Prohibitin as Target for Cancer Therapy

Abstract

The present invention relates to pharmaceutical compositions comprising inhibitors of Prohibitin (PHB) for the prevention or/and treatment of hyperproliferative disorders.

Description

The present invention relates to pharmaceutical compositions comprising inhibitors of Prohibitin (PHB) for the prevention or/and treatment of hyperproliferative disorders. Further, the present invention concerns PHB inhibitors and screening methods for identification of PHB inhibitors, which inhibitors are suitable for prevention or/and treatment of hyperproliferative disorders.

Ras-MAPK cascade has been activated in almost all tumours and plays a very important role in proliferation, tumour cell migration, invasion of the extracellular matrix, resistance to apoptosis and angiogenesis (ability to induce new blood vessel formation). There are at least 20 new therapeutic agents in clinical trials at present exploiting the interaction and activation of several components of this highly conserved pathway. This includes a range of Farnesyl transferase inhibitors (FTIs) to block the translocation of Ras to the plasma membrane, for example the drug R115777 (Zanestra is in clinical trail phase III), SCH66336 (SARASAR), to antisense oligonucleotides to Raf and H-Ras (ISIS5132 and ISIS25403) from Isis pharmaceuticals. Apart from targeting the Ras-MAPK cascade there are also several drugs targeting upstream pathways most importantly the EGF receptor family members. As EGFR or its family members is found to be highly expressed in more than 50% of carcinomas. Most importantly Her-2, an EGFR family member is amplified and therefore the causative for breast cancer. The drugs which target these members range from kinase inhibitors like the OSI-774 (Tarceva) to the well known Herceptin (Transtuzumab, a humanized antibody ) which is now licensed for use on breast cancer. The major aim of these drugs is to reduce the hyper activation of Ras-MAPk kinase cascade and sensitize them to chemotherapeutic cancer drugs. As we have identified PHB as a new modulator of this known pathway, targeting PHB could be a potential alternative in all these cases.

Ras proteins control signalling pathways responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins initiating the mitogen-activated protein (MAP) kinase cascade leading to the activation of transcription factors via ERK. Here we show, that prohibitin (PHB), a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf-MAP kinase pathway by Ras. Raf kinase fails to interact with active Ras induced by epidermal growth factor (EGF) in the absence of prohibitin. Prohibitin and Raf kinase are enriched in caveolae and after depletion of prohibitin, Raf kinase is lost from this compartment. Constitutively active Raf kinase induces ERK activation independent of prohibitin. Interestingly, we find a prominent role of the prohibitin dependent branch of the Ras signalling pathway in epithelial cell adhesion and migration. In prohibitin deficient cells the adherent complex proteins cadherin and β-catenin relocalise to plasma membrane and thereby stabilize adherent junctions. Our data show an unexpected role of prohibitin in the activation of the Ras-Raf signalling pathway and in modulating epithelial cell adhesion and migration.

Prohibitin (PHB) is involved in diverse cellular processes like proliferation and energy metabolism and is found in different cellular compartments including the nucleus and mitochondria (1). We identified PHB in a RNA interference based loss of function screen for proteins involved in the regulation of apoptosis (Machuy et al., in press). Besides the slight sensitisation for apoptotic stimuli observed (not shown), the most prominent phenotype of HeLa cells transfected with siRNAs to suppress PHB expression (siPHB) was reduced spreading and increased intercellular adhesion to form tiny islands of densely packed cells in place of a uniform monolayer (FIG. 1). The observed phenotype correlated with prohibitin expression because, due to the transient effect of siRNAs, PHB expression was normal 6 days post transfection and the growth of epithelial cell as monolayer was restored (data not shown). In order to exclude unspecific effects of the siRNA used, an additional siRNA designed to target the coding region (siPHB-2) and one to target the 3′ untranslated region (siPHB-3) of the prohibitin mRNA were used. All siRNA, siPHB (FIG. 1A, B), siPHB-2 (FIG. S1) and siPHB-3 (FIG. 1E, F) efficiently interfered with the expression of prohibitin and induced a similar phenotype as siPHB (FIG. S1C, FIG. 1C, D). PHB expression was then complemented in siPHB-3 transfected cells by co-transfecting an expression plasmid harbouring the cloned PHB gene. Complemented cells formed monolayers suggesting that the formation of small islands depended on the lack of PHB (FIG. 1D). An similar phenotype could also be detected in other cell types like human larynx carcinoma (HEp-2) and gastric cancer cells (AGS) (not shown).

To investigate the nature of intercellular adhesion we performed scanning and transmission electron microscopy studies on cells with silenced PHB expression. FIG. 2(A-D) shows the SEM and TEM images of the control transfected and siPHB transfected cells. As observed previously, cells transfected with siPHB have almost no intercellular spaces and packed compactly when compared to the control transfected cells. To check if the increased intercellular adhesion was due to the formation and/or stabilization of adherens junctions (AJs), we performed confocal microscopy analysis in the PHB knock down cells. The formation and/or the stabilization of the AJs can be studied by monitoring the intracellular localizations of AJ complex proteins like cadherins or β-catenin. In contrast to control cells, PHB knock down cells exhibited a strong staining for both cadherins and β-catenin at the plasma membrane indicating a predominant lateral localization of these junctional proteins resulting in stabilized adherens junctions (FIG. 2) (E-J).

The control of adherens junction formation is an important step of malignant transformation in epithelial cells and has previously been shown to be controlled by receptor tyrosine kinases like the epithelial growth factor receptor (EGFR) and the human EGFR-related 2 (Her-2). Both receptors are deregulated in several epithelial tumours leading to constitutive kinase activity (2) und malignant transformation (3). Stimulation of these receptors with epidermal growth factor (EGF) results in the enhanced migration on collagen (4) or extracellular matrix a widely used in vitro model for studying cancer metastasis (5). HeLa cells lacking PHB expression were defective in such a migration assay (FIG. 2K) suggesting that PHB is required for EGF-induced migration.

We tested then whether down regulation of the Her-2 or EGFR resulted in the formation of cell clusters as previously observed for cells lacking PHB expression. Cells depleted of Her-2 (FIG. S2) or EGFR (not shown) formed cell clusters similar to those observed in cells with reduced PHB expression. Transfection of siPHB had no effect on the amount of surface exposed EGFR (not shown) or Her-2 (FIG. S2), suggesting that PHB is involved in a signalling pathway downstream of EGFR and Her-2.

EGFR and Her-2 signal via Ras proteins to different pathways involved in cell proliferation, migration, differentiation including the Raf-MAPK-ERK or the phosphoinositide 3-kinase (PI3K)-AKT pathway (2,6). Interestingly, depletion of Her-2 or PHB strongly reduced the phosphorylation of ERK but not of AKT suggesting a specific involvement of PHB in the Ras-Raf-MAPK pathway (FIG. 3A). Neither the protein levels of Ras, Raf-1, ERK2 (FIGS. 3 and 4) or MEK-1 (not shown) were reduced in PHB depleted cells ruling out an unspecific inhibitory effect of the transfected siRNA. Furthermore, expression of the cloned PHB gene in siPHB transfected cells restored the EGF-dependent phosphorylation of ERK showing a specific function of PHB in signalling from RTK receptors to ERK (FIG. 3H).

Raf-1 kinase is regulated by phosphorylation at several amino acids. Ser259 is the major target for inhibitory phosphorylation and dephosphorylation of Ser259 generally precedes the activating phosphorylation at the Ser338 (7). As expected, stimulation of HeLa cells with EGF caused an increased phosphorylation of Raf-1 at Ser338 (FIG. 3D). Interestingly, cells lacking PHB lacked the basal as well as the EGF induced phosphorylation at Ser338 indicative of a block in Raf-1 kinase activation by PHB depletion (FIG. 3D). Moreover, in the absence of PHB, no alteration or relatively high levels of Raf-1 phosphorylated at Ser259 were detected (FIG. 3D). Restoration of PHB expression by complementing the siPHB transfected cells with the cloned PHB gene restored the basal as well as EGF-dependent activation of Raf-1, apparent by high Raf-1 pSer338 and low Raf-1 pSer259 levels (FIG. 3D). Thus, PHB is required for EGF-induced Raf-1 activation.

If PHB is involved in the activation of Raf-1-MEK-ERK, cell clusters induced by reduced motility and stabilized adherens junctions should be affected by direct activation of Raf-1 or the expression of a constitutively active Raf-1 derivative. Treatment of siPHB transfected cells with phorbol myristate acetate (PMA), an activator of Raf-1 (8), rapidly resolved the cell clusters formed upon PHB depletion in between 2 to 4 hours upon addition (FIG. 3E; see also supplementary movie). As expected, PMA treated cells showed increased ERK phosphorylation irrespective of the presence or absence of PHB (FIG. 3F). Likewise, expression of a constitutive active Raf-1 mutant RafBXB (9) was sufficient to prevent clusters formation in cells with silenced PHB expression (FIG. 3G, H). RafBXB expression stimulated the phosphorylation of ERK in siPHB-3 transfected cells to a similar extent as the transgenic expression of the cloned PHB gene (FIG. 3G, H). Taken together, these data demonstrate a direct role of PHB in the activation of Raf-1.

Activation of Raf-1 requires the Ras-dependent recruitment to the plasma membrane (10,11) where both proteins reside in special caveolin rich patches called caveolae (12). PHB has previously been shown to localise to the plasma membrane and further a direct interaction of PHB and Raf-1 was also reported in U937 cells (13). We have also detected Raf-1, as well as Raf-1 pS338 in the PHB immunoprecipitates from Hela cells treated with or without EGF (FIG. S3). Interestingly, we also found PHB in caveolae together with Raf-1 (FIG. 4A). However, Raf-1 was not detected in the caveolae of siPHB treated cells (FIG. 4A), suggesting that PHB is required at a step prior to membrane recruitment of Raf-1.

The GTP-bound active form of Ras proteins directly binds and activates Raf-1 (14). We therefore asked whether Ras and Raf still interact in PHB depleted cells. As expected, endogenous Raf-1 interacted with Ras in EGF treated cells transfected with control siRNAs but not in untreated cells (FIG. 4B) demonstrating the activation dependent binding of Ras to Raf-1. Interestingly, the binding of Ras and Raf-1 was completely abrogated in cells lacking PHB (FIG. 4B) suggesting a direct role of PHB in the interaction of Ras and Raf-1. To confirm that PHB interferes with activation of Raf-1 by Ras we transfected constitutively active HA-Ras(G12V) in HeLa cells and either co-transfected with siPHB or control siRNAs. Interestingly, overexpressed active Ras(G12V) induced the activation of Raf-1 and ERK in control transfected cells but not in cells depleted of PHB (FIG. 4D). These results clearly show that PHB is required for the interaction of Raf-1 and Ras, a prerequisite for membrane translocation and activation of Raf-1.

We have demonstrated an unexpected role of PHB in the activation of Raf-1 by Ras. Moreover, the branch of the Ras signalling cascade controlled by PHB plays an important role in the motility of the cell. In fact, tumour cells with reduced PHB expression showed a dramatic redistribution of cadherin and β-catenin to the plasma membranes indicative of a conversion of the tumour cells from a transformed to a non-transformed phenotype (15). Moreover, prohibitin has been shown to be over expressed In gastric carcinoma, neoplastic thyroid cancer, hepatocellular carcinoma, hyperplasia, adenocarcinoma, and bladder carcinoma (16-20) indicating that prohibin may play a prominent role in the progression of neoplastic carcinoma. The facts that activating Ras mutations are found in more than 20% of all tumours (21) and that the highly conserved PHB is essential for signalling via Ras imposes PHB as a possible target for tumour therapy. One strategy could depend on the specific interference with the binding of PHB to Raf kinases in order to block activation by oncogenic Ras proteins and cellular transformation.

