cells to chemotherapeutic compounds. In some such methods, the effective amount of a chemotherapeutic compound to treat a subject can be reduced.
The role of the Hedgehog pathway has been documented in several cancer histotypes (e.g., Watkins, D. N., et al. (2003) Nature 422, 313-317). The activities of this pathway have been attributed to several mediators, such as platelet-derived growth factor (Xie, J., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 9255-9259), fibroblast growth factor (Sun, X., et al. (2000) Nat. Genet. 25, 83-86), bone morphogenic protein (Yu, J., et al. (2002) Development 129, 5301-5312), Notch (Hallahan, A. R., et al. (2004) Cancer Res. 64, 7794-7800), and Wnt (Madison, B. B., et al. (2005) Development 132, 279-289), which have been identified as Hedgehog target genes in various models. However, few universal target genes have been identified across different systems and much work still needs to be done to determine how Hedgehog overexpression contributes to tumorigenesis.
Studies provided herein indicate that signaling via the Hedgehog pathway can transcriptionally up-regulate OPN, an oncogene that has been widely reported to promote tumorigenesis, tumor progression, and metastasis in several cancer types. The regulation of OPN by the transcription factor GLI1 is integral to the malignant behavior of cancer cells as evidenced by the impaired ability of tumor cells to migrate, invade, and grow in vivo as xenografts when endogenous GLI1 expression is silenced. OPN is a secreted protein that influences multiple downstream signaling events that allows cancer cells to resist apoptosis, invade through extracellular matrix, evade host immunity (Bellahce'ne, A., et al. (2008) Nat. Rev. Cancer 8, 212-226), and influence growth of indolent tumors (McAllister, S. S., et al. (2008) Cell 133, 994-1005). OPN induces integrin and CD44-mediated migration via hepatocyte growth factor, its receptor, Met, and epidermal growth factor and enhances the invasive ability of cells by inducing the expression of proteases such as MT1-matrix metalloproteinase, matrix metalloproteinase-2, and urokinase plasminogen activator (Tuck, A. B., et al. (2003) Oncogene 22, 1198-1205). Clinically, OPN expression is up-regulated in several malignancies including breast cancer, melanoma, prostate cancer, colorectal cancer, and head and neck cancer (Coppola, D., et al. (2004) Clin. Cancer Res. 10, 184-190). OPN constitutes a component of the secretome of several melanoma-derived cell lines and is expressed in metastatic breast cancer cell lines (Riker, A. I., et al. (2008) BMC Med. Genomics 1, 13). Studies have reported an increase in levels of OPN in melanoma-derived cell lines (Rangaswami, H., et al. (2007) Oncol. Rep. 18, 909-915).
Expression of OPN is 13-fold higher when comparing thin melanomas to metastatic melanomas. Expression of GLI1 increases notably as cutaneous cancer progresses from a stage of melanoma in situ to intermediate and thick melanoma to metastatic melanoma. The increase in GLI1 expression is paralleled by an increase in OPN levels. Overall, such observations underscore the role of enhanced Hedgehog signaling via increased GLI1 transcriptional activity in potentiating the malignant behavior of melanoma cells and contributing to disease progression.
The Hedgehog pathway is aberrantly active in several cancer types, including breast cancer and melanoma (Xuan, et al. (2009) J. Cancer Res. Clin. Oncol. 135, 235-240). Hedgehog pathway components were detected in nevi, melanoma, and lymph node metastases of melanoma (Stecca, B., et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 5895-5900). Overexpression of GLI1 induced the expression of Snail, whereas blockade of Hedgehog signaling by the inhibitor cyclopamine suppressed pancreatic cancer invasion and metastasis by inhibiting EMT (Li, X., Deng, et al. (2007) Oncogene 26, 4489-4498). Experiments described herein are consistent with such findings in view of the observation of a loss of mesenchymal markers by abrogating GLI1 expression. EMT-related genes (N-cadherin, OPN, and osteonectin) have been reported to contribute to the promotion of the metastatic phenotype in primary cutaneous malignant melanomas by supporting specific adhesive, invasive, and migratory properties (Alonso, S. R., et al. (2007) Cancer Res. 67, 3450-3460). Moreover, these findings support observations provided herein showing that GLI1 silencing attenuates malignancy-associated attributes, such as invasion, migration, and motility. Results provided herein show that GLI1 silencing retards the tumor (xenograft) rate in the early phase. In the experiments described herein, after day 11 post-injection, the growth of GLI1-silenced tumors proceeded at the same rate as that of controls. This data has multiple implications, for example, it is likely that over time, a revertant population outgrew the GLI1-silenced cells. These revertants may have either lost the effects of RNA interference or may have by-passed the requirement for GLI1 signaling. In this case, the cells may have utilized other signaling pathways to up-regulate OPN. In addition, trace levels of OPN secreted by GLI1-silenced cells (
The MDA-MB-435 cell line was used as a model as it produces abundant levels of OPN (Samant, R. S., et al. (2007) Mol. Cancer 6:6). This model system, which endogenously expresses high levels of OPN, supports a role for the Hedgehog pathway in regulating malignant cell behavior. Moreover, findings provided herein can have implications on multiple cancer histotypes that overexpress OPN (Brown, L. F., et al. (1994) Am. J. Pathol. 145, 610-623).
Hedgehog ligands and OPN, the signaling intermediate of the active Hedgehog pathway, are secreted molecules. This allows them to influence the behavior of cells in the tumor microenvironment. OPN has been documented to influence the behavior of cells in a paracrine manner. Although serum OPN influences the migratory behavior of melanoma cells and tumor-derived OPN inhibits nitric-oxide synthase activity of macrophages, OPN produced by fibroblasts is able to influence growth of pre-neoplastic cells (Hayashi, C., et al. (2007) J. Cell. Biochem. 101, 979-986). Thus, active Hedgehog signaling in a subset of cancer cells can potentially be amplified by secretion of OPN into the tumor microenvironment. The secreted OPN, in turn, can promote malignant behavior in neighboring cancer cells, regardless of the status of the Hedgehog pathway.
In addition, whereas OPN is capable of long-range signaling, the secreted Hedgehog ligand proteins participate in short-range signaling and can move many cell diameters from their source of production and often control developmental outcomes in a concentration-dependent manner. For example, during ventral spinal cord patterning, SHH forms a ventral-to-dorsal gradient with different concentrations specifying distinct pools of neural progenitors (Stamataki, D., et al. (2005) Genes Dev. 19, 626-641). It is likely that such a situation also prevails in a tumor; in which case, the Hedgehog ligands produced by a subpopulation of cells within a tumor can trigger activation of the pathway in the recipient cell.
Hedgehog Pathway and Bone MetastasisLevels of OPN are significantly elevated in the tumors and plasma of patients with metastatic breast cancer and are notably associated with decreased patient survival (Cook, A. C., et al. (2005). Mol Carcinog 43, 225-236). Tumor cells upregulate OPN synthesis and secretion by osteoblasts and cause pathologic activation of osteoclasts, resulting in a net loss of bone (Nemoto H, R. S., et al. (2001). J Bone Miner Res 16, 652-659). Thus, OPN enhances metastasis of breast cancer to bone. OPN is a transcriptional target of Gli1. Gli1 is a transcription factor of the Hedgehog pathway that activates transcription of Hedgehog-target genes. The Hedgehog pathway is activated in a variety of cancer types, thus making it a putative therapeutic target.
Hedgehog Pathway and Osteoclastogenesis and OsteolysisBreast cancer cells preferentially metastasize to the bone. Once within bone, an interaction ensues between breast cancer cells and the cells within the bone microenvironment. Breast cancer cells secrete various factors that stimulate osteoblasts and osteoclasts and other cells within the bone; these in turn secrete factors that stimulate the tumor cells, creating a vicious cycle that nurtures the development and propagation of bone metastases. OPN forms a component of a “bonemetastasis signature” of breast cancer cells i.e., breast cancer cells that metastasized to bone had increased OPN expression. Furthermore, OPN functionally enhanced incidence of bone metastases by breast cancer cells in concert with interleukin-11. OPN is one of the abundant non-collagenous proteins in bone. It is a bone matrix protein that promotes osteoclast function and is consistently overexpressed in highly metastatic cells. Ultrastructural immunocytochemical studies show that the most prominent accumulation of OPN is seen at cement lines in remodeling bone, and at laminae limitantes at bone surfaces. It is localized to cell-matrix and matrix-matrix interfaces in mineralized tissue, where it is deposited as the result of osteoclast action. Moreover, OPN appears to be an important component in the communication between osteoclasts and osteoblasts, and there is strong evidence for the involvement of OPN in the formation, migration and attachment of osteoclasts and in their resorptive activity. Importantly, interfering with the adhesion of osteoclasts to osteopontin by RGD-peptides abolishes their resorptive activity.
Nearly 42% of primary breast tumors express moderate to strong levels of OPN and 83% of bone metastases resulting from these tumors express OPN. OPN expression, specifically within the tumor cells, reciprocally correlates with patient survival. Clinical studies have revealed a correlation between plasma OPN, tumor burden and prognosis in patients with breast cancer metastasis. The levels of OPN in plasma of patients with breast cancer are significantly higher in those with bone metastasis compared to those who do not have bone metastasis. Moreover, the level of OPN increases with progression of the disease. Among these women, those with highest levels of OPN (more than 2000 μg/ml) show poor survival compared to those with OPN levels between 1000-1500 μg/ml. Whether the circulating OPN impacts ‘homing’ of breast cancer cells to bone is still not known. Functionally, OPN expression is vital to the tumorigenic ability of cells. Expression of OPN in OPN-negative breast cancer cells increases their adhesion to bone marrow cells and OPN knock-out mice display significantly lower incidence of bone metastases. In bone, tumor derived OPN plays a vital role in the establishment of vasculature by mediating adhesion to endothelial cells, co-operating with VEGF, and preventing apoptosis of the endothelial cells.
Expression of OPN is regulated, in part, by the Hedgehog (Hh) pathway. The Hh pathway has been reported to be aberrantly activated in breast cancer. In the absence of the ligand, Desert hedgehog (DHH) or Indian hedgehog (IHH) or Sonic hedgehog (SHH), the Hh signaling pathway is inactive. Ligand molecules bind to the receptor Patched (PTCH) thereby alleviating PTCH-mediated suppression of smoothened (SMOH), leading to activation of the pathway through the transcription of target genes mediated by the GLI transcription factors. As described herein, breast cancer cells express Hh ligands. These ligands can mediate a crosstalk directly with osteoclasts and activate expression of OPN in the osteoclasts; this promotes osteoclast maturation and resorptive activity. As such, breast cancer cells can directly influence osteoclast development and activity.
Hedgehog Pathway and Resistance to ChemotherapyCells may develop resistance to a range of chemotherapeutic compounds by reverting to a stem cell-like state. The Hedgehog pathway including genes such as GLI1 play a role in the development and maintenance of a stem cell-like phenotype (Peacock C D, et al. Proc Natl Acad Sci USA 104:4048-4053, 2007). Work on the proximal molecular causes of cellular and clinical resistance to platinum compounds has focused on DNA damage and repair, the nucleotide excision repair (NER) pathway, and ERCC1.
Nucleotide Excision RepairMore than 30 genes are involved in the NER process, which includes activities such as DNA damage recognition, helicase functions of XPB and XPD, damage excision, and gap-filling and ligation (Reed, E. Cisplatin and platinum analogs. in: Cancer Principles and Practice of Oncology; 8th Edition. Lippincott, Williams, and Wilkins; Philadelphia, pp 419-26, 2008). Understanding the molecular and pharmacologic control of NER, allows development of new platinum anticancer agents, and non-platinum agents that damage DNA and/or modulate DNA repair.
In NER, the DNA damage excision step is rate-limiting to the process, where the last sub-step is the 5′ incision into the DNA strand, relative to the site of covalent damage. This 5′ incision occurs after the 3′->5′ and 5′->3′ helicase functions of the repairosome, and after the 3′ incision. The 5′ incision is executed by the ERCC1-XPF heterodimer.
ERCC1ERCC1 is highly conserved across organisms (Xu H, et al. The Plant Journal 13:823-829, 1998). The E. coli homologue is UvrC, which performs the 5′ incision during the conduct of the NER process. Exon VIII of ERCC1 has high homology with uvrC of E coli (Lin J-J, et al. J Biol Chem 267:17688-17692, 1992). In E. coli, the uvrABC protein complex executes NER-types of DNA repair. Within the E. coli complex, uvrC executes the 5′ and the 3′ DNA strand-cutting steps that excise platinum-DNA damage (Verhoeven E E A, et al. J Biol Chem 275:5120-5123, 2000). In mammalian NER, exon VIII of ERCC1 may serve the same DNA-strand cutting function as uvrC in E. coli. An alternatively spliced form of ERCC1 exists in human malignant and non-malignant tissues, which lacks exon VIII. This alternatively spliced variant of ERCC1 may possibly have an inhibitory role for NER, in human cells. These NER activities appear to be distinct from the ERCC1 roles in double strand break repair (Ahmad A, et al. Mol Cell Biol 28:5082-5092, 2008).
Some studies have utilized paired Chinese hamster ovary (CHO) cells having functional ERCC1 or non-functional ERCC1 (Lee, K. B., et al. Carcinogenesis, 14:2177-2180, 1993). CHO cells lacking a functional ERCC1 (43:3B cells) were super-sensitive to IC50 doses of cisplatin, and showed no detectable ability to repair cisplatin-DNA adduct. In CHO cells having a functional ERCC1 (83:J5 cells), cisplatin-DNA adduct repair capability was intact and there was an increased level of cellular resistance to cisplatin.
ERCC1 is biomarker for the overall activity of NER in human cell lines and tissues (Reed E. New Eng J. Med 355:1054-1055, 2006; Reed E. Clinical Cancer Research, 11:6100-6102, 2005). Non-functionality of ERCC1 results in a severe DNA repair deficit phenotype, in vitro or in vivo.
In NER, the ERCC1-XPF heterodimer executes the 5′ incision into the DNA strand, freeing the DNA segment that has covalent bulky DNA damage (Ahmad A, et al. Mol Cell Biol 28:5082-5092, 2008). The ERCC1-XPF heterodimer also plays a role in drug-cross-link induced double-strand break repair, via an end-joining mechanism that is Ku86-independent. Detailed structure-function analyses of both proteins show that XPF is a scaffold protein (Al-Minawi A Z, et al. Nucleic Acids Res 27 Aug. 2009).
Cisplatin damages DNA by inducing double strand breaks, single strand breaks, platinum-DNA adducts, and DNA-platinum-protein adducts. ERCC1 plays a role in cellular resistance to cisplatin including excision of platinum-DNA damage from cellular DNA, and repair of double-strand breaks (Altaha R et al. Int J Mol Med, 14:959-970, 2004).
In human ovarian cancer tissues, high levels of ERCC1 mRNA have been observed in tissues from patients that were clinically resistant to platinum therapy; and low levels of ERCC1 mRNA have been observed in tissues from patients that were clinically sensitive to platinum therapy (Dabholkar, M., et al. J Nat'l Cancer Inst, 84:1512-1517, 1992).
In human ovarian cancer cells, ERCC1 is up-regulated from 1 hr treatment with cisplatin (Li Q, et al. J Biol Chem, 273:23419-23425, 1998). Subsequent to treating cells with IC50 dose of cisplatin, A2780-CP70 human ovarian cancer cells increase expression of mRNA and protein for c-jun and c-fos, with mRNA levels peaking at 1-2 hr and c-jun protein levels peaking at 3-5 hr after treatment. Phosphorylation of c-jun protein has been observed to be greatly enhanced at 1 hr after cisplatin treatment, and peaks at 15-fold over baseline at 3-5 hr after cisplatin treatment. Phosphorylation activates c-jun protein, which in turn activates AP1, which leads to increased transcription of ERCC1. ERCC1 mRNA levels peak levels at 3-4 hr. ERCC1 protein levels begin to rise within 1 hr, and peak at 24 hr.