In one embodiment of the present invention, cancers with high expression of EGFR or Ras point mutations (pancreas, lung adenocarcinoma, colorectal, thyroid, bladder, liver and kidney) can be treated or prevented by decreasing the PHB activity in the method of the present invention for treating or/and prevention of hyperprolerative disorders. In some of these cancers, PHB is overexpressed. Since increased PHB levels are found in metastases, metastatic tumours can also be treated by decreasing PHB activity.

Subject of the present invention is also treatment of EGFR overexpressing tumours, Her-2 family overexpressing tumours, Herceptin resistant tumours, B-Raf transformed tumours, or/and Raf-1 transformed tumours. Since PHB signals downstream of EGFR and Her-2, also EGFR overexpressing tumours and Her-2 family overexpressing tumours can be treated by inhibition of PHB or/and downregulation of PHB.

Since Herceptin acts on Her-2, PHB inhibition may be an alternative treatment strategy in Herceptin resistant tumours, acting e.g. via EGFR signalling which is a path alternative to the Her-2 path.

The involvement of PHB in the signalling cascade of Raf-1 or/and B-Raf leads to the conclusion that also Raf-1 (C-Raf) transformed tumours or/and B-Raf transformed tumours can be treated by inhibition or/and downregulation of PHB.

As we have identified that reducing the activity of PHB in tumour cells block the Raf-Mapk kinase cascade directly and efficiently, RNA interference, antisense nucleic acids or/and a chemical based approach can be employed to reduce PHB expression in tumour cells in the method of the present invention for treating or/and prevention of hyperprolerative disorders.

PHB inhibition in the context of the present invention includes downregulation of PHB transcription or/and translation. While not wishing to be bound to theory, the mechanism of PHB action may be targeting of PHB to membranes combined with targeting of a Raf kinase, in particular Raf-1 (C-Raf) to membranes. Further, posttranslational modification may be important for the proper action of PHB. Therefore, PHB inhibition of the present invention also includes inhibition of targeting of PHB to membranes, inhibition of targeting of a Raf kinase, in particular Raf-1 (C-Raf) to membranes, and inhibition of posttranslational modification of PHB, in particular of posttranslational modifications required for PHB targeting to membranes or/and required for Raf kinase, in particular Raf-1 targeting to membranes.

The PHB-Raf interaction is mediated by a sequence of about 20-30 amino acids, indicating that the interaction between PHB and Raf can be interrupted with either a small molecule inhibitor or a small peptide.

In one embodiment, the inhibitor of PHB of the present invention is a nucleic acid, which can be

• • (i) an RNA molecule capable of RNA interference,
  • (ii) a precursor of the RNA molecule (i), or
  • (iii) a DNA molecule encoding the RNA molecule (i) or the precursor (ii).

RNA molecules capable of RNA interference are described in WO 02/44321 which is included by reference herein.

Preferably, the inhibitor of the present invention, in particular the nucleic acid of the present invention, is used in a pharmaceutical composition.

The antibody of the present invention may be a monoclonal or polyclonal antibody, a chimeric antibody, a chimeric single chain antibody, a Fab fragment or a fragment produced by a Fab expression library.

Techniques of preparing antibodies of the present Invention specific for PHB are known by a skilled person. Monoclonal antibodies may be prepared by the human B-cell hybridoma technique or by the EBV-hybridoma technique (Köhler et al., 1975, Nature 256:495-497, Kozbor et al., 1985, J. Immunol. Methods 81,31-42, Cote et al., PNAS, 80:2026-2030, Cole et al., 1984, Mol. Cell Biol. 62:109-120). Chimeric antibodies (mouse/human) may be prepared by carrying out the methods of Morrison et al. (1984, PNAS, 81:6851-6855), Neuberger et al. (1984, 312:604-608) and Takeda et al. (1985, Nature 314:452-454). Single chain antibodies may be prepared by techniques known by a person skilled in the art.

Recombinant immunoglobulin libraries (Orlandi et al, 1989, PNAS 86:3833-3837, Winter et al., 1991, Nature 349:293-299) may be screened to obtain an antibody of the present invention which are specific against PHB. A random combinatory immunoglobulin library (Burton, 1991, PNAS, 88:11120-11123) may be used to generate an antibody with a related specifity having a different idiotypic composition.

Another strategy for antibody production is the in vivo stimulation of the lymphocyte population.

Furthermore, antibody fragments (containing F(ab′)₂ fragments) of the present invention can be prepared by protease digestion of an antibody, e.g. by pepsin. Reducing the disulfide bonding of such F(ab′)₂fragments results in the Fab fragments. In another approach, the Fab fragment may be directly obtained from an Fab expression library (Huse et al., 1989, Science 254:1275-1281).

Polyclonal antibodies of the present invention may be prepared employing PHB or immunogenic fragments thereof as antigen by standard immunization protocols of a host, e.g. a horse, a goat, a rabbit, a human, etc., which standard immunization protocols are known by a person skilled in the art.

Fragments of polypeptides or/and peptides, in particular immunogenic fragments of SEQ.ID.NO:2 or a Raf kinase may have a length of at least 5 amino acid residues, preferably at least 10, more preferably at least 20 amino acid residues. The length of said fragments may be 200 amino acid residues at the maximum, preferably 100 amino acid residues at the maximum, more preferably 60 amino acid residues at the maximum, most preferably 40 amino acid residues at the maximum.

It can easily be determined by a skilled person if a gene is upregulated or downregulated. In the context of the present invention, upregulation of gene expression may be an upregulation by a factor of at least 2, preferably at least 4. Downregulation in the context of the present invention may be a reduction of gene expression by a factor of at least 2, preferably at least 4. Most preferred is essentially complete inhibition of gene expression.

“Reduction (increase) of the amount” may be a downregulation (upregulation) of gene expression by a factor of at least 2, preferably at least 4. In the case of reduction, essentially complete inhibition of gene expression is most preferred. Examples are reduction of the amount of PHB or reduction (increase) of the gene product amount of the genes of Table 1 or/and 2.

“Reduction (increase) of the activity” may be a decrease (increase) of activity of a gene or gene product by a factor of at least 2, preferably at least 4. In the case of activity reduction, essentially complete inhibition of activity is most preferred. An example relevant for the present invention is reduction of PHB activity or reduction (increase) of activity of the genes of Table 1 or/and 2.

A “target” or “target gene” for treatment of hyperproliferative disorders in the context of the present invention is a gene the expression of which is influenced by prohibitin inhibition. Prohibition inhibition may be provided by downregulation of PHB expression or by inhibition of PHB activity by an inhibitor of the present invention as discussed above.

The surprising finding of the present invention that PHB inhibition is a promising approach for treatment of hyperproliferative disorders leads to the conclusion that also genes which act downstream of PHB in the signalling cascade may be suitable targets for treatment of hyperproliferative disorders. Therefore, subject of the present invention is a method for identification of target genes for treatment of hyperproliferative disorders, based upon inhibition of PHB.

The term “target” also includes a gene product (RNA, in particular mRNA, tRNA, rRNA, a polypeptide or/and a protein) of the target gene. Preferred gene products of a target gene are selected from mRNA and a polypeptide or a protein encoded by the target gene. The most preferred gene product is a polypetide or protein encoded by the target gene. A protein or polypeptide of the present invention may be posttranslationally modified or not.

In the context of the present invention, “activity” of the gene or/and gene product includes transcription, translation, posttranslational modification, modulation of the activity of the gene product by ligand binding, which ligand may be an activator or inhibitor, etc.

In the case of PHB, “activity” also includes targeting of PHB to membranes, targeting of a Raf kinase, in particular of Raf-1 to membranes, posttranslational modification of PHB, in particular required for PHB or/and Raf kinase, in particular Raf-1 targeting to membranes.

Examples of genes which are upregulated by PHB inhibition are described in Table 1. Examples of genes which are downregulated by PHB inhibition are described in Table 2. The targets of Table 1 or/and 2 may be used for identification of new compounds for treatment or/and prophylaxis of hyperproliferative disorders. Therefore, yet another subject of the present invention is a screening method for identification of a compound suitable for treatment of a hyperproliferative disorder based upon the genes of Table 1 and 2.

The compound to be identified by the method of the present invention is a compound which increases the amount or/and the activity of the gene product of the at least one gene of Table 1 or decreases the amount or/and activity of the gene product of the at least one gene of Table 2. Therefore, the compound to be identified may be an inhibitor or an activator of a target gene or a gene product thereof.

The inhibitor or activator of a target gene or a gene product thereof may be selected from the group of nucleic acids, nucleic acid analogues such as ribozymes, peptides, polypeptides, and antibodies. A nucleic acid inhibitor or activator of a target gene or gene product thereof can be

• • (i) an RNA molecule capable of RNA interference,
  • (ii) a precursor of the RNA molecule (i), or
  • (iii) a DNA molecule encoding the RNA molecule (i) or the precursor (ii).

The antibody may be an antibody specific for a gene product of a target gene, in particular an antibody specific for a polypeptide or protein encoded by a target gene. Production of a suitable antibody is described above in the context of prohibitin.

The inhibitor or activator of a target gene or gene product thereof may be used for the manufacture of a pharmaceutical composition for treatment or/and prophylaxis of a hyperproliferative disorder.

In the method of the present invention for identification of a compound suitable for treatment of a hyperproliferative disorder based upon the genes of Table 1 and 2, preferably employed are genes listed in Table 1 and 2 involved in cancer signalling, angiogenesis, adhesion, invasion or/and metastasis formation.

Most preferred genes listed in Table 1 and 2 are genes involved in cancer signalling: NM_—004419, DUSP5 dual specificity phosphatase 5, direct target of p53; U21049, DD96, epithelial protein up-regulated in carcinoma, membrane associated protein 17; NM_—005130 HBP17 (heparin-binding growth factor binding protein), which binds to HB-EGF which activates the RAS-signaling pathway.

Further most preferred genes listed in Table 1 and 2 are genes encoding angiogenis factors: NM_—003370, VASP, vasodilator-stimulated phosphoprotein, direct angiogenic activity, which plays also a role in cell motility and metastasis formation; NM_—139314, ANGPTL4, Homo sapiens angiopoietin-like 4 (ANGPTL4), transcript variant 1; NM_—006108, SPON1, Homo sapiens spondin 1, extracellular matrix protein (SPON1), having a potential function as an angiopoietin or/and as spondin.

Further most preferred genes listed in Table 1 and 2 are genes involved in adhesion, invasion and metastasis: NM_—003255, TIMP2, tissue inhibitor of metalloproteinase 2, which plays an important role in invasion and metastasis formation by inhibiting metalloproteases; NM_—006108, SPON1, Homo sapiens spondin 1, extracellular matrix protein (SPON1); NM_—000213, ITGB4, integrin beta 4, Extracellular matrix binding and signaling, plays an important role in adhesion and invasion; NM_—012385 P8, p8 protein (candidate of metastasis 1); NM_—004360, CDH1, cadherin 1, type 1, E-cadherin (epithelial), plays an important role in adhesion and invasion as a protein involved in cell-cell contact formation; NM_—017717, MUCDHL, mucin and cadherin-like; NM_—005130 HBP17 (heparin-binding growth factor binding protein), which binds to HB-EGF which activates the RAS-signaling pathway; NM_—002272, KRT4, Homo sapiens keratin 4 (KRT4), which is involved in adhesion and wound healing; NM_—004363 CEACAM5 (Homo sapiens carcinoembryonic antigen-related cell adhesion molecule 5) and NM_—002483 CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6). Carcinoembryonic antigen (CEA) is one of the most frequently used serum tumor markers for carcinoma, particularly in colorectal cancer. The role of CEACAM in tumor growth is complex, with apparently conflicting effects on tumor growth seen in different tumor models. Studies in breast and prostate cancer models suggest that CEACAM may be a tumor suppression gene, whereas other models suggest CEACAM may be involved in tumor invasion and metastasis.

The invention is further illustrated by the following figures, tables and examples.

FIGURE AND TABLE LEGENDS

FIG. 1: Knock down of prohibitin induces changes in epithelial cell morphology. Transfection of siPHB reduces the mRNA level (A) and the protein level (B) of prohibitin compared to control transfections (siLuc or siGFP, respectively). Knock down of prohibitin (siPHB) induces the aggregation and reduced migration of epithelia cancer cells not seen in control cells (siGFP) (C). Transfection of siPHB-3 reduces the PHB mRNA (D) and protein level (E). Expression of the cloned PHB gene pPHB-c in siPHB-3 transfected cells restores PHB expression. PHB-c harbours a N-terminal Flag-tag and has therefore a slightly increased molecular mass compared to endogenous PHB. Expression of the cloned prohibitin pPHB-c in siPHB-3 transfected cells prevents cell cluster formation. For details, see Materials and Methods.

FIG. 2: Knock down of prohibitin induces the formation of adherens junctions and changes in epithelial morphogenesis. Knock down of prohibitin (siPHB) induces the formation of multilayered epithelial cell clusters (A, B) whereas control cells (siLuc) form monolayers (C, D). Samples were analyzed by scanning (SEM, A+C) and transmission electron microscopy (TEM, B+D).

Confocal immunofluorescence analysis of prohibitin knock down cells (E, F, G) and controls (H, I, J). Cells with reduced prohibitin expression show a strong membrane staining for pan-cadherin (E) and β-catenin (F) while staining of control cells reveals a reduced membrane signal and a diffuse cytoplasmic pattern (H, I). G and J show the respective overlays including a DNA stain. G′, J′ and G″,J″ are XZ and YZ reconstructions of confocal Z stacks. The reconstructions clearly reveal that the cell clusters of prohibitin knock down cells are multilayered. Induction of cell migration on collagen by EGF is impaired in prohibitin knock down cells. Shown are the individual frames from various time points of a time-lapse video on the control and prohibitin knock down cells with EGF.

FIG. 3:

Prohibitin is required during EGF induced Raf-MAPK activation Hela cells transfected with control siRNA (siLam), siHer-2 and siPHB were treated with EGF and the phosphorylation of ERK, AKT (A) and Raf-1 (B) was determined by immunoblot analysis with phospho-specific antibodies. Complementation of siPHB transfected HeLa cells restores Raf-1 activation (D). Shown are the levels of Raf-1 pS259 and Raf-1 pS338 blots revealing an increase in Raf-1 pS259 phosphorylation in cells transfected with siPHB-3 (D).

Addition of Phorbol ester (PMA) leads to rapid resolution of cell clusters and the activation of ERK irrespective of PHB levels (E, F). Shown are different time points from a time lapse microscopy of siPHB transfected cells treated with PMA (E). Co-expression of constitutively active RafBXB (G, H) or the cloned PHB gene restore ERK activation and cell cluster formation in siPHB transfected cells.

FIG. 4:

PHB is required for the activation of Raf-1 by Ras.

Prohibitin and Raf-1 were found in the caveolin rich fraction of the plasma membrane in cells with normal prohibitin expression (A; siLuc). Raf-1 is lost from the caveolae in cells with reduced levels of PHB (A; siPHB). The different fractions and the respective concentration of sucrose obtained after gradient centrifugation are indicated. Caveolin-1 (Cav-1) as marker for caveolae as well as PHB and Raf-1 were detected by immuno blotting. Ras-Raf-1 interaction is impaired in PHB knock down cells (B). HeLa cells transfected with control siRNA (siLuc) or siPHB were treated with or without EGF. Ras was then immunoprecipitated using pan-Ras antibody and co-precipitating Raf-1 was detected by immunoblotting (IP). Amounts of PHB and Raf-1 were verified in the whole cell lysates (lysate). Active Ras fails to activate Raf-1 and ERK in PHB knock down cells (D). HeLa cells transfected with control siRNA (siLuc) or siPHB and expression plasmids for H-Ras or H-Ras(G12V) were treated with EGF or buffer. Activation of Raf-1 and ERK was tested by immunoblot analysis using phoshpo-specific antibodies as indicated. Expression of Ras constructs was verified by using anti-HA tag antibody.

FIG. 5:

Nucleotide Sequence encoding PHB (GenBank-Accession-No. NM_—002634, SEQ.ID.NO:1). The coding region is given in bold letters.

FIG. 6:

Amino acid sequence of PHB (GenBank-Accession-No. NM_—002634, SEQ.ID.NO:2).

FIG. S1: Formation of cell clusters as consequence of silencing PHB expression in HEp-2 cells. Transfection of siPHB-2 reduces the mRNA level (A) and the protein level (B) of prohibitin compared to control transfections (siLuc or siGFP, respectively). Knock down of prohibitin (siPHB-2) induces the aggregation and multilayered organisation of HEp-2 cells not seen in control cells (siGFP) (C).

FIG. S2: Knock down of Her-2 results in the formation of cell clusters. Validation of siHer-2 by quantitative realtime PCR indicates reduced Her-2 mRNA levels of about 80% of control (siLuc) (A). Transfection of siHer-2 induces the formation of cell clusters (B). Transfection of siHer-2 but not of siPHB reduces the surface exposure of Her-2 (C). Shown is a FACS analysis of cells transfected with siHer-2 (No. 1), siPHB (No. 3) or siLuc (No. 4). Cells were stained with anti-Her-2 antibody (Nos. 1, 3, 4) or a isotype control (No. 2). Relative expression of prohibitin, Her-2 and EGFR in cells transfected with siLuc (Luci) or siPHB (PHB) (D).

FIG. S3: Endogenous Raf-1 interacts with PHB

Endogenous PHB was precipitated from EGF or buffer treated HeLa cells and co-precipitating Raf-1 was determined. Raf-1 and Raf-1 pS338 strongly interact with PHB. IP with HA-antibody was used as an isotype control.

Table 1: Genes up-regulated in prohibitin depleted cells

Table 2: Genes down-regulated in prohibitin depleted cells

EXAMPLE 1

Materials and Methods

Cell Culture

HeLa cells, HEp-2 cells, AGS cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco BRL, Karlsruhe, Germany), and penicillin (100 U/ml)/streptomycin (100 pg/ml) (Gibco BRL, Karlsruhe, Germany) at 37° C. in 5.0% CO₂. Cells were serum starved for 4-6 h prior to the addition of EGF (Promocell) at a final concentration of 20 ng/ml. MEK-1 inhibitor PD98059 was purchased from (Calbiochem).

Transfection of siRNAs

In order to silence expression of prohibitin, 50,000 cells/well were seeded in a 12 well plate at least 20 h prior to transfection. siRNAs were transfected using the Transmessenger transfection kit and RNAifect transfection kit (Qiagen). Two days post transfection, the nearly confluent cells were trypsinized and one half of the cells was seeded on glass cover slips in a 12 well plate for immunofluorescence analysis while the other half was used for western blot analysis. The following siRNAs were employed in this study:

siGFP
5′-AAGUUCAGCGUGUCCGGCGAG-3′,   (SEQ.ID.NO:3)
siLuc-
5′-AACUUACGCUGAGUACUUCGA-3′,   (SEQ.ID.NO:4)
siPHB
5′UGUCAACAUCACACUGCGCdTdT3′    (SEQ.ID.NO:5)
and
GCGCAGUGUGAUGUUGACAdTdT        (SEQ.ID.NO:6)
siPHB-2
AGCCAGCTTCCTCGCATCTdTdT,       (SEQ.ID.NO:7)
and
AGATGCGAGGAAGCTGGCTdGdG,       (SEQ.ID.NO:8)
siPHB-3
5′-CCCAGAAAUCACUGUGAAAdTdT-3′, (SEQ.ID.NO:9)
and
TTTCACAGUGAUUUCUGGGdTdT.       (SEQ.ID.NO:10)

For complementation experiments, cells grown to 80% confluence were transfected with the siPHB-3 siRNA (80 nM) and the full length PHB open reading frame cloned in pCDNA3-myc vector (0.5 μg) or the RafBxB construct (0.5 μg) (a kind gift from Ulf Rapp) using RNAifect transfection kit as mentioned before. The control cells were transfected with the empty vector and a siRNA directed against luciferase (siLuc).

Validation of mRNA Levels by Quantitative Realtime PCR

20,000 cells/well were seeded in a 96 well plate one day prior to transfection. Transfection was performed with 0.25 μg siRNA directed against PHB and Luciferase as control and 2 μl Transmessenger per well according to manufacturer's instructions. After 48 h, RNA was isolated using the RNeasy® 96 BioRobot® 8000 system (Qiagen). The relative amount of PHB mRNA was determined by real time PCR using Quantitect™ SYBR® Green RT-PCR Kit from Qiagen following manufacturer's instructions. The expression level of PHB mRNA was normalized against the internal standard GAPDH. The following primers were used:

PHB-5′:
5′-CTTTGACTGCCGTTCTCGAC-3′,    (SEQ.ID.NO:11)
PHB-3′:
5′-TGGGTGGATTAGTTCTCCAGC-3′,   (SEQ.ID.NO:12)
and
GAPDH-5′:
5′-GGTATCGTGGAAGGACTCATGAC-3′, (SEQ.ID.NO:13)
GAPDH-3′:
5′-ATGCCAGTGAGCTTCCCGTTCAG-3′. (SEQ.ID.NO:14)

Preparation of Caveolae Rich Fractions

Detergent extraction and floatation were performed as described previously (22). Shortly, ME-180 or HeLa cells transfected with siRNAs were solubilized in 1% Triton X-100, MBS [Mes-buffered saline; 0.25 M NaCl and 25 mM Mes (pH 6.8)] and a cocktail of protease inhibitors on ice for 1 h without agitation. The cell lysates were adjusted to 45% sucrose in MBS, overlaid with 7 ml of 35% and 2 ml 5% sucrose in MBS, and centrifuged for 18 h at 36,000 rpm in a SW40Ti rotor (Beckman Instruments). Twelve 1 ml fractions were collected from the top of the gradient and were assayed for protein content, caveolin-1, Raf-1, Ras and prohibitin.

SDS-PAGE and Western Blot

For SDS-PAGE, cells were lysed in single detergent buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 μgml⁻¹ Aprotinin, 0.5 μgml⁻¹ Leupeptin, 1 mM Pefabloc, 10 μM Pepstatin) for 15-20 min on ice and sonicated twice for 15 sec. Lysates were cleared by centrifugation for 10 min at 13,000 rpm, Sample buffer was added to these lysates and boiled at 90° C. for 2 min before loading onto the gels. After separation the proteins were transferred to PVDF-membranes. For immunoblot analysis membranes were blocked with 3% BSA in TBS with 0.5% Tween-20 for 2 h and incubated with anti-prohibitin (Neomarkers), anti-alpha tubulin antibody (Sigma), anti-E-cadherin (Zymed labs), anti-Pan-cadherin (Sigma) or anti-beta-catenin (Sigma) anti-caveolin (N20, Santa Cruz Biotechnology), c-Raf rabbit polyclonal antibody (Santa Cruz Biotechnology), c-Raf mouse monoclonal antibody (Pharmingen), Caveolin mouse monoclonal antibody, anti-Raf-1 pS338 and anti-Raf-1 pS259 rabbit monoclonal antibody (Cell signalling), phospho-MEK (Cell signalling), anti-beta actin monoclonal antibody (Sigma), anti-Her-2 rabbit polyclonal antibody from. (Cell signalling) (anti-pan-Ras monoclonal antibody (Pharmingen), phosphor-ERK, Anti-ERK2 antibody, EGFR-antibody and anti-Myc 9E10 mouse monoclonal antibody (Santa Cruz). Antigen antibody complexes were detected by horseradish peroxidase coupled antibodies (Pharmingen) followed by enhanced chemiluminescence (NEN).