In cisplatin-treated cells, ERCC1 mRNA degrades with a half-life of 24 hr, in contrast to a half-life of 14 hr in untreated cells. This suggests mechanisms that are activated in response to DNA damage that prolong the period during which ERCC1 may be active.
More studies suggest that ERCC1 up-regulation through AP1 may occur through the JNK/SAPK pathway, or the ERK pathway (Li Q, et al. Cellular and Molecular Life Sciences, 55:456-466, 1999). The ERK pathway can be activated by cell exposure to phorbol ester.
Modulating expression levels of ERCC1 may modulate DNA repair activities in a cell, and alter cellular sensitivity to agents that affect DNA repair, such as cisplatin. In one study, a dominant negative AdA-FOS construct to inhibit AP1 binding was transfected into the human ovarian cell line, A2780-CP70, prior to cisplatin exposure (Li Q, et al. Effect of interleukin-1 and tumor necrosis factor on cisplatin-induced ERCC1 mRNA expression in a human ovarian carcinoma cell line. Anticancer Research, 18: 2283-2287, 1998). ERCC1 up-regulation was severely blunted after cisplatin exposure, platinum-DNA adduct repair was severely reduced, and cells were several-fold more sensitive to cisplatin treatment. A series of compounds were assessed for their ability to blunt ERCC1 up-regulation. All agents that blunted ERCC1 up-regulation, also inhibited platinum-DNA adduct repair and enhanced sensitivity to cisplatin. The following compounds blunted ERCC1 up-regulation: heavy metals including platinum, chromium; cycloheximide; α-amanitin; actinomycin D; interleukin 1-α; lactacystin; N-acetyl-leucyl-leucyl-norleucinal; SU5416; cyclosporin A; and herbimycin A.
The ERCC1 gene is alternatively spliced. One splice variant lacks exon VIII, an exon which has high homology to uvrC in E. coli. The occurrence of the variant lacking exon VIII, correlates to a decrease in the ability of cells to repair platinum-DNA adduct, namely, the higher the percent alternatively spliced ERCC1, the lower the DNA adduct repair capability. Another splice variant of ERCC1 mRNA involves the 5′ UTR, and may involve transcriptional regulation by the gene RFX1 (Yu J J, et al. Oncogene, 20:7694-7698, 2001).
A specific polymorphism at codon 118 of the NER gene ERCC1 in exon IV may be clinically relevant (Yu J J, et al. International Journal of Oncology, 16:555-560, 2000). This polymorphism is associated with reduced mRNA expression of the gene, reduced protein expression, reduced platinum-DNA adduct repair, enhanced cellular sensitivity to cisplatin, and more favorable clinical outcomes from platinum-based chemotherapy in patients with cancers including ovarian cancer, lung cancer, and colorectal cancer.
Loss of heterozygosity may occur in some ovarian cancer cells and tissues for the 19q region that contains ERCC1 (Yu, J J, et al. Cancer Letters, 151:127-132, 2000). This has also been observed in malignant gliomas, with changes in gene copy number for ERCC1 and for XPD (Liang, B. C., et al. J Neuro Oncol, 26:17-23, 1995). These changes do not appear to correlate with alterations in mRNA or protein expression of these genes, or with observed clinical outcomes. Indeed, variations in XPA mRNA expression have been observed in the absence of mutations or changes in gene copy number (States, J. C. et al. Cancer Letters, 108:233-237, 1996).
Coordinate Expression of NER Genes in Human Ovarian Cancer TissuesERCC1, XPA, XPB, and XPD of the NER repairosome appear to be coordinately up-regulated and down-regulated in tissues such as human ovarian cancer, non-malignant bone marrow, and human brain (e.g., Dabholkar, M., et al. J Clin Invest, 94:703-708, 1994).
In a study using human ovarian cancer, genes of the NER pathway including ERCC1, XPA, XPB, and CSB were examined, along with the genes, MDR1 and MT-II. The NER genes were up-regulated in platinum-resistant tissues together in the absence of upregulation of MDR1 and of MT-II. Tissues that responded to chemotherapy, namely, platinum-sensitive tissues, consistently showed low level expression of these NER genes. In another study, human ovarian cancer tumors were examined for coordinate mRNA expression of ERCC1, XPB, and XPD. Five different histological types were investigated: clear cell, endometrioid, serous, mucinous, and undifferentiated tumors. Clear cell tumors of the ovary are known for being particularly chemoresistant. In this study, clear cell tumors had consistently higher mRNA levels of ERCC1, XPB, and XPD, and the degree of coordinate expression was statistically significantly greater in clear cell tumors, than in any of the other histologies.
In another study, evidence of coordinate expression of NER genes was investigated in human brain tissues using malignant, and adjacent non-malignant specimens. In high grade gliomas, there was strong coordinate mRNA expression of ERCC1 and XPA, as assessed by linear regression analysis. When malignant and non-malignant glial tissues were assayed from the same patients, there was poor coordinate expression of ERCC1 mRNA. This suggests that during the conversion of cells from the normal to the malignant state, ERCC1 is altered and possibly all of NER is altered. This type of circumstance has been confirmed using different DNA repair genes in an examination of direct reversal of DNA damage caused by methylating agents.
In sum, genes in the NER pathway seem to display several common essential characteristics in human malignant tissues that have some degree of clinical sensitivity to cisplatin and other platinum analogues. For example, higher levels of expression of mRNA and of protein are seen in platinum-resistant tissues, as compared to platinum-sensitive tissues. In addition, the degree to which NER is tightly coordinated between the various genes involved in the process, contributes to that tissue's ability to repair platinum-DNA damage and resist platinum-based therapy.
Base Excision RepairThe base excision repair pathway is an evolutionarily conserved mechanism for repair of several types of DNA lesions, including oxidative lesions, alkylation, and incorporation of inappropriate bases (Hegde, M. L., et al. (2008) Cell Res. 18, 27-47). The primary source of these lesions is reactive oxygen species, whether generated endogenously or due to genotoxic agents. The base excision repair pathway functions to maintain genomic integrity via a high fidelity repair process and is thus anti-mutagenic and anti-carcinogenic (D'Errico, M., et al. (2008) Mutat Res. 659, 4-14). This pathway usually has four or five enzymatic steps, involving a DNA glycosylase, such as OGG1 or NTH1, an AP endonuclease (REF1/APE1), a DNA polymerase, such as POLB and POLD, and a DNA ligase (LIG1 or LIG3) (Mitra, S., et al. (1997) Mol Cells. 7, 305-312). DNA glycosylases such as OGG1 and NTH1, recognize specific subsets of damaged bases, excise the damaged base, and may also incise at the site of the excised base due to an intrinsic lyase activity. REF1/APE1 (an example of a mammalian AP endonuclease) then cleaves the abasic site to form a 3′-OH end and a 5′ deoxyribose phosphate end. The remaining steps may utilize a “long patch” or “short patch” pathway involving repair DNA synthesis and strand ligation by different sets of proteins. The preferred substrates for the DNA glycosylases OGG1 (8-oxoguanine glycosylase 1) and NTH1 (homolog of E. coli endonuclease III) are 8-oxoguanine (8-OxoG) and thymine glycol (TG) lesions, respectively. REF1/APE1 (redox factor 1/apurinic endonuclease 1) is a multifunctional enzyme with apurinic/apyrimidinic (AP) endonuclease activity and 3′,5′-exonuclease, 3′-diesterase, and 3′-phosphatase activities. REF1/APE1 also has transcriptional regulatory activity independently of its function in base excision repair (Izumi, T., et al. (2003) Toxicology. 193, 43-65; Kelley, M. R., et al. (2001) Antioxid Redox Signal. 3, 671-683). Finally, the XRCC1 protein (X-ray repair, cross-complementing defective, in Chinese hamster, 1) associates with several other proteins—polynucleotide kinase (PNK), DNA polymerase-β (POLB), and DNA ligase III (LIG3)—to form a complex that repairs the single-strand DNA breaks generated during the base excision repair process.
Cancer Stem Cells and Drug ResistanceHuman ovarian cancer cells grown under conditions that support a subpopulation that grows in spheroids selects for cells that have a more potent ability to form new independent cancers (Zhang S, et al. Cancer Res 68:4311-4320, 2008). As few as 100 spheroid forming cells could form new independent tumors when transferred to nude mice, while as many as 100,000 cells grown in monolayer, were unable to form independent tumors. The cancer initiating cells (cancer stem cells) become much more drug resistant to a variety of agents, including platinum compounds such as cisplatin and paclitaxel. Such cells may also express a set of molecular markers that differ from the same cell line, grown in monolayer, such as CD117, CD44, and Nestin. Cancer initiating cells have been investigated in other malignancies including prostate cancer, breast cancer, and lung cancer (Zietarska M, et al. Molecular Carcinogenesis 46:872-885, 2007; Burleson K et al. Gynecologic Oncology 93: 170-181, 2004; Casey R C, et al. Am J of Pathology 159:2071-2080, 2001).
Ovarian cancer stem cells may be responsible for persistent low volume disease after induction of a clinical complete response. The inability to eradicate such cells may be a function of cell dormancy, the relative inability of any chemotherapy to have a meaningful effect on cells in the dormant state or these cells may represent a state of extreme drug resistance at the molecular level.
Methods and Compositions to Reduce Activity of the Hedgehog PathwaySome embodiments relate to compositions and/or methods for reducing activity of the Hedgehog pathway. In some embodiments, the level of GLI1 protein, such as the GLI1-130 isoform, or the level of a nucleic encoding GLI1, such as a nucleic acid encoding the GLI1-130 isoform, can be reduced in the cell of a subject. Methods to reduce the level of GLI1 protein, such as the GLI1-130 isoform, or the level of a nucleic encoding GLI1, such as a nucleic acid encoding the GLI1-130 isoform, in a cell or a subject can be useful to kill or retard the growth of a cell or can be useful to treat or ameliorate certain disorders in a subject.
In some embodiments, the methods or compositions described herein result in a decrease of the amounts of GLI1 protein, such as the GLI1-130 isoform, or a nucleic acid encoding GLI1, such as a nucleic acid encoding the GLI1-130 isoform, within a cell, such as endogenous GLI1, or an mRNA encoding GLI1. In some embodiments, the methods or compositions described herein provide a decrease in GLI1 protein, such as the GLI1-130 isoform, or a decrease in a nucleic acid encoding GLI1, such as a nucleic acid encoding the GLI1-130 isoform, within a cell of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 100%.
The level of GLI1 protein, such as the GLI1-130 isoform, or the level of a nucleic encoding GLI1, such as a nucleic acid encoding the GLI1-130 isoform, can be reduced using RNA interference or antisense technologies. RNA interference is an efficient process whereby double-stranded RNA (dsRNA), also referred to herein as siRNAs (small interfering RNAs) or ds siRNAs (double-stranded small interfering RNAs), induces the sequence-specific degradation of targeted mRNA in animal or plant cells (Hutvagner, G. et al. (2002) Curr. Opin. Genet. Dev. 12:225-232); Sharp, P. A. (2001) Genes Dev. 15:485-490).
In mammalian cells, RNA interference can be triggered by various molecules, including 21-nucleotide duplexes of siRNA (Chiu, Y.-L. et al. (2002) Mol. Cell. 10:549-561. Clackson, T. et al. (1991) Nature 352:624-628; Elbashir, S. M. et al. (2001) Nature 411:494-498), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which can be expressed in vivo using DNA templates with RNA polymerase III promoters (Zheng, B. J. (2004) Antivir. Ther. 9:365-374; Paddison, P. J. et al. (2002) Genes Dev. 16:948-958; Lee, N. S. et al. (2002) Nature Biotechnol. 20:500-505; Paul, C. P. et al. (2002) Nature Biotechnol. 20:505-508; Tuschl, T. (2002) Nature Biotechnol. 20:446-448; Yu, J.-Y. et al. (2002) Proc. Natl. Acad. Sci. USA 99(9):6047-6052; McManus, M. T. et al. (2002) RNA 8:842-850; Sui, G. et al. (2002) Proc. Natl. Acad. Sci. USA 99(6):5515-5520, each of which are incorporated herein by reference in their entirety).
The scientific literature is replete with reports of endogenous and exogenous gene expression silencing using siRNA, highlighting their therapeutic potential (Gupta, S. et al. (2004) PNAS 101:1927-1932; Takaku, H. (2004) Antivir Chem. Chemother 15:57-65; Pardridge, W. M. (2004) Expert Opin. Biol. Ther. 4(7):1103-1113; Shen, W.-G. (2004) Chin. Med. J. (Engl) 117:1084-1091; Fuchs, U. et al. (2004) Curr. Mol. Med. 4:507-517; Wadhwa, R. et al. (2004) Mutat. Res. 567:71-84; Ichim, T. E. et al. (2004) Am. J. Transplant 4:1227-1236; Jana, S. et al. (2004) Appl. Microbiol. Biotechnol. 65:649-657; Ryther, R. C. C. et al. (2005) Gene Ther. 12:5-11; Chae, S-S. et al. (2004) J. Clin. Invest 114:1082-1089; de Fougerolles, A. et al. (2005) Methods Enzymol. 392:278-296, each of which is incorporated herein by reference in its entirety).
Therapeutic silencing of endogenous genes by systemic administration of siRNAs has been described in the literature (Kim, B. et al. (2004) American Journal of Pathology 65:2177-2185; Soutschek, J. et al. (2004) Nature 432:173-178; Pardridge, W. M. (2004) Expert Opin. Biol. Ther. 4(7):1103-1113, each of which is incorporated herein by reference in its entirety).
siRNAs induce a sequence-specific reduction in expression of a gene by the process of RNAi. Thus, siRNA is the intermediate effector molecule of the RNAi process. Some nucleic acid molecules or constructs provided herein include dsRNA molecules comprising 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA of GLI1 and the other strand is identical or substantially identical to the first strand. However, it will be appreciated that the dsRNA molecules may have any number of nucleotides in each strand which allows them to reduce the level of GLI1 protein, such as the GLI1-130 isoform, or the level of a nucleic acid encoding GLI1. The dsRNA molecules provided herein can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from, e.g., shRNA. The dsRNA molecules can be designed using any method known in the art.
An example method for designing dsRNA molecules is provided in the pSUPER RNAi SYSTEM™ (OligoEngine, Seattle, Wash.). The system provides inducible expression of a siRNA in a transfected cell. To effect silencing of a specific gene, a pSUPERIOR vector is used in concert with a pair of custom oligonucleotides that include a unique 19-nt sequence derived from the mRNA transcript of the gene targeted for suppression (the “N-19 target sequence”). The N-19 target sequence corresponds to the sense strand of the pSUPER-generated siRNA, which in turn corresponds to a 19-nt sequence within the mRNA. In the mechanism of RNAi, the antisense strand of the siRNA duplex hybridizes to this region of the mRNA to mediate cleavage of the molecule. These forward and reverse oligonucleotides are annealed and cloned into the vector so that the desired siRNA duplex can be generated. The sequence of the forward oligonucleotide includes the unique N-19 target in both sense and antisense orientation, separated by a 9-nt spacer sequence. The resulting transcript of the recombinant vector is predicted to fold back on itself to form a 19-base pair stem-loop structure. The stem-loop precursor transcript is quickly cleaved in the cell to produce a functional siRNA (T. R. Brummelkamp, et al, Science 296, 550 (2002)). More example methods are provided in Taxman D. J. et al. (2006) BMC Biotechnol. 6:7; and McIntyre G. J. et al. (2006) BMC Biotechnol. 6:1, each of which is incorporated by reference in its entirety.