FACS Analysis

For surface staining of Her-2, Hela cells transfected with siRNAs were detached by A cutase treatment 60 h post transfection. The cells were washed once with PBS and incubated in PBS with 2% BSA for 20 min at room temperature. The cells were once again washed with PBS and stained with FITC coupled anti-Her-2 antibody (Bender med systems) diluted in BSA containing PBS at a final dilution of 1:50 and incubated at room temperature for 40 min. At the end of the incubation the cells were washed twice with PBS and the labelled cells were detected using a Becton Dickinson flow cytometer.

Immunoprecipitation

Cells were washed in ice cold PBS and lysed in RIPA buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 0.5% Triton X-100, 1 mM NaVO₃, 10 mM Na-pyrophosphate, 1 mM NaF, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 1 μg/ml Aprotinin, 0.5 μg/ml Leupeptin, 1 mM Pefabloc, 10 μM Pepstatin for 30 min on ice and sonicated twice for 10 sec. Lysates were cleared by centrifugation for 15 min at 13,000 rpm. Supernatants were incubated with agarose-coupled anti-H-Ras (Santa Cruz Biotechnology) or Raf-1 (C-12, Santa Cruz Biotechnology) for over night. In the latter case the antigen-antibody complexes were pull down by Sepharose coupled protein A/G beads.

In Vitro Kinase Assay

For in vitro kinase assays, Hela cells transfected with siRNAs were treated with or without EGF and cell lysates were prepared for immunoprecipitations as mentioned before. The immunoprecipitated Raf-1 was washed twice with RIPA buffer and kinase buffer w/o ATP (25 mM Hepes-NaOH pH 7.5, 5 mM MgCl₂, 4 mM MnCl₂). The beads were incubated at 30° for 30 min in the presence of [γ-³²P] ATP in kinase buffer supplemented with 30 μM ATP with or without 2 μg of MEK-1 full length protein (Santa Cruz) in a total volume of 40 μl. Kinase reactions were terminated by addition of SDS sample buffer and boiling. The proteins were separated by SDS-PAGE and analyzed by autoradiography.

Transmission Electron Microscopy

siRNA transfected Hela cells were trypsinized and seeded on glass coverslips at 48 h post transfection. After 60 h, cells were fixed with 2.5% glutaraldehyde, postfixed with 0.5% osmium tetroxide and contrasted using tannic acid and uranyl acetate. Specimens were dehydrated in a graded ethanol series and embedded in Polybed. Ultrathin sections were analysed in a Leo 906E transmission electron microscope (Leo Oberkochen).

Scanning Electron Microscopy

Samples grown on glass coverslips were fixed in 2.5% glutaraldehyde, dehydrated in a graded ethanol series, critical-point dried and coated with a layer of 2 nm platinum/carbon. Specimens were analysed in a Leo 1550 field emission scanning electron microscope (Leo, Oberkochen).

Immunofluorescence Microscopy

siRNA transfected cells were seeded on glass coverslips at 48 h post transfection and fixed with 4% PFA/PBS at 60 h post transfection. The fixed cells were washed once with PBS and permeabilised with 1% Triton/PBS for 1 min and blocked (1% BSA, 5% NGS, 0.05% Tween 20 in PBS for 1 h. The samples were incubated with antibodies against Pan-Cadherin (Sigma) and β-Catenin (Sigma) in blocking buffer. After washing, bound antibodies were detected using goat anti-mouse Cy2 and goat anti rabbit Cy3 secondary antibodies. Nuclei were stained with Draq 5 (Bilostatus LTD). Tri colour Z-stacks were generated using a Leica TCS-SP confocal microscope (Leica Microsystems). For 3-dimensional analysis, stacks were processed using Volocity software (Improvision).

EXAMPLE 2

Microarray Method

In a microarray analysis, target genes were identified which are downregulated or upregulated in prohibitin depleted cells. Results are summarized in Tables 1 and 2.

Microarray experiments were carried out as two-color dye-reversal ratio hybridizations on a 44.000 Whole Human Genome Oligo Microarray AMADID 012391 with 37327 records and 43931 features (Agilent Technologies, Palo Alto, Calif., USA). RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 4 μg total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent anti-sense cRNA was synthesized with either cyanine 3-CTP (Cy3-CTP) or cyanine 5-CTP (Cy5-CTP) and T7 polymerase. The fluorescent-labeled anti-sense cRNA was precipitated over night with LiCl, ethanol washed and resuspended in water. The purified products were quantified at A_{552 nm} for Cy3-CTP and A_{650 nm} for Cy5-CTP and labeling efficiency was verified with a Nanodrop photometer (Kisker, Steinfurt, Germany). Before hybridization, 1.25 μg labeled cRNA of each product were fragmented and mixed with control targets and hybridization buffer according to the supplier's protocol (Agilent Technologies). Hybridizations were done over night for approximately 17 h at 60° C. The slides were washed according to the manufacturer's manual and scanning of microarrays was performed with 5 μm resolution using a DNA microarray laser scanner (Agilent Technologies). In order to compensate dye specific effects, and to ensure statistically relevant data (G. A. Churchill, Fundamentals of experimental design for cDNA microarrays, Nat Genet 32 Suppl (2002) 490-495), a color swap dye reversal was performed. Features were extracted with an image analysis tool Version A 6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmatics platform Resolver Built 4.0. Ratio profiles were generated from raw scan data by a processing pipeline which includes pre-processing (Feature Extraction) and post-processing (Rosetta Resolver) of data and error model adjustments to the raw scan data. Ratio profiles were combined in an error-weighted fashion (Rosetta Resolver) to create ratio experiments, and ratio experiments consisted of one or more ratio profiles. Expression patterns were identified using stringent analysis criteria of 2-fold expression cut-offs of the ratio experiments and an anti-correlation of the dye reversal ratio profiles. Anti-correlation was determined by using the ‘compare function’ to match two color-swap dye-reversal hybridizations and to decide how similar or dissimilar they were. In this way, only anti-correlated spots that had on the one array a red colour and on the other one a green colour and vice versa were selected. We compared color-swap dye-reversal hybridizations of individual 2 two-channel hybridizations resulting in unchanged genes, query signature genes, target signature genes, common signature genes and anti-correlated genes. By combining the first and the second criteria of analysis we filtered out data points with low P-value (P-value<0.01), making the analysis robust and reproducible. Additionally, by using this strategy we did the data selection independent of error models implemented in the Rosetta Resolver system.

REFERENCE LIST

1. McClung, J. K., Jupe, E. R., Liu, X. T. & Dell'Orco, R. T. Prohibitin: potential role in senescence, development, and tumor suppression. Exp. Gerontol. 30, 99-124 (1995).

2. Harari, D. & Yarden, Y. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene 19, 6102-6114 (2000).

3. Gschwind, A., Fischer, O. M. & Ullrich, A. The discovery of receptor tyrosine kinases: targets for cancer therapy. Nat. Rev. Cancer 4, 361-370 (2004).

4. Spencer, K. S., Graus-Porta, D., Leng, J., Hynes, N. E. & Klemke, R. L. ErbB2 is necessary for induction of carcinoma cell invasion by ErbB family receptor tyrosine kinases. J. Cell Biol. 148, 385-397 (2000).

5. Klemke, R. L. et al. Regulation of cell motility by mitogen-activated protein kinase. J. Cell Biol. 137, 481-492 (1997).

6. Downward, J. Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11-22 (2003).

7. Dhillon, A. S., Meikle, S., Yazici, Z., Eulitz, M. & Kolch, W. Regulation of Raf-1 activation and signalling by dephosphorylation. EMBO J. 21, 64-71 (2002).

8. Howe, L. R. et al. Activation of the MAP kinase pathway by the protein kinase raf. Cell 71, 335-342 (1992).

9. Bruder, J. T., Heidecker, G. & Rapp, U. R. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev. 6, 545-556 (1992).

10. Leevers, S. J., Paterson, H. F. & Marshall, C. J. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369, 411-414 (1994).

11. Marais, R., Light, Y., Paterson, H. F. & Marshall, C. J. Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. EMBO J. 14, 3136-3145 (1995).

12. Prior, I. A. et al. GTP-dependent segregation of H-ras from lipid rafts is required for biological activity. Nat. Cell Biol. 3, 368-375 (2001).

13. Wang, S., Nath, N., Fusaro, G. & Chellappan, S. Rb and prohibitin target distinct regions of E2F1 for repression and respond to different upstream signals. Mol. Cell Biol. 19, 7447-7460 (1999).

14. Shields, J. M., Pruitt, K., McFall, A., Shaub, A. & Der, C. J. Understanding Ras: ‘it ain't over ‘til it's over'. Trends Cell Biol. 10, 147-154 (2000).

15. Hirohashi, S. & Kanai, Y. Cell adhesion system and human cancer morphogenesis. Cancer Sci. 94, 575-581 (2003).

16. Wang, K. J., Wang, R. T. & Zhang, J. Z. Identification of tumor markers using two-dimensional electrophoresis in gastric carcinoma. World J. Gastroenterol. 10, 2179-2183 (2004).

17. Srisomsap, C. et al. Detection of cathepsin B up-regulation in neoplastic thyroid tissues by proteomic analysis. Proteomics. 2, 706-712 (2002).

18. Seow, T. K. et al. Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated proteins by mass spectrometry. Electrophoresis 21, 1787-1813 (2000).

19. Byrjalsen, I. et al. Two-dimensional gel analysis of human endometrial proteins: characterization of proteins with increased expression in hyperplasia and adenocarcinoma. Mol. Hum. Reprod. 5, 748-756 (1999).

20. Asamoto, M. & Cohen, S. M. Prohibitin gene is overexpressed but not mutated in rat bladder carcinomas and cell lines. Cancer Lett. 83, 201-207 (1994).

21. Bos, J. L. ras oncogenes in human cancer: a review. Cancer Res. 49, 4682-4689 (1989).

22. Smart, E. J., Foster, D. C., Ying, Y. S., Kamen, B. A. & Anderson, R. G. Protein kinase C activators inhibit receptor-mediated potocytosis by preventing internalization of caveolae. J. Cell Biol. 124, 307-313 (1994).

TABLE 1
Genes up-regulated in prohibitin depleted cells
Fold Change	Accession #	Sequence Name(s)	Sequence Description
4.91215	NM_032160	NM_032160	Homo sapiens NCAG1 (NCAG1), mRNA
4.39232	BC016329	BC016329	Homo sapiens, clone IMAGE: 4103364, mRNA
3.49996	NM_017831	FLJ20456	hypothetical protein FLJ20456
3.41678	NM_004038	AMY1A	amylase, alpha 1A; salivary
3.35519	U49436	EIF5	eukaryotic translation initiation factor 5
3.34253	NM_018038	FLJ10246	hypothetical protein FLJ10246
3.12768	A_32_P160186	A_32_P160186	Unknown
3.10957	AF445027	AF445027	Homo sapiens clone 114 tumor rejection antigen mRNA, complete cds
3.04008	NM_018357	FLJ11196	hypothetical protein FLJ11196
3.03416	NM_017638	FLJ20045	hypothetical protein FLJ20045
2.97146	NM_004923	MTL5	metallothioneln-like 5, testis-specific (tesmin)
2.94765	NM_007193	ANXA10	annexin A10
2.92751	THC1969492	THC1969492	Unknown
2.91389	THC1847341	THC1847341	Unknown
2.89299	NM_001195	BFSP1	beaded filament structural protein 1, filensin
2.83127	NM_020428	CTL2	CTL2 gene
2.78717	NM_012385	P8	p8 protein (candidate of metastasis 1)
2.64887	BC021677	BC021677	Homo sapiens, clone IMAGE: 4045663, mRNA
2.62683	AK095791	AK095791	Homo sapiens cDNA FLJ38472 fis, clone FEBRA2022148
2.61456	NM_020801	ARRDC3	Homo sapiens arrestin domain containing 3 (ARRDC3), mRNA
2.57308	NM_032229	SLITRK6	Homo sapiens SLIT and NTRK-like family, member 6 (SLITRK6), mRNA
2.56148	AK024085	FLJ20069	hypothetical protein FLJ20069
2.51701	NM_005572	LMNA	lamin A/C
2.51405	THC1919609	THC1919609	Unknown
2.50532	AK098124	AK098124	Homo sapiens cDNA FLJ40805 fis, clone TRACH2009060
2.4728	NM_000426	LAMA2	laminin, alpha 2 (merosin, congenital muscular dystrophy)
2.45336	AK057267	AK057267	Homo sapiens cDNA FLJ32705 fis, clone TESTI2000600, weakly similar to RESTIN
2.44739	NM_133646	NM_133646	Homo sapiens sterile alpha motif and leucine zipper containing kinase AZK (ZAK), mRNA
2.38943	ENST00000333505	ENST00000333505	Unknown
2.38742	NM_005269	GLI	glioma-associated oncogene homolog (zinc finger protein)
2.37759	AL117598	AL117598	Homo sapiens mRNA; cDNA DKFZp564H1663 (from clone DKFZp564H1663)
2.35368	AL832786	AL832786	Homo sapiens mRNA; cDNA DKFZp667D2123 (from clone DKFZp667D2123)
2.3507	NM_004083	DDIT3	DNA-damage-inducible transcript 3
2.34769	AK002152	STAU2	staufen (Drosophila, RNA-binding protein) homolog 2
2.34765	BX647764	BX647764	Homo sapiens mRNA; cDNA DKFZp686E0352 (from clone DKFZp686E0352)
2.33447	BC032558	BC032558	Homo sapiens distal-less homeo box 2, mRNA
			(cDNA clone MGC: 45154 IMAGE: 5562689), complete cds
2.31817	THC1924048	THC1924048	Unknown
2.28953	NM_017651	FLJ20069	hypothetical protein FLJ20069
2.28536	NM_001326	CSTF3	cleavage stimulation factor, 3′ pre-RNA, subunit 3, 77 kD
2.28313	THC1923482	THC1923482	Unknown
2.25252	NM_024699	NM_024699	Homo sapiens hypothetical protein FLJ14007 (FLJ14007), mRNA
2.24907	A_24_P913835	A_24_P913835	Unknown
2.24753	NM_016447	LOC51678	MAGUK protein p55T; Protein Associated with Lins 2
2.23333	NM_001172	ARG2	arginase, type II
2.22362	NM_013448	BAZ1A	bromodomain adjacent to zinc finger domain, 1A
2.21632	BX640843	BX640843	Homo sapiens mRNA; cDNA DKFZp686B14224 (from clone DKFZp686B14224)
2.21413	THC1839547	THC1839547	Unknown
2.21208	NM_003467	CXCR4	chemokine (C-X-C motif), receptor 4 (fusin)
2.2102	NM_152486	NM_152486	Homo sapiens hypothetical protein MGC45873 (MGC45873), mRNA
2.20894	THC1957808	THC1957808	Unknown
2.20499	THC1808814	THC1808814	Unknown
2.20266	NM_004405	DLX2	distal-less homeo box 2
2.18355	AK023854	AK023854	Homo sapiens cDNA FLJ13792 fis, clone THYRO1000072, weakly similar
			to MYOSIN LIGHT CHAIN KINASE,
			SMOOTH MUSCLE AND NON-MUSCLE ISOZYMES (EC 2.7.1.117)
2.17592	AK075052	AK075052	Homo sapiens cDNA FLJ90571 fis, clone OVARC1001725,
			highly similar to Homo sapiens patched related protein TRC8 (TRC8) gene
2.17336	AF001540	CS-1	calcineurin-binding protein calsarcin-1
2.17033	NM_007361	NID2	nidogen 2
2.16647	A_32_P115707	A_32_P115707	Unknown
2.16614	NM_006756	TCEA1	transcription elongation factor A (SII), 1
2.16487	NM_152346	NM_152346	Homo sapiens hypothetical protein MGC34680 (MGC34680), mRNA
2.16351	BC062599	BC062599	Homo sapiens cDNA clone MGC: 74441 IMAGE: 5248209, complete cds
2.15172	BC015814	BC015814	Homo sapiens KIAA1799 protein, mRNA
			(cDNA clone MGC: 9635 IMAGE: 3915942), complete cds
2.15113	AL832398	AL832398	Homo sapiens mRNA; cDNA DKFZp667M2212 (from clone DKFZp667M2212)
2.1504	NM_148169	FBXO26	Homo sapiens F-box only protein 26 (FBXO26), transcript variant 1, mRNA
2.14271	AL832820	AL832820	Homo sapiens mRNA; cDNA DKFZp667D0824 (from clone DKFZp667D0824)
2.13359	BC042535	BC042535	Homo sapiens F-box only protein 33, mRNA (cDNA clone IMAGE: 4751694), partial cds
2.1333	AK026078	AK026078	Homo sapiens cDNA; FLJ22425 fis, clone HRC08686
2.13303	BC062758	BC062758	Homo sapiens cDNA clone IMAGE: 4081583, partial cds
2.13231	NM_030899	ZNF323	Homo sapiens zinc finger protein 323 (ZNF323), mRNA
2.12847	AK000939	AK000939	Homo sapiens cDNA FLJ10077 fis, clone HEMBA1001864
2.1237	NM_024332	NM_024332	Homo sapiens c6.1A (C6.1A), mRNA
2.1212	BC038180	BC038180	Homo sapiens O-linked N-acetylglucosamine (GlcNAc) transferase
			(UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase),
			transcript variant 1, mRNA (cDNA clone MGC: 39117 IMAGE: 5017795), complete cds
2.12077	AF143879	AF143879	Homo sapiens clone IMAGE: 120631 mRNA sequence.
2.12068	NM_012446	SSBP2	single-stranded-DNA-binding protein
2.11775	AK097639	AK097639	Homo sapiens cDNA FLJ40320 fis, clone TESTI2030770
2.11499	AL110141	AL110141	Homo sapiens mRNA; cDNA DKFZp564D0164 (from clone DKFZp564D0164).
2.1033	NM_018725	IL17BR	IL-17B receptor
2.09966	NM_181711	GRASP	Homo sapiens GRP1
			(general receptor for phosphoinositides 1)-associated scaffold protein (GRASP), mRNA
2.09541	NM_012258	HEY1	hairy/enhancer-of-split related with YRPW motif 1
2.09496	NM_021204	MASA	Homo sapiens E-1 enzyme (MASA), mRNA.
2.09345	AW511222	AW511222	ESTs, Moderately similar to
2.08172	NM_006759	UGP2	UDP-glucose pyrophosphorylase 2
2.07536	NM_004734	DCAMKL1	doublecortin and CaM kinase-like 1
2.06927	NM_017996	FLJ10103	hypothetical protein FLJ10103
2.05569	NM_000538	RFXAP	regulatory factor X-associated protein
2.05446	THC1871361	THC1871361	Unknown
2.05396	A_24_P516728	A_24_P516728	Unknown
2.04781	A_24_P545200	A_24_P545200	Unknown
2.04682	NM_018698	P15-2	hypothetical protein P15-2
2.03828	NM_138433	NM_138433	Homo sapiens hypothetical protein BC009980 (LOC113730), mRNA
2.03462	NM_001969	EIF5	eukaryotic translation initiation factor 5
2.03183	NM_183013	CREM	Homo sapiens cAMP responsive element modulator (CREM), transcript variant 19, mRNA
2.02975	NM_024786	ZDHHC11	Homo sapiens zinc finger, DHHC domain containing 11 (ZDHHC11), mRNA
2.02721	BC026998	BC026998	Homo sapiens, Similar to envoplakin, clone IMAGE: 5107188, mRNA
2.02703	NM_003084	SNAPC3	small nuclear RNA activating complex, polypeptide 3, 50 kD
2.01497	A_32_P118522	A_32_P118522	Unknown
2.01111	THC2005339	THC2005339	Unknown
2.0072	BC030611	BC030611	Homo sapiens cDNA clone MGC: 27088 IMAGE: 4827474, complete cds
2.00265	AF026526	AF026526	Homo sapiens unknown mRNA downregulated by Induced
			differentiation with 13-cis retinoic acid.