Nucleic acids provided herein can include both unmodified siRNAs and modified siRNAs as known in the art. For example, in some embodiments, siRNA derivatives can include siRNA having two complementary strands of nucleic acid, such that the two strands are crosslinked. For example, a 3′ OH terminus of one of the strands can be modified, or the two strands can be crosslinked and modified at the 3′ OH terminus. The siRNA derivative can contain a single crosslink (e.g., a psoralen crosslink). In some embodiments, the siRNA derivative has at its 3′ terminus a biotin molecule (e.g., a photocleavable biotin), a peptide (e.g., a Tat peptide), a nanoparticle, a peptidomimetic, organic compounds (e.g., a dye such as a fluorescent dye), or dendrimer. Modifying siRNA derivatives in this way can improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.
Nucleic acids provided herein can include nucleic acids that can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability, and/or half-life. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert, G. et al. (2001) Drug Deliv. Rev. 47(1): 99-112 (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fatal et al. (1998) J. Control Release 53(1-3): 137-43 (describes nucleic acids bound to nanoparticles); Schwab et al. (1994) Ann. Oncol. 5 Suppl. 4:55-58 (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard, G. et al. (1995) Eur. J. Biochem. 232(2):404-10 (describes nucleic acids linked to nanoparticles). Because RNAi is believed to progress via at least one single stranded RNA intermediate, the skilled artisan will appreciate that ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also be designed as described herein and utilized according to the claimed methodologies.
Synthetic siRNAs can be delivered to cells by methods known in the art, including cationic liposome transfection and electroporation. However, these exogenous siRNA generally show short term persistence of the silencing effect (4 to 5 days in cultured cells), which may be beneficial in certain embodiments. To obtain longer term suppression of expression for targeted genes, such as GLI1, and to facilitate delivery under certain circumstances, one or more siRNA duplexes, e.g., ds siRNA, can be expressed within cells from recombinant DNA constructs. Such methods for expressing siRNA duplexes within cells from recombinant DNA constructs to allow longer-term target gene suppression in cells are known in the art, including mammalian Pol III promoter systems (e.g., H1 or U6/snRNA promoter systems (Tuschl, T. (2002) Nature Biotechnol. 20:446-448) capable of expressing functional double-stranded siRNAs; (Lee, N. S. et al. (2002) Nature Biotechnol. 20:500-505; Miyagishi, M. and Taira, K. (2002) Nature Biotechnol. 20:497-500; Paul, C. P. et al. (2002) Nature Biotechnol. 20:505-508; Yu, J.-Y. et al. (2002) Proc. Natl. Acad. Sci. USA 99(9):6047-6052; Sui, G. et al. (2002) Proc. Natl. Acad. Sci. USA 99(6):5515-5520). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence. The siRNA is complementary to the sequence of the target gene in 5′-3′ and 3′-5′ orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by an H1 or U6 snRNA promoter can be expressed in cells, and can inhibit target gene expression. Constructs containing siRNA sequence(s) under the control of a T7 promoter also make functional siRNAs when co-transfected into the cells with a vector expressing T7 RNA polymerase (Jacque J.-M. et al. (2002) Nature 418:435-438). A single construct may contain multiple sequences coding for siRNAs, such as multiple regions of the GLI1 gene, such as a nucleic acid encoding the GLI1 mRNA, and can be driven, for example, by separate Pol III promoter sites.
Nucleic acids provided herein can include micro RNA (miRNAs) which can regulate gene expression at the post transcriptional or translational level. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop, probably by Dicer, an RNase III-type enzyme, or a homolog thereof. By substituting the stem sequences of the miRNA precursor with miRNA sequence complementary to the target mRNA, a vector construct that expresses the novel miRNA can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zheng, B. J. (2004) Antivir. Ther. 9:365-374). When expressed by DNA vectors containing polymerase III promoters, micro-RNA designed hairpins can silence gene expression, such as GLI1 expression.
Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al. (2002) Nature Biotechnol. 20(10):1006-10). In vitro infection of cells by such recombinant adenoviruses allows for diminished endogenous target gene expression. Injection of recombinant adenovirus vectors into transgenic mice expressing the target genes of the siRNA results in in vivo reduction of target gene expression. In an animal model, whole-embryo electroporation can efficiently deliver synthetic siRNA into post-implantation mouse embryos (Calegari, F. et al. (2002) Proc. Natl. Acad. Sci. USA 99(22):14236-40). In adult mice, efficient delivery of siRNA can be accomplished by the “high-pressure” delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA containing solution into animal via the tail vein (Lewis, D. L. (2002) Nature Genetics 32:107-108). Nanoparticles, liposomes and other cationic lipid molecules can also be used to deliver siRNA into animals. A gel-based agarose/liposome/siRNA formulation is also available (Jiamg, M. et al. (2004) Oligonucleotides 14(4):239-48).
Nucleic acids provided herein can include an antisense nucleic acid sequence selected such that it is complementary to the entirety of GLI1 or to a portion of GLI1. In some embodiments, a portion can refer to at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, and at least about 80%, at least about 85%, at least about 90%, at least about 95%. In some embodiments, a portion can refer up to 100%. An example mRNA sequence (SEQ ID NO:11) of human GLI1 is shown in TABLE 1.
An antisense oligonucleotide can have a length of at least about 5 nucleotides, at least about 7 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, and at least about 100 nucleotides. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation, namely, RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest. The antisense nucleic acid molecules can be administered to a subject (e.g., systemically or locally by direct injection at a tissue site, or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding GLI1 to thereby inhibit its expression. Alternatively, antisense nucleic acid molecules can be modified to target particular cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to particular cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter can be used.
In some embodiments, antisense oligonucleotide include α-anomeric nucleic acid molecules. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier, C. et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide, or a chimeric RNA-DNA analogue (Inoue, H. et al. (1987) Nucleic Acids Res. 15:6131-6148; Inoue, H. et al. (1987a) FEBS Lett. 215:327-330).
Additional methods or compositions described herein to reduce the level of GLI1 protein, such as the GLI1-130 isoform, or a nucleic acid encoding GLI1, such as a nucleic acid encoding the GLI1-130 isoform, within a cell, such as endogenous GLI1, or an mRNA encoding GLI1, can utilize ribozymes. In general, a ribozyme is a catalytic RNA molecule that cleaves RNA in a sequence specific manner. Ribozymes that cleave themselves are known as cis-acting ribozymes, while ribozymes that cleave other RNA molecules are known as trans-acting ribozymes. The term “cis-acting ribozyme sequence” as used herein refers to the sequence of an RNA molecule that has the ability to cleave the RNA molecule containing the cis-acting ribozyme sequence. A cis-acting ribozyme sequence can contain any sequence provided it has the ability to cleave the RNA molecule containing the cis-acting ribozyme sequence. For example, a cis-acting ribozyme sequence can have a sequence from a hammerhead, axhead, or hairpin ribozyme. In addition, a cis-acting ribozyme sequence can have a sequence from a hammerhead, axhead, or hairpin ribozyme that is modified to have either slow cleavage activity or enhanced cleavage activity. For example, nucleotide substitutions can be made to modify cleavage activity (Doudna and Cech, Nature, 418:222-228 (2002)). Examples of ribozyme sequences that can be used with the methods and compositions described herein include those described in U.S. Pat. No. 6,271,359, and U.S. Pat. No. 5,824,519, incorporated by reference in their entireties. One example method for preparing a ribozyme is to synthesize chemically an oligodeoxyribonucleotide with a ribozyme catalytic domain (approximately 20 nucleotides) flanked by sequences that hybridize to the target mRNA. The oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers. The amplified product is cloned into a eukaryotic expression vector. A ribozyme can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ohkawa et al., Nucleic Acids Symp. Ser., 27: 15-6 (1992); Taira et al., Nucleic Acids Res., 19: 5125-30 (1991); Ventura et al., Nucleic Acids Res., 21, 3249-55 (1993).
Methods of TreatmentSome embodiments relate to compositions and/or methods for treating or ameliorating disorders related to an increased activity of the Hedgehog pathway. In some embodiments, treating such disorders can include decreasing the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding the GLI1-130 isoform, in the cell of a subject. In some embodiments, a composition can include an isolated nucleic acid having activity to reduce the levels of GLI1, such as a nucleic acid having activity to reduce the levels of the GLI1-130 isoform, in a cell of a subject. Examples of such nucleic acids are described herein and include a sequence encoding GLI1 or a fragment thereof, or a sequence encoding antisense GLI1 or a fragment thereof. Such nucleic acids can be useful for RNA interference or antisense technologies. A fragment of a polynucleotide sequence will be understood to include any nucleotide fragment having, for example, at least about 5 successive nucleotides, at least about 12 successive nucleotides, at least about 15 successive nucleotides, at least about 18 successive nucleotides, or at least about 20 successive nucleotides of the sequence from which it is derived. An upper limit for a fragment can include, for example, the total number of nucleotides in a full-length sequence encoding a particular polypeptide. Methods to select for nucleic sequences that have activity to reduce the level of a protein, such as GLI1 protein, including the GLI1-130 isoform, or the level of a nucleic acid encoding a polypeptide, such as an mRNA encoding GLI1, including an mRNA encoding the GLI1-130 isoform, in a cell or a subject, are also provided herein.
In some embodiments, a nucleic acid having activity to reduce GLI1 protein expression, such as the GLI1-130 isoform protein expression, or the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding the GLI1-130 isoform, in a cell of a subject is further operably linked to a regulatory sequence. Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), the disclosure of which is incorporated herein by reference in its entirety. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue specific promoters may be used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non-targeted tissues. For example, promoters such as the PSA, probasin, prostatic acid phosphatase or prostate-specific glandular kallikrein (hK2) may be used to target gene expression in the prostate. Similarly, promoters as follows may be used to target gene expression in other tissues. Examples of more tissue specific promoters include in (a) pancreas: insulin, elastin, amylase, pdr-I, pdx-I, glucokinase; (b) liver: albumin PEPCK, HBV enhancer, a fetoprotein, apolipoprotein C, α-I antitrypsin, vitellogenin, NF-AB, Transthyretin; (c) skeletal muscle: myosin H chain, muscle creatine kinase, dystrophin, calpain p94, skeletal α-actin, fast troponin 1; (d) skin: keratin K6, keratin KI; (e) lung: CFTR, human cytokeratin IS (K 18), pulmonary surfactant proteins A, B and C, CC-10, Pi; (f) smooth muscle: sm22 α, SM-α-actin; (g) endothelium: endothelin-I, E-selectin, von Willebrand factor, TIE, KDR/flk-I; (h) melanocytes: tyrosinase; (i) adipose tissue: lipoprotein lipase, adipsin, acetyl-CoA carboxylase, glycerophosphate dehydrogenase, adipocyte P2; (j) blood: P-globin; and (k) mammary: MMTV, and whey acidic protein (WAP).
In certain embodiments, it may be desirable to activate transcription at specific times after administration of a vector comprising a nucleic acid having activity to reduce GLI1 protein level, such as the level of the GLI1-130 isoform, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, in a cell. This may be done with such promoters as those that may be regulated by hormone or cytokine. For example, in a gonadal tissue where specific steroids are produced or routed to, use of androgen or estrogen regulated promoters may be advantageous. Such promoters that are hormone regulatable include MMTV, MT-1, ecdysone and RuBisco. Other hormone regulated promoters such as those responsive to thyroid, pituitary and adrenal hormones are expected to be useful with the nucleic acids described herein. Cytokine and inflammatory protein responsive promoters that could be used include K and T Kininogen, c-fos, TNF-α, C-reactive protein, haptoglobin, serum amyloid A2, C/EBP α, IL-1, IL-6, Complement C3, IL-8, α-1 acid glycoprotein, α-1 antitrypsin, lipoprotein lipase, angiotensinogen, fibrinogen, c-jun (inducible by phorbol esters, TNF α, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothionein (heavy metal and glucocorticoid inducible), Stromelysin (inducible by phorbol ester, interleukin-1 and EGF), α-2 macroglobulin and α-I antichymotrypsin. It is envisioned that any of the promoters described herein, alone or in combination with another, may be useful depending on the action desired.
Nucleic acid constructs having activity to reduce GLI1 protein levels, such as the level of GLI1-130 isoform, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, in a cell and described herein can be introduced in vivo as naked DNA plasmids, for example, using transfection, electroporation (e.g., transcutaneous electroporation), microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (Wu et al. J. Biol. Chem., 267:963-967, 1992; Wu and Wu J. Biol. Chem., 263:14621-14624, 1988; and Williams et al. Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991). A needleless delivery device, such as a BIOJECTOR® needleless injection device can be utilized to introduce nucleic acid constructs in vivo. Receptor-mediated DNA delivery approaches can also be used (Curiel et al. Hum. Gene Ther., 3:147-154, 1992; and Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987). Methods for formulating and administering naked DNA to mammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466, both of which are herein incorporated by reference in their entireties. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931), the disclosures of which are incorporated herein by reference in their entireties.
Alternatively, electroporation can be utilized conveniently to introduce nucleic acid constructs, having activity to reduce GLI1 protein levels, such as the level of the GLI1-130 isoform, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, in a cell and described herein, into cells. Electroporation is well known by those of ordinary skill in the art (see, for example: Lohr et al. Cancer Res. 61:3281-3284, 2001; Nakano et al. Hum Gene Ther. 12:1289-1297, 2001; Kim et al. Gene Ther. 10:1216-1224, 2003; Dean et al. Gene Ther. 10:1608-1615, 2003; and Young et al. Gene Ther 10:1465-1470, 2003). For example, in electroporation, a high concentration of vector DNA is added to a suspension of host cell (such as isolated autologous peripheral blood or bone marrow cells) and the mixture shocked with an electrical field. Transcutaneous electroporation can be utilized in animals and humans to introduce heterologous nucleic acids into cells of solid tissues (such as muscle) in vivo. Typically, the nucleic acid constructs are introduced into tissues in vivo by introducing a solution containing the DNA into a target tissue, for example, using a needle or trochar in conjunction with electrodes for delivering one or more electrical pulses. For example, a series of electrical pulses can be utilized to optimize transfection, for example, between 3 and ten pulses of 100 V and 50 msec. In some cases, multiple sessions or administrations are performed.
Another well known method that can be used to introduce nucleic acid constructs, having activity to reduce GLI1 protein levels, such as the GLI1-130 isoform protein levels, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, in a cell and described herein, into host cells is biolistic transformation. One method of biolistic transformation involves propelling inert or biologically active particles at cells, e.g., U.S. Pat. Nos. 4,945,050, 5,036,006; and 5,100,792, the disclosures of which are hereby incorporated by reference in their entireties. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the plasmid can be introduced into the cell by coating the particles with the plasmid containing the exogenous DNA. Alternatively, the target cell can be surrounded by the plasmid so that the plasmid is carried into the cell by the wake of the particle.
Alternatively, nucleic acid constructs, having activity to reduce GLI1 protein levels, such as the GLI1-130 isoform protein levels, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, in a cell and described herein, can be introduced in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et al. Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackey, et al. Proc. Natl. Acad. Sci. USA 85:8027-8031, 1988; Ulmer et al. Science 259:1745-1748, 1993, the disclosures of which are incorporated herein by reference in their entireties). The use of cationic lipids can promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Felgner and Ringold Science 337:387-388, 1989, the disclosure of which is incorporated by reference herein in its entirety). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127, incorporated herein by reference in their entireties.