TABLE 2
Genes down-regulated in prohibitin depleted cells
Fold Change	Accession #	Sequence Name(s)	Sequence Description
−2.00202	NM_001935	DPP4	dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing protein 2)
−2.00298	AL080111	AL080111	Homo sapiens mRNA; cDNA DKFZp586G2222 (from clone DKFZp586G2222)
−2.00413	THC1971019	THC1971019	Unknown
−2.00532	BC036187	BC036187	Homo sapiens serine/arginine repetitive matrix 1, mRNA
			(cDNA clone MGC: 39488 IMAGE: 4827738), complete cds
−2.00654	NM_000873	ICAM2	Intercellular adhesion molecule 2
−2.00705	U17077	BENE	BENE protein
−2.00718	AK027134	AK027134	Homo sapiens cDNA: FLJ23481 fis, clone KAIA03003.
−2.00915	THC1960903	THC1960903	Unknown
−2.01233	BC026020	BC026020	Homo sapiens, Similar to KIAA0562 gene product, clone IMAGE: 4875334, mRNA
−2.01781	BC029358	BC029358	Homo sapiens G protein-coupled receptor 110, mRNA
			(cDNA clone IMAGE: 4812868), containing frame-shift errors
−2.01879	X97261	MT1L	metallothioneln 1L
−2.01979	A_32_P99533	A_32_P99533	Unknown
−2.02275	NM_014889	MP1	metalloprotease 1 (pitrilysin family)
−2.0241	NM_005186	CAPN1	Homo sapiens calpain 1, (mu/l) large subunit (CAPN1), mRNA.
−2.02523	NM_030812	NM_030812	Homo sapiens actin like protein (LOC81569), mRNA
−2.02584	NM_021180	NM_021180	Homo sapiens sister-of-mammalian grainyhead (SOM), transcript variant 1, mRNA
−2.02612	THC1957838	THC1957838	Unknown
−2.02672	NM_001144	AMFR	autocrine motility factor receptor
−2.02765	NM_016229	LOC51700	cytochrome b5 reductase b5R.2
−2.02802	AK055065	AK055065	Homo sapiens cDNA FLJ30503 fis, clone BRAWH2000494
−2.0296	A_24_P118361	A_24_P118361	Unknown
−2.02984	BC004277	BC004277	Homo sapiens cDNA clone MGC: 10837 IMAGE: 3615489, complete cds
−2.03347	ENST00000330567	ENST00000330567	Unknown
−2.03436	THC1910374	THC1910374	Unknown
−2.03465	BC007117	BC007117	Homo sapiens, clone IMAGE: 4333276, mRNA, partial cds
−2.0355	THC1940812	THC1940812	Unknown
−2.0366	AK074229	AK074229	Homo sapiens cDNA FLJ23649 fis, clone COL06395, highly
			similar to Oryctolagus cuniculus lacrimal lipase mRNA
−2.03806	NM_000346	SOX9	SRY (sex-determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal)
−2.03887	ENST00000330875	ENST00000330875	Unknown
−2.04904	NM_002612	PDK4	pyruvate dehydrogenase kinase, isoenzyme 4
−2.05042	BC012091	BC012091	Homo sapiens hairy and enhancer of split (Drosophila) homolog 2,
			mRNA (cDNA clone MGC: 20048 IMAGE: 4834002), complete cds
−2.05083	THC1958676	THC1958676	Unknown
−2.05132	A_24_P247303	A_24_P247303	Unknown
−2.05232	A_24_P230466	A_24_P230466	Unknown
−2.05305	NM_080430	NM_080430	Homo sapiens selenoprotein M (SELM), mRNA
−2.05578	NM_032012	C9orf5	Homo sapiens chromosome 9 open reading frame 5 (C9orf5), mRNA
−2.05599	NM_000696	ALDH9	aldehyde dehydrogenase 9 (gamma-aminobutyraldehyde dehydrogenase, E3 isozyme)
−2.05617	A_24_P247233	A_24_P247233	Unknown
−2.05929	NM_006108	SPON1	Homo sapiens spondin 1, extracellular matrix protein (SPON1), mRNA
−2.06232	NM_014256	TMEM3	transmembrane protein 3
−2.06567	A_24_P792988	A_24_P792988	Unknown
−2.06675	NM_199293	TH	Homo sapiens tyrosine hydroxylase (TH), transcript variant 3, mRNA
−2.06697	NM_025080	ASRGL1	Homo sapiens asparaginase like 1 (ASRGL1), mRNA
−2.06789	THC1991570	THC1991570	Unknown
−2.06805	AK024513	AK024513	Homo sapiens cDNA; FLJ20860 fis, clone ADKA01632.
−2.06874	THC1881737	THC1881737	Unknown
−2.06884	A_24_P358857	A_24_P358857	Unknown
−2.07022	AK092260	AK092260	Homo sapiens cDNA FLJ34941 fis, clone NT2RP7007480
−2.07198	AK075362	AK075362	Homo sapiens cDNA PSEC0048 fis, clone NT2RP2000028,
			highly similar to Homo sapiens serine protease mRNA
−2.07486	A_34_P471242	A_24_P471242	Unknown
−2.0763	NM_018584	PRO1489	hypothetical protein PRO1489
−2.07713	ENST00000333197	ENST00000333197	Unknown
−2.07923	NM_004360	CDH1	cadherin 1, type 1, E-cadherin (epithelial)
−2.07957	AK002195	AK002195	Homo sapiens cDNA FLJ11333 fis, clone PLACE1010616
−2.08301	A_24_P530977	A_24_P530977	Unknown
−2.08542	ENST00000327707	ENST00000327707	Unknown
−2.08675	A_24_P186746	A_24_P186746	Unknown
−2.09026	NM_002230	JUP	Junction piakogiobin
−2.09252	NM_000693	ALDH6	aldehyde dehydrogenase 6
−2.09611	THC1881942	THC1881942	Unknown
−2.09756	NM_138768	MYEOV	Homo sapiens myeloma overexpressed gene
			(In a subset of t(11; 14) positive multiple myelomas) (MYEOV), mRNA
−2.09924	NM_007267	LAK-4P	expressed in activated T/LAK lymphocytes
−2.10043	AK024480	FLJ00074	Homo sapiens mRNA for FLJ00074 protein, partial cds
−2.103	AK022268	AK022268	Homo sapiens cDNA FLJ12206 fis, clone MAMMA1000941
−2.10499	NM_018192	FLJ10718	hypothetical protein FLJ10718
−2.10701	A_24_P84970	A_24_P84970	Unknown
−2.1111	NM_000112	SLC26A2	solute carrier family 26 (sulfate transporter), member 2
−2.11277	NM_014292	CBX6	chromobox homolog 6
−2.11301	A_24_P109661	A_24_P109661	Unknown
−2.11559	NM_004753	SDR1	short-chain dehydrogenase/reductase 1
−2.12023	A_24_P375360	A_24_P375360	Unknown
−2.12417	BC036649	BC036649	Homo sapiens Sec23 homolog A (S. cerevisiae),
			mRNA (cDNA clone MGC: 26267 IMAGE: 4821858), complete cds
−2.12449	BC007034	BC007034	Homo sapiens metallothionein 2A, mRNA
			(cDNA clone MGC: 12397 IMAGE: 4051220), complete cds
−2.12555	AK021884	NPEPPS	aminopeptidase puromycin sensitive
−2.13148	A_24_P350136	A_24_P350136	Unknown
−2.13248	NM_020402	CHRNA10	cholinergic receptor, nicotinic, alpha polypeptide 10
−2.13329	AF161448	HSPC016	hypothetical protein
−2.13427	AL833323	AL833323	Homo sapiens mRNA; cDNA DKFZp686G1128 (from clone DKFZp686G1128)
−2.13627	AK095472	AK095472	Homo sapiens cDNA FLJ38153 fis, clone DFNES1000083
−2.13814	NM_005737	ARL7	ADP-ribosylation factor-like 7
−2.14183	A_24_P780319	A_24_P780319	Unknown
−2.14398	NM_025214	NM_025214	Homo sapiens CTCL tumor antigen se57-1 (SE57-1), mRNA
−2.14628	A_32_P80809	A_32_P80809	Unknown
−2.14944	BC002759	BC002759	Homo sapiens hypothetical protein FLJ20489, mRNA
			(cDNA clone IMAGE: 3632656), complete cds
−2.14967	BC049193	BC049193	Homo sapiens KIAA1277, mRNA (cDNA clone IMAGE: 4445131), partial cds
−2.15143	A_24_P922101	A_24_P922101	Unknown
−2.15502	NM_005953	MT2A	metallothionein 2A
−2.15502	L32537	L32537	Homo sapiens (clone XP6G6B) mRNA, partial EST.
−2.1592	A_32_P182135	A_32_P182135	Unknown
−2.16029	NM_032409	PINK1	Homo sapiens PTEN induced putative kinase 1 (PINK1), mRNA
−2.16178	THC1953717	THC1953717	Unknown
−2.1701	THC1940902	THC1940902	Unknown
−2.17121	NM_006460	HIS1	HMBA-inducible
−2.17172	NM_015894	SCLIP	SCG10-like-protein
−2.17557	AK096428	AK096428	Homo sapiens cDNA FLJ39109 fis, clone NTONG2005137, highly similar to
			[PYRUVATE DEHYDROGENASE(LIPOAMIDE)]
			KINASE ISOZYME 4, MITOCHONDRIAL PRECURSOR (EC 2.7.1.99)
−2.17744	AK055851	AK055851	Homo sapiens cDNA FLJ31289 fis, clone KIDNE2007328
−2.17823	NM_004878	PTGES	prosteglandin E synthase
−2.17957	NM_002276	KRT19	keratin 19
−2.18837	ENST00000332472	ENST00000332472	Unknown
−2.19207	AK125106	AK125106	Homo sapiens cDNA FLJ43116 fis, clone CTONG3002127, weakly similar to
			Mus musculus synaptotagmin-like 4 (Sytl4)
−2.1944	THC1993191	THC1993191	Unknown
−2.19924	NM_001848	COL6A1	Homo sapiens collagen, type VI, alpha 1 (COL6A1), mRNA
−2.19977	BC035496	BC035496	Homo sapiens Cbp/p300-Interacting transactivator,
			with Glu/Asp-rich carboxy-terminal domain,
			4, mRNA (cDNA clone MGC: 19748 IMAGE: 3622207), complete cds
−2.1999	NM_198473	NM_198473	Homo sapiens FLJ46111 protein (FLJ46111), mRNA
−2.20097	AF092095	TIP30	Tat-Interacting protein (30 kD)
−2.20981	BC004295	BC004295	Homo sapiens, clone IMAGE: 3622356, mRNA, partial cds
−2.21584	THC1952994	THC1952994	Unknown
−2.21861	NM_006651	CPLX1	complexin 1
−2.2225	NM_002614	PDZK1	PDZ domain containing 1
−2.22477	X16896	IL1R1	Interleukin 1 receptor, type I
−2.22745	THC1846762	THC1846762	Unknown
−2.22906	NM_003370	VASP	vasodilator-stimulated phosphoprotein
−2.23039	ENST00000311197	ENST00000311197	Unknown
−2.23181	AK025062	AK025062	Homo sapiens cDNA: FLJ21409 fis, clone COL03924
−2.23286	NM_003500	ACOX2	acyl-Coenzyme A oxidase 2, branched chain
−2.24005	BC036885	BC036885	Homo sapiens, clone IMAGE: 5399920, mRNA
−2.24334	NM_206827	RASL11A	Homo sapiens RAS-like, family 11, member A (RASL11A), mRNA
−2.24932	NM_005952	MT1X	Homo sapiens metallothionein 1X (MT1X), mRNA.
−2.25159	AK027217	LIM	LIM protein (similar to rat protein kinase C-binding enigma)
−2.25366	AB017116	AD 1	Homo sapiens mitochondrial mRNA for AD 1, partial cds.
−2.25688	U06469	SLC1A1	solute carrier family 1 (neuronal/epithelial high affinity glutamate
			transporter, system Xag), member 1
−2.25716	NM_022369	FLJ12541	Homo sapiens hypothetical protein FLJ12541 similar to Stra6 (FLJ12541), mRNA.
−2.26077	A_24_P928250	A_24_P928250	Unknown
−2.26084	THC1860502	THC1860502	Unknown
−2.26869	NM_138448	ACYP2	Homo sapiens acylphosphatase 2, muscle type (ACYP2), mRNA
−2.26924	NM_004695	SLC16A5	solute carrier family 16 (monocarboxylic acid transporters), member 5
−2.26993	A_32_P104469	A_32_P104469	Unknown
−2.27435	Z34282	MUC5AC	H. sapiens (MAR11) MUC5AC mRNA for mucin (partial)
−2.27521	NM_014452	DR6	death receptor 6
−2.28033	A_24_P315594	A_24_P315594	Unknown
−2.28449	A_24_P917810	A_24_P917810	Unknown
−2.28898	L02922	tropomyosin	Human tropomyosin isoform hTM5a mRNA, exon 1.
−2.29203	AK091178	AK091178	Homo sapiens cDNA FLJ33859 fis, clone CTONG2006223,
			moderately similar to KERATIN, TYPE II CYTOSKELETAL 8
−2.2968	ENST00000322533	ENST00000322533	Unknown
−2.30396	NM_139314	ANGPTL4	Homo sapiens angiopoietin-like 4 (ANGPTL4), transcript variant 1, mRNA
−2.3124	NM_022119	PRSS22	Homo sapiens protease, serine, 26 (PRSS22), mRNA.
−2.32359	NM_138967	SCAMP5	Homo sapiens secretory carrier membrane protein 5 (SCAMP5), mRNA
−2.32421	NM_017878	FLJ20556	hypothetical protein FLJ20556
−2.32808	NM_002646	PIK3C2B	Homo sapiens phosphoinositide-3-kinase, class 2, beta polypeptide (PIK3C2B), mRNA.
−2.32922	AK074567	AK074567	Homo sapiens cDNA FLJ90086 fis, clone HEMBA1005145
−2.3301	NM_016201	LCCP	Leman coiled-coil protein
−2.34004	NM_182494	NM_182494	Homo sapiens hypothetical protein LOC119395 (LOC119395), mRNA
−2.34802	BF129169	BF129169	ESTs
−2.352	X91221	NCX1	H. sapiens mRNA for NCX1 protein 3′UTR.
−2.35349	BC032946	BC032946	Homo sapiens aquaporin 5, mRNA
			(cDNA clone MGC: 33163 IMAGE: 5269384), complete cds
−2.36509	A_24_P843309	A_24_P843309	Unknown
−2.3684	NM_020353	LOC57088	phospholipid scramblase 4
−2.37251	NM_017458	MVP	major vault protein
−2.3833	A_24_P844100	A_24_P844100	Unknown
−2.38554	NM_005476	GNE	UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase
−2.38839	NM_004635	MAPKAPK3	mitogen-activated protein kinase-activated protein kinase 3
−2.39088	NM_003740	KCNK5	potassium channel, subfamily K, member 5 (TASK-2)
−2.39301	NM_031311	CPVL	Homo sapiens carboxypeptidase, viteilogenic-like (CPVL), transcript variant 1, mRNA
−2.39894	A_32_P152999	A_32_P152999	Unknown
−2.40969	NM_000142	FGFR3	fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism)
−2.41275	THC1905270	THC1905270	Unknown
−2.41493	THC1944835	THC1944835	Unknown
−2.42522	ENST00000328006	ENST00000328006	Unknown
−2.42726	NM_002273	KRT8	keratin 8
−2.42838	NM_002305	LGALS1	lectin, galactoside-binding, soluble, 1 (galectin 1)
−2.43097	BF512676	BF512676	ESTs
−2,43469	A_32_P202708	A_32_P202708	Unknown
−2.43629	NM_014471	PEC-60	gastrointestinal peptide
−2.43751	NM_001897	CSPG4	chondroltin sulfate proteoglycan 4 (melanoma-associated)
−2.43756	NM_003896	SIAT9	sialyltransferase 9 (CMP-NeuAc: lactosylceramide alpha-2,3-sialyltransferase; GM3 synthase)
−2.44725	A_24_P306644	A_24_P306844	Unknown
−2.45084	BC009792	BC009792	Homo sapiens cleavage stimulation factor, 3′ pre-RNA, subunit 3, 77 kDa,
			mRNA (cDNA clone IMAGE: 4274695), complete cds
−2.4533	BQ362073	BQ362073	Unknown
−2.45467	A_24_P15973	A_24_P15973	Unknown
−2.45903	BC040110	BC040110	Homo sapiens, Similar to fer-1-like 3, myoferlin
			(C. elegans), clone IMAGE: 3882757, mRNA, partial cds
−2.46008	A_24_P418216	A_24_P418216	Unknown
−2.467	NM_170726	ALDH4A1	Homo sapiens aldehyde dehydrogenase 4 family,
			member A1 (ALDH4A1), nuclear gene encoding mitochondrial protein,
			transcript variant P5CDhS, mRNA
−2.4732	NM_017451	BAIAP2	BAI1-associated protein 2
−2.47787	THC1893261	THC1893261	Unknown
−2.47944	NM_003255	TIMP2	tissue Inhibitor of metelloproteinase 2
−2.4888	NM_005368	MB	myoglobin
−2.50196	NM_001548	IFIT1	Interferon-Induced protein with tetratricopeptide repeats 1
−2.51182	NM_002275	KRT15	keratin 15
−2.51385	NM_005971	FXYD3	FXYD domain-containing Ion transport regulator 3
−2.5213	NM_000716	C4BPB	complement component 4-binding protein, beta
−2.52213	NM_182741	MUC1	Homo sapiens mucin 1, transmembrane (MUC1), mRNA
−2.52676	NM_001549	IFIT4	Interferon-Induced protein with tetratricopeptide repeats 4
−2.5292	NM_016619	LOC51316	hypothetical protein
−2.533	AK075564	AK075564	Homo sapiens cDNA PSEC0264 fis, clone NT2RP3002337
−2.53403	A_24_P7021	A_24_P7021	Unknown
−2.54393	NM_182532	NM_182532	Homo sapiens hypothetical protein LOC199964 (LOC199964), mRNA
−2.54549	A_24_P83111	A_24_P93111	Unknown
−2.56068	A_24_P7750	A_24_P7750	Unknown
−2.56166	A_24_P281605	A_24_P281605	Unknown
−2.57194	BX374774	BX374774	Unknown
−2.57249	BC014851	BC014851	Homo sapiens lunatic fringe homolog (Drosophila), mRNA
			(cDNA clone MGC: 22145 IMAGE: 4453156), complete cds
−2.58019	BC040210	BC040210	Homo sapiens, aldo-keto reductase family 1, member C1
			(dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid
			dehydrogenase), clone IMAGE: 4825338, mRNA
−2.58072	A_24_P478940	A_24_P478940	Unknown
−2.59118	AK097804	AK097804	Homo sapiens cDNA FLJ40485 fis, clone TESTI2043857,
			moderately similar to Homo sapiens nolp mRNA
−2.59217	A_24_P213336	A_24_P213336	Unknown
−2.59289	A_23_P120545	A_23_P120545	Unknown
−2.60575	NM_173481	C19orf21	Homo sapiens chromosome 19 open reading frame 21 (C19orf21), mRNA
−2.61387	NM_032943	SYTL2	Homo sapiens synaptotagmin-like 2 (SYTL2), transcript variant a, mRNA
−2.62052	AF034803	PPFIBP2	PTPRF interacting protein, binding protein 2 (liprin beta 2)
−2.62447	AK074149	AK074149	Homo sapiens mRNA for FLJ00222 protein
−2.62707	NM_002966	S100A10	S100 calcium-binding protein A10 (annexin II ligand, calpactin I, light polypeptide (p11))
−2.62858	NM_020792	NM_020792	Homo sapiens KIAA1363 protein (KIAA1363), mRNA
−2.6377	A_24_P341546	A_24_P341546	Unknown
−2.64372	NM_152353	NM_152353	Homo sapiens hypothetical protein MGC33839 (MGC33839), mRNA
−2.64623	NM_013321	SNX8	Homo sapiens sorting nexin 8 (SNX8), mRNA
−2.65233	NM_152864	C20orf58	Homo sapiens chromosome 20 open reading frame 58 (C20orf58), mRNA
−2.66028	M59979	PTGS1	prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)
−2.6608	BC005987	BC005987	Homo sapiens interferon-induced protein with tetratricopeptide repeats 2,
			mRNA (cDNA clone IMAGE: 3838493), containing frame-shift errors
−2.67518	A_24_P195974	A_24_P195974	Unknown
−2.67611	NM_004669	CLIC3	chloride intracellular channel 3
−2.69138	Y12878	ncx1	Homo sapiens NCX1 mRNA alternative 5′end, exon 1d, 1c and 2.
−2.69364	NM_019894	TMPRSS4	Homo sapiens transmembrane protease, serine 4 (TMPRSS4), transcript variant 1, mRNA
−2.69856	NM_004170	SLC1A1	solute carrier family 1 (neuronal/epithelial high affinity glutamate
			transporter, system Xag), member 1
−2.70809	X90980	aml1	Homo sapiens mRNA for an acute myeloid leukaemia protein (486 bp).
−2.71286	A_32_P112546	A_32_P112546	Unknown
−2.71657	NM_013434	CSEN	Calsenilin, presenilin-binding protein, EF hand transcription factor
−2.7278	A_24_P401124	A_24_P401124	Unknown
−2.7392	NM_003739	AKR1C1	aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1;
			20-alpha (3-alpha)-hydroxysteroid dehydrogenase)
−2.77465	A_32_P75141	A_32_P75141	Unknown
−2.81822	AK095421	AK095421	Homo sapiens cDNA FLJ38102 fis, clone D3OST2000618,
			moderately similar to Drosophila melanogaster slingshot mRNA
−2.82133	NM_144587	C10orf87	Homo sapiens chromosome 10 open reading frame 87 (C10orf87), mRNA
−2.82251	AB083038	AB083038	Homo sapiens RDH-E2 mRNA for retinal short chain dehydrogenase reductase, complete cds
−2.82589	NM_032034	SLC4A11	Homo sapiens solute carrier family 4, sodium bicarbonate
			transporter-like, member 11 (SLC4A11), mRNA
−2.8323	A_24_P247074	A_24_P247074	Unknown
−2.84091	U21049	DD96	epitheilal protein up-regulated in carcinoma, membrane associated protein 17
−2.84421	NM_000891	KCNJ2	potassium inwardly-rectifying channel, subfamily J, member 2
−2.85298	NM_001353	AKR1C1	aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1;
			20-alpha (3-alpha)-hydroxysteroid dehydrogenase)
−2.86724	NM_031419	NM_031419	Homo sapiens molecule possessing ankyrin repeats induced
			by lipopolysaccharide (MAIL), homolog of mouse (MAIL), mRNA
−2.88219	BC018597	BC018597	Homo sapiens, clone IMAGE: 3869276, mRNA
−2.9014	NM_016434	LOC51750	helicase-like protein NHL
−2.91156	NM_024625	ZC3HAV1	Homo sapiens zinc finger CCCH type, antiviral 1 (ZC3HAV1), transcript variant 2, mRNA
−2.94339	NM_024306	FA2H	Homo sapiens fatty acid 2-hydroxylase (FA2H), mRNA
−2.94648	AB028955	KIAA1032	KIAA1032 protein
−2.95121	NM_002274	KRT13	keratin 13
−2.97481	NM_138632	NM_138632	Homo sapiens Tara-like protein (HRIHFB2122), transcript variant 2, mRNA
−2.98332	BC029496	BC029496	Homo sapiens protein tyrosine phosphatase, receptor type,
			S, mRNA (cDNA clone IMAGE: 5272670), complete cds
−3.00058	THC1927382	THC1927382	Unknown
−3.0138	NM_006123	IDS	iduronate 2-sulfatase (Hunter syndrome)
−3.01734	NM_003733	OASL	Homo sapiens 2′-5′oligoadenylate synthetase-like (OASL), mRNA.
−3.03167	NM_004363	CEACAM5	Homo sapiens carcinoembryonic antigen-related cell adhesion
			molecule 5 (CEACAM5), mRNA.
−3.05601	NM_003225	TFF1	trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in)
−3.10811	U64094	IL1R2	interleukin 1 receptor, type II
−3.11205	NM_001547	IFIT2	Homo sapiens interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), mRNA
−3.12631	NM_004419	DUSP5	dual specificity phosphatase 5
−3.16603	BC007533	BC007533	Homo sapiens neuropilin 1, mRNA
			(cDNA clone MGC: 15383 IMAGE: 2958475), complete cds
−3.17057	NM_004368	CNN2	calponin 2
−3.19544	AJ001402	MUC5AC	mucin 5, subtypes A and C, tracheobronchial/gastric
−3.22204	BC069216	BC069216	Homo sapiens cDNA clone MGC: 78739 IMAGE: 6376829, complete cds
−3.29685	NM_000213	ITGB4	,integrin, beta 4
−3.24972	NM_005101	ISG15	interferon-stimulated protein, 15 kDa
−3.25168	NM_005423	TFF2	trefoil factor 2 (spasmolytic protein 1)
−3.25561	U91543	CHD3	chromodomain helicase DNA binding protein 3
−3.30115	AK056142	AK056142	Homo sapiens cDNA FLJ31580 fis, clone NT2RI2002041
−3.33043	AF311286	AF311286	Homo sapiens tissue transglutaminase (TGM2) mRNA, partial cds
−3.3437	THC2003438	THC2003438	Unknown
−3.3638	NM_003541	H4FD	H4 histone family, member D
−3.37813	NM_021968	H4FE	Homo sapiens H4 histone family, member E (H4FE), mRNA.
−3.42093	A_24_P614940	A_24_P614940	Unknown
−3.42714	NM_005672	PSCA	prostate stem cell antigen
−3.48275	NM_000962	PTGS1	Homo sapiens prostaglandin-endoperoxide synthase 1
			(prostaglandin G/H synthase and cyclooxygenase) (PTGS1), mRNA.
−3.49519	THC1891572	THC1891572	Unknown
−3.52709	NM_003225	TFF1	trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in)
−3.54417	A_23_P66347	A_23_P66347	Unknown
−3.73647	NM_144777	SCEL	Homo sapiens sciellin (SCEL), transcript variant 2, mRNA
−3.78901	BC015940	BC015940	Homo sapiens 5′-nucleotidase, ecto (CD73), mRNA
			(cDNA clone IMAGE: 3920680), complete cds
−4.03972	THC1941189	THC1941189	Unknown
−4.07695	NM_017717	MUCDHL	mucin and cadherin-like
−4.09543	NM_148672	CCL28	Homo sapiens chemokine (C-C motif) ligand 28 (CCL28), transcript variant 2, mRNA
−4.09875	BC017859	BC017859	Homo sapiens trefoil factor 3 (intestinal), mRNA
			(cDNA clone MGC: 22588 IMAGE: 4696566), complete cds
−4.09911	NM_003226	TFF3	trefoil factor 3 (intestinal)
−4.21393	A_32_P115077	A_32_P115077	Unknown
−4.21838	NM_018414	SIAT7A	Homo sapiens sialyltransferase 7 ((alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-
			N-acetyl galactosaminide alpha-2,6-sialyltransferase) A (SIAT7A), mRNA
−4.33931	NM_002634	PHB	prohibitin
−4.34628	A_24_P349606	A_24_P349606	Unknown
−4.35457	NM_024508	ZBED2	Homo sapiens zinc finger, BED domain containing 2 (ZBED2), mRNA
−4.3942	NM_032689	ZNF607	Unknown
−4.39683	A_24_P50639	A_34_P50639	Unknown
−4.58843	ENST00000329070	ENST00000329070	Unknown
−4.65917	NM_002193	INHBB	inhibin, beta B (activin AB beta polypeptide)
−4.69951	A_23_P113692	A_23_P113692	Unknown
−4.69982	BC014228	BC014228	Homo sapiens cDNA clone MGC: 20874 IMAGE: 4547239, complete cds
−4.71106	BC013401	BC013401	Homo sapiens prohibitin, mRNA
			(cDNA clone MGC: 3926 IMAGE: 3010198), complete cds
−4.90254	NM_019601	BK65A6.2	Homo sapiens Sushi domain (SCR repeat) containing (BK65A6.2), mRNA.
−4.90852	A_24_P238896	A_24_P238896	Unknown
−4.93829	NM_005130	HBP17	heparin-binding growth factor binding protein
−5.06624	NM_004633	IL1R2	interleukin 1 receptor, type II
−5.08793	A_24_P203984	A_24_P203984	Unknown
−5.41703	BC018706	BC018706	Homo sapiens C-terminal tensin-like, mRNA
			(cDNA clone MGC: 31757 IMAGE: 5013235), complete cds
−5.44197	NM_002483	CEACAM6	carcinoembryonic antigen-related cell adhesion
			molecule 6 (non-specific cross reacting antigen)
−5.52721	NM_002272	KRT4	Homo sapiens keratin 4 (KRT4), mRNA
−5.67766	A_24_P152845	A_24_P152845	Unknown
−5.71755	ENST00000331608	ENST00000331608	Unknown
−5.78911	NM_020299	AKR1B10	Homo sapiens aldo-keto reductase family 1,
			member B10 (aldose reductase) (AKR1B10), mRNA
−7.96627	BC005008	BC005008	Homo sapiens carcinoembryonic antigen-related cell adhesion
			molecule 6 (non-specific cross reacting antigen),
			mRNA (cDNA clone MGC: 10467 IMAGE: 3640231), complete cds