In some embodiments, the nucleic acid constructs, having activity to reduce GLI1 protein levels, such as the GLI1-130 isoform protein levels, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, in a cell and described herein, are viral vectors. Methods for constructing and using viral vectors are known in the art (See e.g., Miller and Rosman, BioTech., 7:980-990, 1992). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In some cases, the replication defective virus retains the sequences of its genome that are necessary for encapsulating the viral particles. DNA viral vectors commonly include attenuated or defective DNA viruses, including, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), Moloney leukemia virus (MLV) and human immunodeficiency virus (HIV) and the like. Defective viruses, that entirely or almost entirely lack viral genes, are preferred, as defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al. Mol. Cell. Neurosci., 2:320-330, 1991, the disclosure of which is incorporated herein by reference in its entirety), defective herpes virus vector lacking a glycoprotein L gene (See for example, Patent Publication RD 371005 A, incorporated herein by reference in its entirety), or other defective herpes virus vectors (See e.g., WO 94/21807; and WO 92/05263, incorporated herein by reference in their entireties); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630 1992; La Salle et al., Science 259:988-990, 1993, the disclosure of which is incorporated herein by reference in its entirety); and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61:3096-3101, 1987; Samulski et al., J. Virol., 63:3822-3828, 1989; and Lebkowski et al., Mol. Cell. Biol., 8:3988-3996, 1988, the disclosures of which are incorporated herein by reference in their entireties).
In some embodiments, the viral vectors, having activity to reduce GLI1 protein levels, such as the GLI1-130 isoform protein levels, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, in a cell and described herein, may be adenovirus vectors. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the disclosure to a variety of cell types. Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present disclosure, to type 2, type 5 or type 26 human adenoviruses (Ad 2 or Ad 5), or adenoviruses of animal origin (See e.g., WO94/26914 and WO2006/020071, the disclosures of which are incorporated herein by reference in their entireties). Those adenoviruses of animal origin that can be used within the scope of the present disclosure include adenoviruses of canine, bovine, murine (e.g., Mav1, Beard et al. Virol., 75-81, 1990, the disclosure of which is incorporated herein by reference in its entirety), ovine, porcine, avian, and simian (e.g., SAV) origin. In some embodiments, the adenovirus of animal origin is a canine adenovirus, such as a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800)).
Some embodiments include pharmaceutical compositions comprising a nucleic acid which reduces GLI1 protein levels, such as the GLI1-130 isoform protein levels, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, and a suitable carrier. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions described herein, the type of carrier will typically vary depending on the mode of administration. Compositions described herein may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration. Carriers for use within such pharmaceutical compositions are biocompatible, and may also be biodegradable. In certain embodiments, the formulation preferably provides a relatively constant level of active component release.
The pharmaceutical compositions described herein can further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions described herein may be formulated as a lyophilizate.
Pharmaceutical compositions described herein can be administered to a subject, such as a mammal, such as a human. Pharmaceutical compositions can be administered at a therapeutically effective amount. A “therapeutically effective amount” is a quantity of a chemical composition (such as a nucleic acid construct, vector, or polypeptide) used to achieve a desired effect in a subject being treated. Pharmaceutical compositions may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs. Pharmaceutical compositions may be administered in combination with at least one additional therapeutic compound, such as a chemotherapeutic compound.
IndicationsMethods and compositions described herein can be used to treat disorders that relate to increased activity of the Hedgehog pathway. Examples of such disorders include cancers, for example, breast cancer, melanoma, prostate cancer, colorectal cancer, head and neck cancer, lung cancer, colon cancer, oesophageal cancer, gastric cancer, testicular cancer cell, and ovarian cancer. More examples include any cancer that may be treated with the therapeutic compounds described herein.
Methods to Increase Sensitivity of Cells to Therapeutic CompoundsIt has been discovered that reducing GLI1 protein levels or the level of a nucleic acid encoding GLI1 in a cell increases the sensitivity of the cell to particular therapeutic compounds. Accordingly, some embodiments relate to methods for increasing the sensitivity of a cell or a subject to a therapeutic compound. As will be understood, increasing the sensitivity of a cell or a subject to a therapeutic compound can decrease the therapeutically effective amount of a therapeutic compound needed to treat the cell or subject.
In some embodiments, a cell or a subject may be treated with an agent that reduces GLI1 protein levels, such as the GLI1-130 isoform protein levels, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform. Reducing GLI1 protein levels, such as the GLI1-130 isoform protein levels, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, in certain cells can increase the sensitivity of those cells to particular therapeutic compounds. Such cells can include cells in which GLI1 expression is increased compared to normal cells, for example, in certain neoplastic cells. More examples include cells in which the activity of DNA repair mechanisms is increased compared to normal cells. Such DNA repair mechanisms can include nucleotide excision repair, and base excision repair.
Therapeutic compounds for which the therapeutic dosage may be reduced can include chemotherapeutic compounds. Examples of chemotherapeutic compounds include platinum-based compounds such as cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, and triplatin tetranitrate, nitrogen mustards such as cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, and ifosfamide, nitrosoureas such as carmustine, lomustine, and streptozocin, alkyl sulfonates such as busulfan, thiotepa, procarbazine, and altretamine. In more embodiments, chemotherapeutic compounds can include compounds in which an increased cellular resistance to the chemotherapeutic compound correlates to an increased expression of ERRC1, XPD, XRCC1, or c-jun such as c-jun (Ser 63). More embodiments, include therapeutic compounds for which increased activity of the base excision repair pathway results in increased cellular resistance to the therapeutic compounds. More embodiments, include therapeutic compounds for which increased activity of the nucleotide excision repair pathway results in increased cellular resistance to the therapeutic compounds.
Methods for Identifying AgentsMore embodiments include methods of identifying compounds and agents useful for the methods and compositions described herein. Some such methods can be useful to evaluate test compounds useful to treat disorders related to increased activity of the Hedgehog pathway. More methods can be useful to evaluate test compounds useful to increase the sensitivity of certain cells to particular therapeutic compounds.
In some embodiments, a test compound is evaluated by contacting the cell with the test compound. A test compound that reduces the level of GLI1 protein, such as the level of the GLI1-130 isoform, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, may be useful to decrease the activity of the Hedgehog pathway. Such a test compound can be useful to treat or ameliorate disorders related to increased activity of the Hedgehog pathway. More methods include comparing the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, or the level of GLI1 protein, such as the level of the GLI1-130 isoform, in a target cell to the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, or the level of GLI1 protein, such as the level of GLI1-130 isoform, in a target cell contacted with the test compound.
More methods can also include selecting a test compound that, in addition to reducing the level of the GLI1 protein, such as the level of the GLI1-130 isoform or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, also reduces the level of c-jun (Ser 63) protein in a target cell, where c-jun (Ser 63) is a c-jun protein phosphorylated at the Serine 63 residue. More methods can also include selecting a test compound that also inhibits or reduces the upregulation of the level of c-jun (Ser 63) protein in a target cell. Upregulation of c-jun (Ser 63) can be in response to a chemical compounds that upregulates c-jun (Ser 63).
More methods can also include selecting a test compound that, in addition to reducing the level of the GLI1 protein, such as the level of the GLI1-130 isoform or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, also reduces the level of ERCC1 protein or a nucleic acid encoding ERCC1 in a target cell. More methods can also include selecting a test compound that also inhibits or reduces upregulation of the level of ERCC1 protein or a nucleic acid encoding ERCC1 in a target cell. The upregulation of ERCC1 protein or a nucleic acid encoding ERCC1 in a target cell can be in response to a chemical compound that upregulates ERCC1.
More methods can also include selecting a test compound that, in addition to reducing the level of the GLI1 protein, such as the level of the GLI1-130 isoform or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, also reduces the level of XPD protein or a nucleic acid encoding XPD in a target cell. More methods can also include selecting a test compound that also inhibits or reduces upregulation of the level of XPD protein or a nucleic acid encoding XPD in a target cell. The upregulation of XPD protein or a nucleic acid encoding XPD in a target cell can be in response to a chemical compound that upregulates XPD.
More methods can also include selecting a test compound that, in addition to reducing the level of the GLI1 protein, such as the level of the GLI1-130 isoform or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, also reduces the level of XRCC1 protein or a nucleic acid encoding XRCC1 in a target cell. More methods can also include selecting a test compound that also inhibits or reduces upregulation of the level of XRCC1 protein or a nucleic acid encoding XRCC1 in a target cell. The upregulation of XRCC1 protein or a nucleic acid encoding XRCC1 in a target cell can be in response to a chemical compound that upregulates XRCC1.
More methods can also include selecting a test compound that while reducing the level of the GLI1 protein, such as the level of the GLI1-130 isoform or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, also does not reduce the level of GLI2 protein or a nucleic acid encoding GLI2 in a cell. More methods can also include selecting a test compound that does not substantially reduce the level of GLI2 protein or a nucleic acid encoding GLI2 in a cell. As used herein the term “not substantially reduce” and grammatical equivalents can refer to a reduction of no more than about 1%, no more than about 2%, no more than about 3%, no more than about 4%, no more than about 5%, no more than about 6%, no more than about 7%, no more than about 8%, no more than about 9%, and no more than about 10%.
Test compounds that do not reduce or substantially reduce the level of GLI2 protein or a nucleic acid encoding GLI2 in a cell, and also have activity that reduces the level of GLI1 protein, such as the GLI1-130 isoform, or the level of a nucleic acid encoding GLI1, such as a nucleic acid encoding the GLI1-130 isoform, are particularly advantageous, as these compounds may selectively inhibit a tumor cell's, for example, a neoplastic cell's, ability to up-regulate those processes/pathways that promote tumor cell survival.
Examples of chemical compounds that can upregulate target cell levels of c-jun (Ser 63) protein, ERCC1 protein, XPD protein, or XRCC1 protein are well known and include chemotherapeutic agents, such as cisplatin. Examples of test compounds can include chemical compounds, nucleic acids, for example, nucleic acids encoding GLI1 or fragments thereof, or antisense GLI1 or fragments thereof.
Methods for Assessing the Effectiveness of a Compound or AgentMore methods include assessing the effectiveness of a compound or agent in treating a disorder. In some such methods, treatment can include methods and/or compositions that reduce the level of GLI1 protein, such as the level of the GLI1-130 isoform, or the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, in a cell of a subject. The effectiveness of the compound or agent can be evaluated by measuring the level of OPN protein or the level of a nucleic acid encoding OPN in a sample from the subject. The measuring can be carried out in vivo or ex vivo. More methods can include comparing the level of a nucleic acid encoding OPN or the level of OPN protein in the sample to the level of a nucleic acid encoding OPN or the level of OPN protein in a subject who does not have the disorder, and/or a subject who has not been contacted with the compound or agent. In some embodiments, a decrease in the level of a nucleic acid encoding OPN or the level of OPN protein is indicative of a favorable prognosis.
More methods include methods for assessing the potential effectiveness of a nucleic acid as a therapeutic agent. Some such methods include determining whether the nucleic acid reduces the level of a nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, or the level of GLI1 protein, such as the level of the GLI1-130 isoform, in a cell. In such methods, the nucleic acid can be identified as having potential effectiveness as a therapeutic agent if the nucleic acid reduces the level of the nucleic acid encoding GLI1, such as the level of a nucleic acid encoding the GLI1-130 isoform, or the level of the GLI1 protein, such as the level of the GLI1-130 isoform, in said cell. In further embodiments, methods can also include determining whether a nucleic acid has no substantial effect on the level of a nucleic acid encoding GLI2 or the level of GLI2 protein in a cell. In some such embodiments, the nucleic acid is identified as having potential effectiveness a therapeutic agent if the nucleic acid has no substantial effect on the level of the nucleic acid encoding GLI2 or the level of the GLI2 protein in said cell.
More embodiments include nucleic acids identified as having potential effectiveness as a therapeutic agent by the methods described herein.
EXAMPLES Example 1—Expression Levels of GLI1 and OPN Increase with the Development and Progression of MelanomaActivation of the Hedgehog pathway results in nuclear translocation of GLI1 transcription factors and up-regulation of target genes. Microarray analysis of genes that were differentially regulated by the Hedgehog pathway revealed that OPN expression was up-regulated. Clinically derived primary cutaneous cancers and melanoma specimens were profiled by gene expression analysis and it was shown that the expression of OPN was increased 67.3-fold in metastatic melanoma samples when compared with primary cancer samples. This data set was queried for the changes in the expression of GLI1 and OPN with disease progression. As seen in
To determine whether OPN expression is regulated by Hedgehog pathway signaling three metastatic melanoma-derived cell lines, MCC012A, MCC012F, and MDAMB-435 were studied. To establish autocrine Hedgehog signaling, the cell lines should express the Hedgehog pathway products, including the receptor PTCH, the ligand SHH, and the transcription factor GLI1. As seen in
The effect of the Hedgehog inhibitor, cyclopamine, was tested on OPN levels (19). As seen in
The decreases in the levels of OPN mRNA are also reflected in the decreased levels of OPN protein in the secretome of cyclopamine-treated cells (
Signaling via the Hedgehog pathway culminates in the transcription of target genes by the GLI transcription factors. The role of transcription factor GLI1 in mediating the effects of the Hedgehog pathway on OPN was tested. The promoter region of human OPN was scanned (up to 1 kb upstream of transcription start site) for GLI1-binding sites using TFSEARCH and identified a putative GLI1 binding site at position −243 to −259 (5′-TGCTGAATGCCCATCCC-3′ (SEQ ID NO:12)).
As shown in
To determine whether GLI1 physically associates with the OPN promoter, cross-linked chromatin from MDA-MB-435 cells was immunoprecipitated with an anti-GLI1 antibody and amplified the region of the OPN promoter that bears the GLI1 recognition sequence (
To evaluate the functional effects of active Hedgehog signaling stable cell lines that were knocked down for GLI1 expression by RNA interference were generated. The efficacy of three shRNA constructs for silencing GLI1 expression was assessed. TABLE 2 shows details of the region in GLI1 transcript that was targeted by the shRNAs used to assess efficacy of silencing GLI1
The two shRNA constructs that demonstrated more effective GLI1 silencing, shRNA-1 and shRNA-2, overlap in the region they target. Using vector construct GLI1 shRNA-1 (
The Hedgehog pathway has been reported to influence the expression of signature proteins that mediate epithelial-mesenchymal transition (EMT). Hence GLI1-knocked down clones KO1 and KO4 for the status of these signature markers were examined. As seen in
GLI1-silenced cells for their in vitro attributes of aggressiveness, viz. cell migration, invasion, and motility were tested. Although GLI1 silencing had no significant effect on cell motility measured with a scratch assay (
To determine the role of OPN in mediating GLI1 effects the effects of OPN in GLI1-KO cells was restored. This was assessed in two ways: (a) the GLI1-knocked down cells were treated with recombinant OPN and (b) the GLI1-knocked down cells were stably transfected with a plasmid construct expressing human OPN (
Similarly, stably restoring the expression of OPN (
Cell Culture—The melanoma cell lines, MCC012A and MCC012F, used were established from two subcutaneous metastatic nodules from the same patient. Cells were grown in a Dulbecco's modified minimum essential medium, F-12 mixture (1:1) (Invitrogen) supplemented with 5% heat inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, Ga.), 200 μM sodium pyruvate (Invitrogen), and 20 μM non-essential amino acids (Invitrogen). All cells were maintained in a humidified 5% CO2 environment. MDA-MB-435 cells were also cultured under similar conditions.
Generation of Stable Transfectants—Endogenous GLI1 from MDA-MB-435 cells was silenced using shRNAs (short hairpin RNA) cloned into pSuperior.neo+gfp plasmid (OligoEngine, Seattle, Wash.) (TABLE 1). Introduction of double-stranded RNA has proven to be a powerful tool to suppress gene expression through a process known as RNA interference (P. A. Sharp, Genes Dev. 13, 139 (1999)). However, in some mammalian cells this can provoke a strong cytotoxic response (T. Hunter, T. Hunt, R. J. Jackson, H. D. Robertson, J. Biol. Chem. 250, 409 (1975)). This non-specific effect can be circumvented by use of synthetic short [21- to 22-nucleotide (nt)] interfering RNAs (siRNAs), which can mediate strong and specific suppression of gene expression (S. M. Elbashir et al., Nature 411, 494 (2001)). shRNAs included a target sequence sense strand, a short hairpin, and a target sequence antisense strand.
Stable vector only and non-targeting (scrambled control) transfectants were also generated. Stable transfectants were selected on medium supplemented with 500 μg/ml Geneticin (Invitrogen). The four GLI1-knocked down clones chosen were based on the extent of GLI1 knockdown and were termed KO1, K02, K03, and K04. The expression of OPN was restored in the KO1 cells by transfecting with pcDNA3.1-OPN. A corresponding vector-only transfectant was also generated. Transfectants were selected on medium containing Geneticin (500 μg/ml) and hygromycin (750 μg/ml). Serum-free conditioned medium harvested from approximately 3.0×106 cells after 24 hr was assayed for OPN by immunoblotting. To test the inhibitory effect of cyclopamine on Hedgehog pathway activation, cells were cultured in Dulbecco's modified minimum essential medium supplemented with 0.5% fetal bovine serum and treated for the indicated time intervals with dimethyl sulfoxide (vehicle control), 10 and 20 μM cyclopamine (Sigma).
Western Blotting Analysis—Whole cell lysates were collected in Nonidet P-40 buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40). Isolation of cytosolic and nuclear fractions was done as previously reported (15). Total protein (30 μg) was resolved by SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. Membranes were immunoblotted overnight at 4° C. with antibodies to OPN (catalog number 905-629; Assay Designs, Ann Arbor, Mich.), GLI1 (sc-20687; Santa Cruz Biotechnology, Santa Cruz, Calif.), CD44 (HCAM) (sc-7946; Santa Cruz Biotechnology), vimentin (sc-32322; Santa Cruz Biotechnology), N-cadherin (catalog 18-0224; Invitrogen), SNAI2 (catalog H00006591-M02; Novus Biologicals, Littleton, Colo.), SHH (sc-1194; Santa Cruz Biotechnology), or PTCH1 (sc-6149; Santa Cruz Biotechnology). Equal loading was confirmed with anti-β-actin (Sigma) antibody. The purity of cytosolic and nuclear fractions was confirmed with anti-β-tubulin (catalog 2146; Cell Signaling, Danvers, Mass.) or anti-HDAC1 (catalog 2062; Cell Signaling) antibodies, respectively. Secreted OPN was assessed by loading an equal quantity of protein from the serum-free conditioned medium. Corresponding horseradish peroxidase conjugated secondary antibodies were used for detection; blots were developed with SuperSignal enhanced chemiluminescence substrate (Pierce) and imaged using a Fuji LAS3000 imager.
Expression Constructs—OPN promoter activity was assessed by a luciferase reporter assay using the human OPN promoter construct (OPN-352) cloned into pGL3-basic vector (Promega) (Samant, R. S., et al. (2007) Mol. Cancer 6: 6). The putative GLI1 binding site (Kinzler, K. W., and Vogelstein, B. (1990) Mol. Cell. Biol. 10, 634-642) (5′-TGCTGAATGCCCATCCC-3′) in the OPN promoter was disrupted using an inside-out PCR and replaced with a NotI site using primers: (SEQ ID NO: 13) forward, 5′-CTCAGCGGCCGCTAATAAATGAAAAAGC-3′ and (SEQ ID NO: 14) reverse, 5′-GTTAGCGGCCGCTGAGAGTTCCAGGAAG-3′. The resultant construct, referred to as OPN-352Mut has a mutated GLI1-binding site.
Luciferase Assay—Cells (40,000) were transfected with pGL3-OPN-352 or pGL3-OPN-352Mut in combination with pLNCX or pLNCX-GLI1 as previously described (16). Empty pGL3 vector was used as control. Hedgehog ligands were added to the well 6 h prior to harvesting the cells (approximately 33 hr of initiation of transfection) for assay. Readings were normalized to total protein content. Each parameter was studied in triplicate and the experiment repeated at least 3 times. The data are represented as percent luciferase activity, which was derived as a percent of the relative light units in treated groups compared with the untreated groups.
Quantitative RT-PCR (qRT-PCR)—cDNA was generated using High Capacity Reverse Transcriptase Kit (Applied Biosystems, Foster City, Calif.). Real time PCR was performed using a Bio-Rad iQ5 Real-Time Detection system (Bio-Rad). All reactions were done as three independent replicates. All assays were done using the TaqMan Gene Expression Assays from Applied Biosystems. OPN (SPP1: Hs 00959010_m1) transcript levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (Hs 99999905_m1) levels (δCT), which was used to calculate changes in OPN expression (2−δδCT). To analyze the effect of cyclopamine treatment on OPN expression, untreated samples were set as calibrator (control) and compared with their respective treated samples. GLI1 and SHH (Hs 00179843_m1) expressions were also similarly assessed with glyceraldehyde-3-phosphate dehydrogenase as an endogenous control. To analyze the knockdown effectiveness, “scrambled transfectants” of MDA-MB-435 was set as calibrator.
Chromatin Immunoprecipitation Assay—MDA-MB-435 cells were utilized for chromatin immunoprecipitation using the ChIP-IT Express enzymatic kit (Active Motif, Carlsbad, Calif.) following the manufacturer's protocol using GLI1 (N-16) X TransCruz antibody (Santa Cruz Biotechnology; sc-6153 X). The recovered DNA was PCR amplified using primers: forward, (SEQ ID NO: 15) 5′-GTTTTTCCCTACTTTCTCCC-3′ and reverse, (SEQ ID NO: 16) 5′-CCAAAAACGCACACACAC-3′ to amplify a 145-bp segment of the OPN promoter containing the putative GLI1 binding site. The specificity of the pull-down was confirmed by amplifying a region approximately 1 kb upstream from the PCR product containing the GLI1 site tested. The primers used were: (SEQ ID NO: 17) 5′-TTCCCCCTACCAAATGTTCA-3′ and (SEQ ID NO: 18) 5′-TGCTGCAAAAGTAATTGTGGTT-3′. The PCR generates a 151-bp product. This segment lacks a predicted GLI1-binding site.
In Vitro Proliferation Assay—Cells (5000) of each cell type were seeded per well in separate 96-well plates. Cells were allowed to grow in complete medium for 6 days. Every day after initial seeding cells were harvested by trypsinization and counted in a hemocytometer. Counting for each cell type for each day was done in triplicate.
Motility Assay—These experiments were performed as previously described (Shevde, L. A., et al. (2006) Clin. Exp. Metastasis 23, 123-133). Images were acquired at T0, the reading at the initial time, and at T10 (10 h later). The experiment was conducted in duplicate and cell motility was calculated as (T0−T10)/T0, which represents the rate of movement over a 10-h period. For OPN add-back experiments the cells were pretreated for 12 h with 100 ng/ml human OPN (recombinant R & D Systems) and the assay conducted in the presence of OPN. Each experimental group was assessed in duplicate and data were recorded in three fields per well. Thus, six data points were recorded and analyzed per experimental group.
Invasion and Migration Assays—These experiments were performed as previously outlined (18) using a modified Boyden chamber assay. OPN add-back experiments for migration and invasion were conducted as described above with an additional step of pre-treating the cells for 24 h with rOPN. OPN was added to the upper and lower chambers (100 ng/ml) to ensure the OPN was present during the entire duration of the experiment. Each experimental group was assessed as three independent replicates.
In Vivo Assay—One million (100 μl) cells were injected into the third mammary fat pad of 6-week-old female athymic nude mice (Harlan Sprague-Dawley, Indianapolis, Ind.). Orthogonal tumor measurements were taken twice a week. The mean tumor diameter was calculated by taking the square root of the product of orthogonal measurements. Spontaneous metastasis was monitored as previously described (18). Eight mice were used for each group and the entire experiment was repeated once. All animals were maintained under the guidelines of the National Institutes of Health and University of South Alabama. All protocols were approved and evaluated by the Institutional Animal Care and Use Committee.
Statistical Analysis—Statistical differences between groups were assessed using the Mann-Whitney test, t test, or analysis of variance, using GraphPad Prism 4 software. Statistical significance was determined if the analysis reached 95% confidence. The precise p values are listed in the corresponding figure legends. In figures, the error bars represent mean±S.E.
Example 7—Molecular Analysis of the Crosstalk Between Breast Cancer Cells, Osteoblasts and Osteoclasts Involving the Hedgehog Pathway and OPNCell lines used: Breast cancer (BC) cells: Two metastatic breast cancer cell lines (i) MDA-MB-231 and (ii) SUM 159. Osteoblasts (OB): Two osteoblast cell lines. (i) hFOB and, (ii) MC3T3-E1 pre-osteoblast cells. Both lines produce OPN. Differentiation of the MC3T3-E1 cells, clone 14 was studied when cultured under conditions that induce differentiation (presence of ascorbic acid and β-glycerophosphate) (Zunich, S. M., et al. (2009) Mol Cancer 8, 12). Osteoclasts (OC): Osteoclasts (i) RAW264.7 cells of the macrophage/monocyte lineage were cultured under conditions to differentiate them into osteoclasts (Narducci, P., et al. (2009) Ann Anat; Voronov, I., Li, K., et al. (2008); and Biochem Pharmacol 75, 2034-2044).
Example 8—Effects of Breast Cancer Cells on OsteoblastsThe role of the Hedgehog pathway in regulating the expression of OPN in the two osteoblastic cell lines, hFOB and MC3T3-E1 was assessed. Referring to
The procedure for differentiation of osteoblasts was standardized. MC3T3-E1 cells were cultured in the presence of ascorbic acid and β-glycerophosphate. Mineralization was examined by Alizarin RedS staining (Ueno, A., et al. (2001). Matrix Biol 20, 347-355; Lee, Y. K., et al. (2004). J Biomed Mater Res A 69, 188-195; Duarte, W. R., et al. (2003). S100A4: a novel negative regulator of mineralization and osteoblast differentiation. J Bone Miner Res 18, 493-501). Referring to
The effect of conditioned culture medium from breast cancer cells on the differentiation of osteoblasts was assessed. Referring to
OPN expression by the SUM1315 cells influences osteoblast differentiation. Referring to
Differentiated osteoblasts were stained for their alkaline phosphatase activity. Alkaline phosphatase is a reliable marker of osteoblast differentiation (Ebisawa, T., et al. (1999). J Cell Sci 112 (Pt 20), 3519-3527; Mori, K., et al. (2008). Cancer Sci 99, 2170-2176; Kitagawa, Y., et al. (2005). Cancer Res 65, 10921-10929; Mercer, R. R., et al. (2004). Clin Exp Metastasis 21, 427-435). Referring to
The expression of sialoprotein and osteocalcin, two bonafide markers of osteoblast differentiation viz. bone was assessed (Wang, J., et al. (2008) J Dent Res 87, 650-654; Lampasso, J. D., et al. (2006) Int J Mol Med 17, 1125-1131; Chou, Y. F., et al. (2005). J Biomed Mater Res B Appl Biomater 75, 81-90; Wang, D., et al. (1999) J Bone Miner Res 14, 893-903; Maeda, T., et al. (2004). J Cell Biochem 92, 458-471). Referring to
Effects of breast cancer cells-conditioned medium on the differentiation of osteoclasts were examined. Pre-osteoblastic RAW cells were cultured in the presence of M-CSF and RANKL. The effects of conditioned medium from breast cancer cells on osteoclast differentiation were also assessed (
Cell lines—Human metastatic breast cancer cells, MDA-MB-231, were cultured in Dulbecco's Modified Eagle's Medium (DMEM/F12; Invitrogen, Carlsbad, Calif.), supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.02 mM non-essential amino acids, 5% fetal bovine serum, FBS (Atlanta Biologicals, Norcross, Ga.), without antibiotics or antimycotics (cDME/F12). SUM1315 and SUM159 cells (DiMeo, T. A., et al. (2009) Cancer Res 69, 5364-5373) (Asterand, Detroit, Mich.) were cultured in DMEM/F12 supplemented with 5 mg/ml insulin, 5% FBS (Atlanta Biologicals) and either 10 ng/ml EGF or 1 ng/ml hydrocortisone, without antibiotics or antimycotics. The SUM1315 cells are derived from a metastasis in a patient with infiltrating ductal carcinoma; SUM159 cells were derived from a primary breast tumor with metaplastic carcinoma. RAW264.7 (ATCC, TIB 71) cells, a murine pre-osteoclastic line capable of differentiation and mineralization in culture (in presence of RANKL and MCSF), were grown in DMEM with L-glutamine (ATCC, 30-2002) supplemented with 10% FBS. A 2× differentiation medium (DM) was formulated for the RAW 264.7 cells comprising RAW264.7 growth medium supplemented with 20% FBS, RANKL (100 ng/ml) and M-CSF (40 ng/ml) (Matsubara, T., et al. J Bone Miner Res 25, 1068-1076). Conditioned media was harvested from breast cancer cells and mixed in a 1:1 ratio with double strength differentiation medium to assess the effect on osteoclast differentiation. lx DM was used as control or wherever SHH (R & D Systems, Minneapolis, Minn.) or OPN (R & D Systems) was used alone. The medium on the RAW264.7 cells was replenished every 48 hours. In order to assess the effect of secreted Hh ligands, the neutralizing 5E1 antibody was used (Developmental Studies Hybridoma Bank, at the University of Iowa, Iowa). The amount of 5E1 antibody used for the studies was determined following titration of the antibody with respect to its effects on osteoclast differentiation. Medium was supplemented with 5E1 (2.5 μg/ml) and was changed on alternate days until the end of the experiment. The effect of OPN on osteoclast activity was assessed by transfecting an OPN shRNA-expressing construct on day 6 post-induction of differentiation. Fresh DM was added the following day and cells were allowed to grow for another 12 hours before termination of experiment. GLI1 expression was silenced in the SUM1315 cells using shRNA targeting GLI1 into pSuperior. gfp+neo (Oligoengine, Wash., USA). Silencing of OPN expression was done using OPN-targeting shRNA cloned into pSuper (Oligoengine)
Osteoclast differentiation and activity assays—Tartarate-resistant acid phosphatase (TRAP) assay was conducted for RAW 264.7 following the manufacturer's protocol (Sigma, St. Louis, Mo.). This assay was indicative of the extent of differentiation. OAAS plates (Osteogenic Core Technologies, Choongnam, Republic of Korea) were utilized to measure osteoclastic activity. RAW 264.7 cells (25×103) were inoculated in 48-well plates. Cells were treated with serum-free conditioned media from breast cancer cells or 100 nM SHH or 100 ng/ml OPN on the following day. Media was changed every two days and the experiments were terminated either on day 6 (day 7 for knockdown experiments, with transfections being done on the sixth day). At the completion, cells were detached with 5% sodium hypochlorite and the wells were observed under a Nikon Eclipse TS 100 microscope at 10× magnification. To test the inhibitory effect of cyclopamine on osteoclast differentiation and activity, RAW264.7 cells were cultured in differentiation medium supplemented with 20 μM cyclopamine (Sigma) dissolved in DMSO (Sigma). Medium containing cyclopamine was changed every 48 hrs. The percentage of area resorbed was calculated using the NIS-Elements BR. 3.1 software.
Western Blotting Analysis—Whole cell lysates were collected in NP-40 buffer (150 mM NaCl, 50 mM Tris, 1% NP-40). Total protein (30 μg) was resolved by SDS-PAGE gel and transferred to PVDF membranes. Membranes were immunoblotted overnight at 4° C. with antibodies to either SHH (Santacruz, Calif., USA) or IHH (Santacruz). Equal loading was confirmed with anti-β-tubulin (Cell Signaling, Danvers, Mass.). To assess OPN expression upon SHH treatment, 105 cells were grown in 6-well plates in the presence of SHH. After 24 hours the cells were lysed in NP-40 buffer and 30 μg of each experimental group assessed by immunoblotting. To assess for the expression of OPN, MMP9 and CTSK, at the end of Day 6 of differentiation, all the different experimental groups were kept in serum free medium for 24 hrs and both, the conditioned media and whole cell lysates were collected and immunoblotted using antibodies to either anti-mouse OPN (Millipore, Bedford, Mass.), or CTSK (SantaCruz Biotech). Equal loading was confirmed with anti-β-actin (Sigma) antibody. Secreted MMP9 (Santa Cruz Biotech) was assessed by loading equal quantity of protein from the serum-free conditioned medium. Corresponding HRP conjugated secondary antibodies were used for detection; blots were developed with SuperSignal enhanced chemiluminescence substrate (Pierce, Rockford, Ill.) and imaged using a Fuji LAS3000 imager. Detection and quantification of proteins was done using Fuji LAS 3000 apparatus and Multigauge V3.1 software (Fujifilm, Valhalla, N.Y.). Band intensities were measured in arbitrary Units (AU). Relative band intensity was obtained as a ratio of individual band intensity to that of the corresponding β-actin band.
Luciferase Assay—Cells were co-transfected with the OPN promoter construct, pGL3-OPNB and pSV-β-galactosidase (Promega) (Das, S., et al. (2009) J Biol Chem 284, 22888-22897). Empty pGL3 vector was used as control. Different concentrations of SHH ligand were added to the well the next day. Cells were lysed in Reporter Lysis buffer (Promega) 24 hrs post addition of SHH and both β-galactosidase assay and luciferase assays were done following manufacturer's protocol. Readings were normalized to β-galactosidase. Each parameter was studied in triplicate and the experiment repeated at least 3 times.
Quantitative RT-PCR (qRT-PCR)—cDNA was generated using High Capacity Reverse Transcriptase Kit (Applied Biosystems, Foster City, Calif.). Real time PCR was performed in two cycles using a BioRad iQ5 Real-Time Detection system (Bio-Rad, Hercules, Calif.): the first cycle of 95° C. for 10 mins followed by 40 repeats of the second cycle comprising 95° C. for 15 sec followed by 60° C. for 1 min. All reactions were done in triplicate. Transcript levels were normalized to GAPDH levels (dCT) which was used to calculate changes in gene expression (2-ddCT). In order to assess levels of OPN, CTSK, and MMP9 cells (50×103) were seeded in each well of 12-well plates. Following the experimental regime as described herein (for Osteoclast differentiation/activity) RNA was isolated using Trizol (Invitrogen) and assessed as described herein. The details of the primers used were as follows: OPN (Spp1; Mm00436767_m1), Matrixmetalloprotease 9 (MMP9; Mm00600163_m1), Cathepsin K (Ctsk; Mm00484036_m1), SHH (Hs00179843_m1), GLI1 (Hs01110766_m1), OPN (Hs00959010_m1), hGAPDH (Hs99999905_m1) and GAPDH (Mm99999915_g1).
Statistical analysis—Statistical differences between groups were assessed using the Mann-Whitney test, t-test or ANOVA, using GraphPad Prism 4 software. Statistical significance was determined if the analysis reached 95% confidence. The precise p values are listed in the corresponding figure legends. In all figures the error bars represent standard error of the mean (S.E.M.).
Hh Signaling Activates OPN Expression in Pre-OsteoclastsIn melanoma, activated Hh signaling culminates in the transcription of OPN by GLI1, the transcription factor of the Hh pathway. The ability of pre-osteoclastic RAW264.7 cells to regulate OPN in response to Hh ligands was assessed. As seen in
The Hh pathway may have a role in bone development and homeostasis, OPN is critical to osteoclast activity, specifically their motility (Sugatani, T., et al. (2003) J Biol Chem 278, 5001-5008). Since both, SHH and OPN are secreted molecules, the effects of recombinant SHH and OPN on influencing differentiation of RAW264.7 cells were evaluated. Osteoclast differentiation was scored by staining the cells for TRAP, an indication of differentiated osteoclasts. multinucleate (>3 nuclei per cell) TRAP-positive cells were scored. As seen in
Breast cancer cells potentiate osteoclast activity leading to increased osteolysis. This is brought about, in part, by factors secreted by the breast cancer cells (Kingsley, L. A., et al. (2007) Mol Cancer Ther 6, 2609-2617). In order to determine the effects of factors secreted by the breast cancer cells on osteoclast differentiation, the conditioned medium of breast cancer cells was mixed in equal proportion with double strength DM and assessed the effects on differentiation of the RAW264.7 cells was assessed. As seen in
Breast cancer cells express SHH and IHH (
In order to determine the effects of Hh signaling on osteoclast activity, the osteoclasts on plates were cultured coated with a mineralized bone matrix. The area resorbed was estimated. As seen in
In order to assess the role of Hh signaling in osteoclasts in determining their maturation and activity, the differentiation medium was supplemented with the SMOH inhibitor, cyclopamine. As seen in
While OPN enhances osteoclast motility and overall activity, activation of osteoclasts is functionally dictated by the expression of proteases such as MMP9 and cathepsin K (CTSK). Thus, in order to assess if Hh signaling initiated by breast cancer cells influences the ability of the differentiated osteoclasts to express these key molecules, the expression of OPN, MMP9 and CTSK was assessed by a real-time quantitative PCR. As seen in
Hh signaling transcriptionally promotes the expression of OPN (
Silencing the expression of endogenous OPN in osteoclasts decreases their ability to express Cathepsin K and MMP9 in response to breast cancer cell conditioned media. RAW254.7 cells were cultured under differentiating conditions for 6 days to allow for complete differentiation. Differentiation conditions included recombinant SHH (100 nM) or conditioned media from breast cancer cells (MDA-MB-231, SUM159 and SUM1315) with or without the 5E1 antibody (2.5 μg/ml). One set of osteoclasts was silenced on day 6 for OPN expression (KO) using shRNA targeting OPN cloned into pSuper. The expression of
As seen in
Hh Signaling in the Breast Cancer Cells Ameliorates their Ability to Influence Osteoclast Differentiation and Activity
While Hh ligands expressed by the breast cancer cells clearly play a role in influencing osteoclast differentiation and activity, the effect of SUM1315 cells that have been abrogated for the expression of the transcription factor, GLI1, was assessed.
RAW264.7 cells were cultured for 6 days in presence of double strength DM supplemented (1:1) with medium from SUM1315 breast cancer cells. Osteoclast differentiation was assessed by TRAP assay and the activity was assessed by culturing the osteoclasts as described above, on OAAS plates. Relative to untransfected SUM1315 cells and SUM1315 cells transfected with a scrambled control (scr1 (pSuperior.gfp+neo) and scr2 (pSuper)), the medium from the cells silenced for GLI1 expression (KD2) and OPN (OPNi) was significantly less efficient in inducing
As seen in
Metastases in the bone occur in 60-80% of advanced breast cancer patients. The bone metastases in breast cancer are predominantly osteolytic, characterized by vigorous bone resorption (bone breakdown). The most common mode of transport of breast cancer cells from the breast to bone is through the vertebral-venous system that allows breast cancer cells to come into contact with the axial skeleton, including the ribs, spine, pelvis, and proximal humorous and femur, which is the main distribution of bone metastases in breast cancer patients. Once malignant cells have migrated to the bone, their ability to colonize is facilitated by various growth factors that are secreted by the bone. The crosstalk between tumor cells and the microenvironment promotes a vicious cycle of tumor growth and bone loss that perpetuates the formation of bony lesions. When the bone is lysed by osteoclasts, the factors released stimulate malignant tumor growth, which then increases the number of cells available to release the factors that stimulate osteoclastic activity, so more bone is resorbed and the cycle continues.
Factors, such as MMPs, chemokine receptor 4 (CXCR4), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) target metastatic tumor cells to bone and facilitate survival within the bone microenvironment. Physical factors within the bone microenvironment, including hypoxia, acidic pH, and extracellular Ca2+, and bone-derived growth factors, such as TGF-β and IGFs, activate tumor expression of osteoblast-stimulatory factors, like vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and Endothelin (ET-1) (Yin, J. J., et al. (2003) Proc Natl Acad Sci USA 100, 10954-10959. Maturation of osteoblasts is coupled with their release of RANKL that can stimulate osteoclastogenesis. Breast cancer cells also express osteoclast-stimulatory factors, such as PTHrP, TGF-β, and IL-11. In fact, expression of IL-11 and OPN by breast cancer cells has been found to be critical for the osteolytic activity of breast cancer cells.
The Hh signaling pathway involves the binding of a Hh ligand to the receptor PTCH, thereby relieving its inhibitory effect on SMOH, permitting transduction of the Hh signal to intracellular components, culminating in transcriptional activation of downstream genes like GLI1. The Hh ligands were initially thought to be a transmembrane, non-diffusible signal for neighboring cells. Further research revealed that the Hh ligands are secreted after being post-translationally modified and participate in short- and long-range signaling. Data provided herein shows that Hh ligands expressed by breast cancer cells can initiate a crosstalk directly with osteoclasts and promote osteoclast differentiation (assessed by multinucleate cells showing TRAP activity) and resorption activity accompanied by increased expression of OPN, CTSK and MMP9. TRAP, a glycosylated monomeric metalloenzyme, is highly expressed in osteoclasts and has been implicated in the detachment of cells necessary for initiating cell migration. It is upregulated during osteoclastogenesis along with CTSK and as such used as a histochemical marker for differentiated osteoclasts. Multinucleation, an essential step in osteoclast differentiation, is a prerequisite for its efficient bone resorbing ability. Mononuclear osteoclasts fuse repeatedly to form giant multinucleated osteoclasts which after the polarization of the membrane and organization of the cytoskeleton result in a mature bone-resorbing osteoclast.
The bone resorbing ability of mature osteoclast has been attributed to cysteine proteinase CTSK and a host of different matrix metalloproteinases, including MMP9, MMP 13 and MMP14. CTSK with its ability to cleave the native helix of collagen at multiple sites has been implicated as the molecule for matrix solubilisation whereas collagenolysis-enhancing MMP 9 has been found to be critical for osteoclast migration. Enhanced osteoclast differentiation and activation elicited by breast cancer cells was concomitant with significantly increased expression of OPN and the cysteine protease CTSK and MMP9. Moreover, Hh ligands expressed by the breast cancer cells play a critical role in inducing changes in the osteoclasts, since neutralizing the activity of these ligands from the conditioned medium of the breast cancer cells reduces the efficacy of the breast cancer cells to elicit osteoclast differentiation and resorptive activity. Hh ligands expressed by breast cancer cells are also essential for the production of OPN, CTSK and MMP9 by the osteoclasts since squelching them with the 5E1 antibody resulted in a significant decrease in expression.
As such, the data shows that OPN expression is upregulated as a downstream event of Hh signaling initiated by the Hh ligands expressed by the breast cancer cells. OPN is particularly abundant at the attachment sites of osteoclasts and is essential for reorganization of the osteoclast cytoskeleton for osteoclast motility. It is not only responsible for activating the bone resorptive ability of osteoclasts but also for their migration. Further, OPN enhances the differentiation of pre-osteoclasts to osteoclasts, resulting in upregulated expression of CTSK and MMP9. Consequently, resorptive activity of the osteoclasts is also negatively impacted by the inability to express OPN. While Hh signaling in osteoclasts influences their activity, the data reveals that Hh signaling in breast cancer cells is also vital to their ability to elicit osteoclast activation. OPN expressed by breast cancer cells also enhances osteoclast activity. Overall, the study indicates a causal role for Hh signaling in promoting breast cancer cell-mediated osteolytic activity.
Hh ligands expressed by breast cancer cells act as conversational molecules and can directly mediate a paracrine crosstalk with osteoclast precursors leading to osteoclastogenesis and the induction of resorptive activity. Osteoclasts respond to the stimulus provided by breast cancer cells by activating Hh signaling and upregulating OPN expression that is vital to their differentiation and resorptive activity. Thus, it is likely that the accumulation of OPN and PTHrP in the bone microenvironment in response to Hh signaling can potentially have a cumulative effect on the osteoclasts resulting in their activation (
In this study, the role of the Hh pathway in the crosstalk between tumor cells and osteoblasts is investigated. Tumor cells are shown to facilitate osteoblast differentiation and deposition of mineralized matrix via Hh signaling. These differentiated osteoblasts express RANKL, that together with OPN and PTHrP tilt the balance in favor of the osteoclasts. As such, these studies highlight the importance of the delicate balance between the activities of osteoblasts and osteoclasts and bring forth the importance of Hh signaling as an important attribute of the tumor cells' ability to cause osteolytic metastases.
Materials and MethodsCell lines—Human fetal osteoblasts, hFOB 1.19 (ATCC, CRL-11372) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM/F12; Invitrogen, Carlsbad, Calif.), supplemented with 2 mM L glutamine, 1 mM sodium pyruvate, 0.02 mM nonessential amino acids, 5% FBS (Atlanta Biologicals, Norcross, Ga.), without antibiotics or antimycotics (DMEM/F12). MC3T3-E1 subclone 14 (ATCC, CRL-2594) murine pre-osteoblast cells capable of differentiation and mineralization in culture (these lines exhibit high levels of osteoblast differentiation after growth in ascorbic acid and 3 to 4 mM inorganic phosphate) were maintained in alpha Minimum Essential Medium (αMEM) (Mediatech, Herndon, Va.) and 10% FBS but devoid of ascorbic acid. RAW 264.7 (ATCC, TIB 71) cells, a murine preosteoclastic line capable of differentiation and mineralization in culture (in presence of RANKL and M-CSF) were grown in DMEM with L-glutamine (ATCC, 30-2002). MDA-MB-231 human metastatic breast cancer cells, SUM1315 (derived from a metastasis in a patient with infiltrating ductal carcinoma), SUM159 cells (derived from a primary breast tumor with metaplastic carcinoma) and MDA-MB-435 (435) cells were cultured as described herein. The generation and culture conditions of 435 cells stably silenced for OPN (OPNi) or GLI1 (K01 and K04) is previously described (Das S, et al. The hedgehog pathway transcription factor GLI1 promotes malignant behavior of cancer cells by up-regulating osteopontin. J Biol Chem 2009; 284:22888-97).
Induction of osteoblastic and osteoclastic differentiation—In order to test the effect of conditioned medium from the tumor cells on osteoblast differentiation, a double-strength differentiation medium (DM) was formulated for MC3T3 E1 Sc-14 cells. It comprised αMEM, 20% FBS, 50 μg/ml ascorbic acid and 20 mM β-glycerophosphate. Conditioned media and the double-strength DM were mixed in a 1:1 ratio. 1×DM was used as control. Similarly a double-strength differentiation medium was formulated for RAW 264.7 cell lines. It consisted of 20% FBS, 50 ng/ml of RANKL and 20 ng/ml of M-CSF added to the growth medium. Conditioned media from the tumor cells was mixed 1:1 with the double-strength DM. 1×DM was used as control. Osteoblast differentiation was assessed by alkaline phosphatase (ALP) activity assay in the perspective of total phosphatase. The functional assessment of osteoblast mineralization was quantified by staining with Alizarin Red S and scoring the number of mineralized nodules.
Apoptosis detection—MC3T3 cells were grown under differentiation conditions along with conditioned media from tumor cells for 21 days with media being changed every 3rd day. At the end of 21 days apoptosis was assayed using the In Situ Cell Death Detection Kit (Roche, Indianapolis, Ind.) following the manufacturers' protocol for initial TUNEL staining. Cells were further stained with DAPI (Vectashield, H-1200, Vector Laboratories, Burlingame, Calif.) and phalloidin coupled with AlexaFluor 555 (Molecular Probes, Invitrogen) to visualize the nuclei and cytoskeleton respectively. The latter staining imparted context to the TUNEL staining. Cells were visualized under the Nikon TE2000 microscope and TUNEL positive cells were counted and expressed as a percentage of total cells in each field of view.
Western Blotting Analysis—was performed as described herein
Studies with Hh inhibitor, cyclopamine—Serum-free conditioned medium (SFM) harvested from ˜3.0×106 cells after 24 hours was assayed for OPN by immunoblotting. To test the inhibitory effect of cyclopamine on the Hh pathway cells were cultured in DMEM supplemented with 0.5 FBS and treated for the indicated time intervals with DMSO (vehicle control) or cyclopamine (Sigma, St. Louis, Mo.).
Luciferase Assay—was performed as described herein.
Quantitative RT-PCR (qRT-PCR)—cDNA was generated using High Capacity Reverse Transcriptase Kit (Applied Biosystems, Foster City, Calif.). Real time PCR was performed using a BioRad iQ5 Real-Time Detection system (Bio-Rad, Hercules, Calif.). All reactions were done in triplicate. OPN transcript levels were normalized to GAPDH levels (dCT) which was used to calculate changes in OPN expression (2-ddCT). To analyze the effect of cyclopamine treatment on OPN expression untreated samples were set as calibrator (control) and compared to their respective treated samples. The primers used included Spp1 (OPN) (Mm 00436767_m1); Bglap (osteocalcin) (Mm 01741771_g1); IBSP (Mm 00492555_m1); PTHrP (Mm 00433057_m1); RANKL (Mm 00441906_m1); GAPDH (Mm 99999915_g1).
Immunohistochemical analyses—Breast tumor tissue microarrays were obtained from the NCI Cooperative Breast Cancer tissue Resource (CBCTR). The tissues were immunohistochemically stained for IHH and GLI1. Immunohistochemical staining was performed using Dako LSAB+ System-HRP reagents in a Dako Autostainer Plus automated immunostainer (Glostrup, Denmark). The intensity of staining was quantitated with computer-assisted image analysis in a Dako ACIS III Image Analysis System (Glostrup, Denmark).
Statistical Analysis—was performed as described herein.
Results Expression of GLI1 and IHH is Upregulated in Breast Cancer.Using immunohistochemical analyses, the expression of the Hh ligand, IHH and the transcription factor GLI1 were assessed in a tissue array comprising 75 breast cancer tissues and 9 tissues representing normal breast. While the staining intensity of IHH was comparable (p>0.05) in normal tissues and in tissues derived from Ductal Carcinoma In Situ (DCIS), the tissues derived from invasive cancer (representing Infiltrating Ductal Carcinoma Grades II-IV) and from metastatic breast cancer exhibited significantly (p<0.0001) increased staining intensity for IHH (
In order to assess the effect of Hh signaling on the formation of osteoblasts, the monopotential cell line, MC3T3-E1, was used. This cellin is a clonal osteoblastic cell line isolated from calvariae of a late stage mouse embryo. These cells express various osteoblast functions including formation of mineralized bone nodules in long-term culture. The addition of Hh ligands, SHH and IHH to the DM of the MC3T3 cells stimulated differentiation as seen by the increase (p<0.0001) in the ALP activity (
Hh signaling induces the expression of OPN. OPN promotes adhesion of osteoblasts allowing them to function in osteogenesis. Two osteoblast-forming cells, hFOB and MC3T3 were treated with two Hh ligands, SHH and IHH and assessed the effect on OPN promoter activity. Both ligands caused an upregulation in OPN promoter activity (p<0.0001) (
Tumor cells express Hh ligands. In order to determine the role of the Hh pathway in mediating the crosstalk between tumor cells and osteoblasts, the effect of conditioned medium from the tumor cells on MC3T3 osteoblast differentiation was assessed after 2 weeks using an ALP activity assay. Relative to DM alone, conditioned medium from the tumor cells caused a significant (p<0.001) increase in the ALP activity in 2 weeks. The 5E1 antibody blocks binding of all three mammalian Hh ligands to Ptc1 with low nanomolar affinity, thereby inhibiting Hh signaling. Depleting the Hh ligands from the conditioned medium of the tumor cells using the neutralizing 5E1 antibody caused a decrease in the ALP activity of the differentiated osteoblasts. While the decrease was apparent, although not statistically significant with respect to the conditioned medium from MDA-MB-231 and MDA-MB-435 cells, the decrease was statistically significant (p<0.05) with respect to conditioned medium from SUM1315 and SUM159 cells (
OPN, a secreted protein expressed by tumor cells, has been implicated as an important regulator of osteoblast differentiation. Both, SUM1315 and MDA-MB-435 cells express OPN. To assess the effect of tumor cell-derived OPN on the osteoblasts, OPN expression was abrogated using shRNA, and the cell-free conditioned medium from these cells was harvested. Osteoblast differentiation was studied in presence of this conditioned medium and the expression of BSP and osteocalcin as indicators of osteoblast differentiation was assessed and measured osteoblast differentiation activity by enumerating the mineralized nodules formed. The conditioned medium from the SUM1315-OPNi and 435-OPNi cells was less efficient (p<0.005) in inducing osteoblast differentiation and mineralization (
Hh Signaling in Tumor Cells Impacts their Ability to Induce Osteoblast Differentiation.
Hh signaling in breast cancer cells also plays a vital role in communication between the breast cancer cells and osteoclasts. In order to assess the role of Hh signaling in tumor cells on their ability to elicit osteoblast differentiation, the expression of GLI1 from the tumor cells was abrogated by shRNA. Conditioned medium from the GLI1-silenced cells was inefficient (p<0.005) in inducing osteoblast differentiation as represented in the expression of BSP, osteocalcin (
The data suggested that soluble factors that include OPN and the Hh ligands secreted by tumor cells enhance osteoblast differentiation and mineralization activity. This starkly contradicts the well-established notion that tumor cells causes osteoblasts to undergo apoptosis (Mastro A M, et al. J Cell Biochem 2004; 91:265-76). Notably, these reported studies conducted osteoblast differentiation for longer time periods i.e. 3 weeks or longer. Thus, in order to capture the full impact of the differentiation conditions on the osteoblasts, parallel experiments that were assessed 3 weeks post induction of differentiation were conducted. While differentiation and mineralization activity were already attained at 14 days, the levels of BSP and osteocalcin plummeted sharply (p<0.001) at 3 weeks relative to their expression at 2 weeks in differentiation conditions comprising conditioned media from tumor cells (
Intuitively, the data suggests that tumor cells initiate osteoblast differentiation and the expression of osteoclastogenic factors as an early event, followed by elimination of osteoblasts later. Thus, the overall microenvironment appears to shift in favor of osteoclastogenesis. In order to investigate the significance of Hh signaling in the tumor cells with respect to osteolytic metastasis, tumor cells were injected via the left ventricle and assessed the incidence of osteolytic metastases at the tibio-femoral junction 4-6 weeks later. In the mice injected with 435-vector control cells, metastasis in 100% of the mice injected was observed. In contrast, the incidence of mice injected with tumor cells stably silenced for GLI1 was reduced to 60% (
The Hh pathway plays an essential function in regulating cell fate and in developmental patterning in animals and humans. This pathway is also important in the formation of the skeleton. During skeletogenesis and endochondral ossification Hh signaling coordinates growth and differentiation. In adult animals, systemic administration of the ligand SHH, resulted in a primary increase in osteoblasts and their precursors. Interestingly, this was accompanied by an enhanced osteoclastogenic potential and decreased bone volume due to upregulation of the PTH/PTHrP receptor. Thus, Hh signaling in the adult bone milieu caused stimulatory effects on osteoprogenitors and osteoblasts resulting in bone remodeling and reduced bone strength because of a secondary increase in osteoclastogenesis.
The bone is a common site of metastasis for several malignancies. The impact of metastasized tumor cells in the bone disrupts the balance between the activities of the osteoclasts and osteoblasts. Radiographically, the bone lesions are classified as being osteolytic (bone loss) or osteosclerotic (bone formation) or mixed. Breast cancer bone metastases are usually osteolytic, characterized by excess bone turnover and consequent bone resorption. This is concomitant with the apoptosis and elimination of osteoblasts. In fact, several papers suggest that breast cancer cells limit osteoblasts by either inducing apoptosis or interfering with normal function and thus facilitating osteolysis through increased osteoclast activity. Paradoxically, it must be noted that the basic trigger for the differentiation of pre-osteoclasts to osteoclasts is supplied by the osteoblasts. Osteoblasts produce M-CSF and RANKL that promote pre-osteoclasts to differentiate into multinuclear, activated osteoclasts that adhere to bone and degrade the bone matrix. RANKL and M-CSF activate a dendritic cell-specific transmembrane protein (DCSTAMP) that facilitates cell-cell adhesion and cytoskeletal re-arrangements resulting in a multinucleate osteoclast. Thus, the availability of differentiated osteoblasts is vital to the development of active osteoclasts. Likewise, osteoclasts express BMPs that promote recruitment and proliferation of osteoblasts at resorption sites.
Thus, given the vital role that osteoblasts play in facilitating osteoclast activity, the elimination of osteoblasts by the tumor cells seems counter intuitive and warrants further understanding of the delicate balance between osteoblast and osteoblasts. In this study, the role of Hh signaling in tumor cells on the interaction between tumor cells and osteoblasts was investigated. Breast cancer cells were determined to express elevated staining intensities for the Hh ligand IHH and the transcription factor, GLI1, indicating that the Hh pathway is activated in breast tumor cells. In order to determine the consequences of the interaction between tumor cells and osteoblasts, osteoblast differentiation at early (14 days) and late (21 days) postinitiation of differentiation was investigated in presence of conditioned media from tumor cells. While Hh ligands expressed by the tumor cells enhanced osteoblastogenesis and mineralization activity as an early event, enhanced expression of osteoclastogenesis-promoting factors viz. RANKL and PTHrP in the differentiated osteoblasts was observed. Likewise, OPN expressed by the tumor cells also stimulated osteoblast differentiation. Tumor cells with a competent Hh pathway were more potent at inducing osteoblast differentiation and expression of RANKL and PTHrP. While the expression of osteoblast differentiation markers, BSP and osteocalcin dwindled at a later event (21 days) characterized by increased apoptosis of the osteoblasts, the expression of RANKL and PTHrP continued to be robust, suggesting that the osteoblasts were expressing factors that would propel osteoclastogenesis. Thus, this data suggests that tumor cells initially enhance the differentiation of osteoblasts that in turn, express osteoclastogenesis enhancing factors. Later, as the osteoblasts get eliminated, the availability of RANKL and PTHrP creates an environment that will stimulate osteoclast differentiation and activity. Thus, an active Hh signaling in the tumor cells facilitates the generation of an osteoclast-stimulating milieu by initially kickstarting osteoblast development. This is apparent in the fact that ablating GLI1 severely compromised the ability of the tumor cells to form osteolytic metastasis in an experimental model of bone metastasis.
A role for osteoblast-derived PTHrP as a physiological regulator of bone remodeling has been previously suggested (Miao D, et al. J Clin Invest 2005; 115:2402-11; Miao D, et al. Endocrinology 2004; 145:3554-62). PTHrP is produced by cells of early osteoblast lineage that do not express PTH-receptor. PTHrP acts on receptor-positive committed preosteoblasts, and these cells respond by differentiating into mature osteoblasts. PTHrP acts directly on mature osteoblasts and osteocytes to prevent their apoptosis and is also required to enhance production of RANKL by PTHR1-positive pre-osteoblasts. As a result, osteoclast formation is promoted by interaction of the membrane molecule, RANKL, with its receptor, RANK. It is surmised that a fine balance or spatiotemporal control mechanisms exist to ensure availability of PTHrP for enhancing osteoblast differentiation, as persistently increased local PTHrP levels would favor increased osteoclast formation, through stimulation of RANKL production resulting in increased bone resorption, and high-turnover osteoporosis (Martin T J. J Clin Invest 2005; 115:2322-4.). In fact, the results herein show a steady expression of PTHrP by osteoblasts (at 21 days) and are supported by the fact that Hh signaling competent tumor cells in fact, cause radiographically evident osteolysis in animal models.
Skeletal integrity is an essential survival function of mammals. The findings herein reveal that the tumor cells can alter the balance between the activities of osteoblasts and osteoclasts via Hh signaling. Thus, given the fact that breast cancer cells express Hh ligands (
MDA-MB-435 (435) and SUM1315 (1315) cells were transfected with either empty vector (vec) or with vector encoding shRNA to GLI1 (shGLI1). Cells were treated with the indicated concentrations of drugs for 24 hours and viability was assessed using an MTS assay (
The paired human ovarian cancer cell lines A2780 and A2780-CP70 are cisplatin-sensitive (IC50 ˜3 μM), and cisplatin-resistant (IC50 ˜40 μM), respectively (Parker, R. J., et al. J Clin Invest, 87:772-777, 1991; Li Q, et al. J Biol Chem, 273:23419-23425, 1998; Bonovich M, et al. Cancer Gene Therapy, 9:62-70, 2002). A2780-CP70 cells were grown in monolayers, harvested in log phase growth, and assessed for the presence of Gli1 in: a) whole cell lysate; b) nuclear fraction; and, c) cytoplasmic fraction using Western blot analysis. Controls included α-tubulin and histone deactylase. Referring to
Gli1 protein expression was assessed in A2780 and A2780-CP70 cells grown in log phase in monolayers using Western blot analysis.). Gli1 would be expected in the cytosol but not in the nucleus in cells in which the Hedgehog pathway is inactive. Referring to
To assess whether the Hedgehog pathway is self-driven in A2780 and A2780-CP70 monolayers, cell lysates were assayed for IHH, SHH, and DHH using Western blot analysis. Referring to
Gli1 protein expression was examined in A2780-CP70 cells treated with the Smoothened inhibitor, cyclopamine, using Western blot analysis. A2780-CP70 cells were grown in log phase and treated with 70 μM cyclopamine for 24 hr, 48 hr, and 72 hr. Protein lysates were obtained form adherent cells. Under these conditions, 70 μM cyclopamine is associated with 50-70% cell killing at 72 hr.
Referring to
Transfection of an anti-GLI1 shRNA construct inhibits the Hedgehog pathway. Reduced Gli1 protein and mRNA levels were observed in the nucleus of transfected cisplatin-resistant A2780-CP70 cells. Gli1 mRNA levels rebounded by 72 hr post-transfection. These observations were similar to those observed in A2780-CP70 cells treated with cyclopamine, an inhibitor of the Hedgehog pathway. In addition, transfected A2780-CP70 cells showed stable mRNA levels of c-jun, increased mRNA levels of c-fos. C-fos mRNA levels peaked between 6 and 24 hr post-transfection. These observations were similar to those observed in A2780-CP70 cells treated with cyclopamine.
Example 17—Sonic Hedgehog (SHH) and Indian Hedgehog (IHH) Protein Expression in Cyclopamine-Treated A2780-CP70 CellsSHH and IHH protein levels were assessed in cyclopamine-treated A2780-CP70 cells by Western blot analysis. Referring to
c-jun protein expression was assessed in cyclopamine-treated A2780-CP70 cells using Western blot analysis. Controls included α-tubulin and histone deacetylase 3. c-jun antibody recognizes non-phosphorylated c-jun protein.
Referring to
c-jun and c-fos mRNA expression was assessed in A2780-CP70 cells treated with cyclopamine using semi-quantitative PCR (
c-jun protein expression was assessed in cisplatin-treated A2780-CP70 cells using Western blot analysis. A2780-CP70 cells were treated with an IC50 dose of cisplatin (Li Q, et al. J Biol Chem, 273:23419-23425, 1998). Referring to
The expression pattern of c-jun and c-fos is similar in A2780-CP70 cells treated with cisplatin or phorbol ester (Li Q, et al. International Journal of Oncology, 13:987-992, 1998; Li Q, et al. Cellular and Molecular Life Sciences, 55:456-466, 1999).
Comparative Example 20—c-Jun Protein Expression in A2780-CP70 Cells Treated with CisplatinPhosphorylation of c-jun protein in A2780-CP70 cells treated with cisplatin was assessed using Western blot analysis (Li Q, et al. J Biol Chem, 273:23419-23425, 1998) (
Protein expression of c-fos and phosphorylation of c-jun in cyclopamine-treated A2780-CP70 cells was assessed using Western blot analysis. Protein levels for c-jun, c-fos, phosphorylated c-jun (Ser 63), phosphorylated c-jun (Ser 73), and phosphorylated c-jun (Thr 91, Thr 93) were tested in cytosolic, nuclear, and whole cell fractions at 0 hr, 6 hr, 24 hr, 48 hr, and 72 hr. Internal controls for cytosolic and nuclear fractions included α-tubulin and histone deacetylase 3.
Referring to
The observed pattern of c-jun expression was distinct from the pattern observed in A2780-CP70 cells treated with cisplatin (Li Q, et al. J Biol Chem, 273:23419-23425, 1998). In response to cisplatin, c-jun (Ser 63/73) protein levels peaked at 3-5 hr after cisplatin exposure, and dropped dramatically below peak levels by 8 hr after exposure.
Accordingly, disruption of the Hedgehog pathway suppresses up-regulation of c-jun. Up-regulation of c-jun is necessary for up-regulation of genes that play a role in resistance to chemotherapeutic agents, such as genes involved in nucleotide excision repair, such as ERCC1 (Reed E. Cisplatin and platinum analogs. in: Cancer Principles and Practice of Oncology; 8th Edition. Lippincott, Williams, and Wilkins; Philadelphia, pp 419-26, 2008; Reed E. Cisplatin, Carboplatin, and Oxaliplatin. in: Cancer Chemotherapy and Biotherapy: Principles and Practice. 4th Edition. Lippincott, Williams & Wilkins, Philadelphia, pp 332-343, 2006; Li Q, et al. AntiCancer Research, 20: 645-652, 2000; Dabholkar, M., et al. J Clin Invest, 94:703-708, 1994; Dabholkar, M., et al. Oncology Reports, 2:209-214, 1995).
Example 22—Transfection of A2780-CP70 Cells with an Anti-GLI1 shRNA ConstructAn anti-GLI1 shRNA construct that incorporated an anti-GLI1 shRNA in a pSUPERIOR GFP neo vector was transfected into A2780-CP70 cells. This construct corresponds to GLI1 shRNA-1 construct of Example 4.
Gli1 protein expression was examined in nuclear and cytoplasmic fractions of A2780-CP70 cells transfected with anti-GLI1 shRNA construct using Western blot analysis. Referring to
GLI1 mRNA expression was examined in A2780-CP70 cells transfected with anti-GLI1 shRNA construct at 6 hr, 24 hr, 48 hr, and 72 hr post-transfection using a semi-quantitative PCR analysis (
Gli2 protein expression was examined in A2780 and A2780-CP70 cells transfected with anti-GLI1 shRNA construct using Western blot analysis. Referring to
c-jun and c-fos mRNA expression was examined in A2780-CP70 cells transfected with anti-GLI1 shRNA construct at 6 hr, 24 hr, 48 hr, and 72 hr post-transfection using semi-quantitative PCR analysis (
These data suggest that the c-jun response in A2780-CP70 cells is similar whether the hedgehog pathway is challenged by inhibiting the plasma transmembrane protein, Smoothened, with cyclopamine, or by reducing the levels of the transcriptional activator, Gli1, by transfecting cells with anti-GLI1 shRNA construct. An increase in c-jun protein expression is suppressed for 6 to 24 hr when the Hedgehog pathway is challenged by methods that result in ˜50% cell killing. In contrast, the c-jun response in cells treated with an IC50 dose of cisplatin is very different. Suppression of the Hedgehog pathway results in suppression of c-jun expression.
The Hedgehog pathway acting through Gli1 may allow for the rapid up-regulation of c-jun after treatment of cells with cisplatin. Rapid up-regulation of c-jun would allow for rapid up-regulation of ERCC1 and other NER genes that play a role in removal of platinum-DNA damage.
The >20-fold higher levels of Gli1 that are observed in cisplatin-resistant A2780-CP70 cells (as compared to cisplatin-sensitive A2780 cells), would allow for greater up-regulation of NER. This greater up regulation of NER would be consistent with previous observations, when comparing these two cell lines for platinum-DNA adduct repair. Such an interaction between Gli1 and Jun has been described on non-NER genes.
Example 25—c-Jun Protein Phosphorylation in A2780-CP70 Cells Transfected with Anti-GLI1 shRNA ConstructPhosphorylation of c-jun in A2780-CP70 cells transfected with anti-GLI1 shRNA construct was assessed using Western blot analysis. Protein levels for c-jun, phosphorylated c-jun (Ser 63), phosphorylated c-jun (Ser 73), and phosphorylated c-jun (Thr 91, Thr 93) were examined at 0 hr, 6 hr, 24 hr, 48 hr, and 72 hr. Referring to
The anti-GLI1 shRNA construct inhibits phosphorylation of c-jun at Ser 63. c-jun (Ser 63) plays a role in cellular resistance to chemotherapeutic agents, and DNA repair, such as ERCC1-related DNA repair. Accordingly, inhibition of Gli1 is likely to block the up-regulation of the c-jun (Ser 63) cascade and inhibit DNA repair mechanisms, such as those in which ERCC1 plays a role.
Gli1 and c-jun may co-operate in the transcriptional regulation of some genes (Laner-Plamberger S, et al. Oncogene 28:1639-1651, 2009). The data presented here suggests that Gli1 and c-jun cooperate in the transcriptional regulation of at least ERCC1 and other DNA repair genes.
Example 26—Sonic Hedgehog (SHH) and Indian Hedgehog (IHH) Expression in A2780 and A2780-CP70 Cells Transfected with Anti-GLI1 shRNA ConstructMonolayers of A2780 cells or A2780-CP70 cells were transfected with anti-GLI1 shRNA construct. SHH and IHH protein expression was examined 24 hr post-transfection on whole cell lysates using Western blot analysis. Referring to
Protein levels of Gli1, SHH, and IHH in A2780-CP70 cells were examined at 0 hr, 6 hr, 24 hr, 48 hr, and 72 hr post-transfection in cytosolic and nuclear fractions and whole cell lysates using Western blot analysis. Referring to
Cellular insults that result in 50% cell killing in A2780-CP70 cells can result in up-regulation of the JUN-kinase pathway and/or the ERK pathway (Li Q, et al. J Biol Chem, 273:23419-23425, 1998; Li Q, et al. International Journal of Oncology, 13:987-992, 1998; Li Q, et al. Cellular and Molecular Life Sciences, 55:456-466, 1999). Up-regulation of these pathways can result in up-regulation of ERCC1 and other essential DNA repair genes. To determine whether the Hedgehog pathway plays a role in up-regulation of ERCC1, the Hedgehog pathway was inhibited in conditions that resulted in 50% cell killing.
A2780-CP70 cells were treated with 70 μM cyclopamine, a concentration that causes 50-70% cell death. mRNA levels for ERCC1 and XPD of the NER pathway, and XRCC1 of the base excision repair pathway were determined at 6 hr, 24 hr, 48 hr, and 72 hr in treated and non-treated cells using semi-quantitative PCR. Referring to
In another experiment, A2780-CP70 cells were transfected with anti-Gli1 shRNA construct. mRNA levels for ERCC1 and XPD of the NER pathway, and XRCC1 of the base excision repair pathway were determined at 6 hr, 24 hr, 48 hr, and 72 hr in transfected and control cells using semi-quantitative PCR. Referring to
In sum, A2780-CP70 cells treated with cyclopamine or transfected with anti-GLI1 shRNA construct at levels that are associated with 50% cell killing had no detected effect on mRNA levels for ERCC1, XPD, or XRCC1. This observation contrasts with previous experiments where ERCC1, XPD, or XRCC1 were up-regulated in A2780-CP70 cells treated with other agents that invoke >50% cell death, including cisplatin and phorbol ester at IC50 doses.
Example 28—ERCC1, XPD, or XRCC1 mRNA Levels in Anti-GLI1 shRNA Construct—Transfected A2780-CP70 Cells Treated with CisplatinA2780-CP70 cells were transfected with anti-GLI1 shRNA construct at levels associated with 50% cell death. Cells were treated 24 hr post-transfection with 40 μM cisplatin for 1 hr (IC50 dose). mRNA levels of ERCC1, XPD, or XRCC1 were measured in adherent cells 6 hr, 24, hr, 48 hr, and 72 hr post-treatment using semi-quantitative PCR. Referring to
In sum, in cells treated with cisplatin, expression levels of ERCC1, XPD, and XRCC1 proteins increase 5-fold. However, in cells pretreated with anti-Gli1 shRNA construct, the expression levels of ERCC1, XPD, and XRCC1 do not increase in response to treatment to cisplatin.
Example 29—CD44, CD117 (c-Kit), and Gli1 Protein Levels in A2780 Cell Monolayers, Spheroids, and Monolayers Derived from SpheroidsA2780 and A2780-CP70 cells can be cultured to have stem cell-like phenotypes by inducing spheroids in spheroid-forming non-adherent culture conditions. CD44, CD117 (c-Kit), and Gli1 protein expression was assessed using Western blot analysis in A2780 cells grown in monolayers (MFC—monolayer forming cells), spheroids, and monolayers derived from spheroids (SFC—spheroid forming cells), monolayers derived from spheroids were cultured as described in (Zhang S, et al. Cancer Res 68:4311-4320, 2008).
Referring to
CD117 (c-Kit), and Gli1 protein expression in A2780 cell monolayers, spheroids, and monolayers derived from spheroids was assessed in cytoplasmic and nuclear fractions using Western blot analysis. Referring to
A2780-CP70 cells were transfected with 0.07 μg/well anti-Gli1 shRNA construct or control. Transfected cells were treated with 0 μM, 10 μM, 30 μM, and 100 μM cisplatin at 24 hr post-transfection. Percent cell growth was determined relative to control cells. Referring to
A2780-CP70 cells were treated with 20 μM cyclopamine for 1 hr, and 0 μM, 10 μM, 30 μM, or 100 μM cisplatin at 24 hr post-transfection. Control cells were treated with cisplatin only. Percent cell growth was determined relative to control cells. Referring to
A2780-CP70 cells were transfected with an IC50 dose of anti-Gli1 shRNA (treated) construct or vector (control). Total intracellular c-jun mRNA and c-jun protein was measured at 6 hr, 24 hr, 48 hr, and 72 hr.
A2780-CP70 cells treated with cisplatin results in upregulation of phosphorylation of c-jun protein (Ser 63/73); upregulation of c-jun (Ser 63/73) results in upregulation of AP-1; upregulation of AP-1 results in upregulation of genes including ERRC1, XPD, XPA, XRCC1, and other NER and BER genes. Upregulation of ERCC1 does not occur if upregulation of c-jun or AP-1 is blocked.
All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.
Claims
1.-139. (canceled)
140. A method of diminishing the levels of osteolysis in a subject having a cancer comprising: reducing the activity of Hedgehog (Hh) in a cancer cell of the subject.
141. The method of claim 140, wherein reducing the activity of Hedgehog (Hh) comprises reducing the level of a nucleic acid encoding OPN or the level of OPN protein in the cancer cell.
142. The method of claim 141, wherein reducing the level of a nucleic acid encoding OPN or the level of OPN protein comprises administering to the subject an isolated nucleic acid selected from the group consisting of a small hairpin RNA (shRNA), a small interfering RNA (siRNA), an artificial micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
143. The method of claim 140, wherein reducing the activity of Hedgehog (Hh) comprises reducing the level of a nucleic acid encoding GLI1 or the level of GLI1 protein in a cancer cell of the subject.
144. The method of claim 143, wherein reducing the level of a nucleic acid encoding GLI1 or the level of GLI1 protein comprises administering to the subject an isolated nucleic acid selected from the group consisting of a small hairpin RNA (shRNA), a small interfering RNA (siRNA), an artificial micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
145. The method of claim 144, wherein the isolated nucleic acid comprises a sequence encoding GLI1 or a fragment thereof, a sequence encoding antisense GLI1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof.
146. The method of claim 145, wherein the isolated nucleic acid comprises a sequence selected from SEQ ID NO.s:1-10.
147. The method of claim 140, wherein the Hedgehog (Hh) comprises Sonic Hedgehog (SHh).
148. The method of claim 140, wherein the cancer is breast cancer.
149. The method of claim 148, wherein the breast cancer is an infiltrating ductal carcinoma or a metaplastic carcinoma.
150. A method of regulating osteoclast differentiation in a subject, comprising reducing the activity of Hedgehog (Hh) in a cancer cell of the subject.
151. The method of claim 150, wherein reducing the activity of Hedgehog (Hh) comprises reducing the level of a nucleic acid encoding OPN or the level of OPN protein in the cancer cell.
152. The method of claim 151, wherein reducing the level of a nucleic acid encoding OPN or the level of OPN protein comprises administering to the subject an isolated nucleic acid selected from the group consisting of a small hairpin RNA (shRNA), a small interfering RNA (siRNA), an artificial micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
153. The method of claim 152, wherein reducing the activity of Hedgehog (Hh) comprises the level of a nucleic acid encoding GLI1 or the level of GLI1 protein in the cancer cell of the subject.
154. The method of claim 153, wherein reducing the level of a nucleic acid encoding GLI1 or the level of GLI1 protein comprises administering to the subject an isolated nucleic acid selected from the group consisting of a small hairpin RNA (shRNA), a small interfering RNA (siRNA), an artificial micro RNA (miRNA), an antisense polynucleotide, and a ribozyme.
155. The method of claim 154, wherein the isolated nucleic acid comprises a sequence encoding GLI1 or a fragment thereof, a sequence encoding antisense GLI1 or a fragment thereof, or an antisense nucleic acid complementary to a sequence encoding GLI1 or a fragment thereof.
156. The method of claim 155, wherein the isolated nucleic acid comprises a sequence selected from SEQ ID NO.s:1-10.
157. The method of claim 150, wherein the Hedgehog (Hh) comprises Sonic Hedgehog (SHh).
158. The method of claim 150, wherein the cancer is breast cancer.
159. The method of claim 158, wherein the breast cancer is an infiltrating ductal carcinoma or a metaplastic carcinoma.
Type: Application
Filed: Nov 30, 2016
Publication Date: May 25, 2017
Inventors: Lalita Samant (Vestavia Hills, AL), Rajeev Samant (Vestavia Hills, AL), Shamik Das (Vestavia Hills, AL), Eddie Reed (Bethsada, MD)
Application Number: 15/365,479