Claims

1-38. (canceled)

39. Use of at least one inhibitor of Prohibitin (PHB) optionally together with pharmaceutically acceptable carriers, adjuvants, diluents or/and additives for the manufacture of a pharmaceutical composition for the treatment or/and prevention of hyperprolerative disorders.

40. Use as claimed in claim 39, wherein the pharmaceutical composition is for the treatment or/and prevention of tumours, metastatic tumours, benign tumours, carcinoma, neoplastic carcinoma, gastric carcinoma, larynx carcinoma, neoplastic thyroid cancer, hepatocellular carcinoma, hyperplasia, adenocarcinoma, bladder carcinoma, EGFR overexpressing tumours, Her-2 family overexpressing tumours, Herceptin resistant tumours, B-Raf transformed tumours, or/and Raf-1 transformed tumours.

41. Use as claimed in claim 39, wherein the at least one inhibitor specifically inhibits the interaction of PHB with a Raf kinase, in particular the binding of PHB to a Raf kinase, preferably Raf-1.

42. Use as claimed in claim 39, wherein the at least one inhibitor inhibits activation of the Ras-Raf signalling pathway, preferably the Ras-Raf-MAPK pathway or/and the PHB dependent branch of the Ras signalling pathway.

43. Use as claimed in claim 39, wherein the at least one inhibitor specifically disrupts a Ras-Raf interaction.

44. Use as claimed in claim 39, wherein the at least one inhibitor inhibits downstream of members of the EGFR family, in-2-particular downstream of EGFR and Her-2.

45. Use as claimed in claim 39, wherein the at least one inhibitor converts tumour cells from a transformed to a non-transformed phenotype at least in vitro and in vivo.

46. Use as claimed in claim 39, wherein the at least one inhibitor prevents angiogenesis in tumours.

47. Use as claimed in claim 39, wherein the at least one inhibitor prevents cell migration in vitro or/and in vivo.

48. Use as claimed in claim 39, wherein the at least one inhibitor downregulates PHB transcription or/and translation, inhibits targeting of PHB to membranes, inhibits targeting of a Raf kinase, in particular Raf-1 (C-Raf) to membranes, or/and inhibits posttranslational modification of PHB, in particular required for the PHB targeting to membranes or/and required for the targeting of a Raf kinase, in particular Raf-1 (C-Raf) to membranes.

49. Use as claimed in claim 39, wherein the at least one inhibitor is selected from the group of nucleic acids, nucleic acid analogues such as ribozymes, peptides, polypeptides, and antibodies.

50. Use as claimed in claim 49, wherein the nucleic acid is

(i) an RNA molecule capable of RNA interference,

(ii) a precursor of the RNA molecule (i), or

(iii) a DNA molecule encoding the RNA molecule (i) or the precursor (ii).

51. Use as claimed in claim 50, wherein the DNA molecule is a vector.

52. Use as claimed in claim 49, wherein the RNA molecule is a double-stranded RNA molecule, preferably a double-stranded siRNA molecule with or without a single-stranded overhang alone at one end or at both ends.

53. Use as claimed in claim 49, wherein the nucleic acid is an antisense nucleic acid.

54. Use as claimed in claim 49, wherein the peptide or/and the polypeptide is biologically inactive, capable of inhibiting the activity of PHB, and comprises a fragment of SEQ.ID.NO:2, a derivative thereof, a fragment to of a Raf kinase or/and a derivative thereof.

55. Use as claimed in claim 49, wherein the antibody is directed against PHB.

56. Use as claimed in claim 55, wherein the antibody is directed against a peptide or polypeptide comprising

(a) the amino acid sequence of SEQ.ID.NO:2,

(b) an amino acid sequence which is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the sequence of (a), or/and

(c) an immunogenic fragment of (a) or (b).

57. A nucleic acid suitable as inhibitor in a pharmaceutical composition as defined in claim 39 comprising

(a) a fragment of the nucleotide sequence of SEQ.ID.NO:1, or

(b) a fragment which is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the sequence of (a).

58. The nucleic acid as claimed in claim 57 having a length of at least 15, preferably at least 17, more preferably at least 19, most preferably at least 21 nucleotides.

59. The nucleic acid as claimed in claim 57, which has a length of at the maximum 29, preferably at the maximum 27, more preferably at the maximum 25, especially more preferably at the maximum 23, most preferably at the maximum 21 nucleotides.

60. The nucleic acid as claimed in claim 57, which is an RNA molecule, preferably a double-stranded RNA molecule, more preferably a double-stranded siRNA molecule with or without a single-stranded overhang alone at one end or at both ends.

61. The nucleic acid as claimed in 57 comprising a sequence selected from the group consisting of SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID,NO:8, SEQ.ID.NO;9, SEQ,ID.NO:10 and fragments thereof which may have a length of at least 15 nucleotides.

62. A screening method for identification of a compound suitable as inhibitor in a pharmaceutical composition defined in claim 39, comprising the steps

(a) providing a cell or/and a transgenic non-human animal capable of expressing, particularly overexpressing PHB,

(b) contacting a compound with the cell or/and the transgenic non-human animal,

(c) determining the amount or/and the activity of PHB, and

(d) selecting a compound which reduces the amount or/and the activity of PHB.

63. The screening method as claimed in claim 62, wherein the activity of PHB in steps (c) and (d) is determined by measuring PHB expression, by prevention of cell migration, by determining targeting of PHB to membranes, by determining targeting of a Raf kinase, in particular Raf-1 to membranes, or/and by determining posttranslational modification of PHB.

64. The screening method as claimed in claim 62, wherein the activity of PHB in steps (c) and (d) is an interaction of PHB with a Raf kinase, in particular the binding of PHB to a Raf kinase, preferably Raf-1, or/and targeting of a Raf kinase, preferably Raf-1, to membranes.

65. A screening method for identification of a compound suitable as inhibitor in a pharmaceutical composition defined in claim 62, comprising the steps

(a) contacting a compound with isolated PHB or/and an isolated Raf kinase,

(b) determining the activity of PHB to interact with Raf kinase, and

(c) selecting a compound which reduces the activity of PHB.

66. The screening method as claimed in claim 65, wherein the interaction is the binding of PHB to a Raf kinase, preferably Ref-1 or/and targeting of a Raf kinase, preferably Raf-1, to membranes.

67. A method for treating or/and prevention of hyperprolerative disorders comprising the administration of a pharmaceutical composition of claim 39 to a subject in need thereof.

68. Use of an inhibitor of Prohibitin for prevention of cell migration in vivo or/and in vitro.

69. A screening method for identification of genes suitable as targets for treatment of a hyperproliferative disorder, comprising the steps

(a) providing a cell or/and a transgenic non-human animal capable of so expressing, particularly overexpressing PHB,

(b) downregulating PHB expression or/and inhibiting PHB activity within the cell or/and the transgenic non-human animal,

(c) determining the expression or/and activity of at least one gene within the cell or/and the animal, and

(d) identifying a target gene of which the expression is upregulated or downregulated by downregulation of PHB expression or/and inhibition of PHB activity.

70. The screening method of claim 69 wherein the downregulation of PHB expression or/and the inhibition of PHB activity in step (b) is performed by an inhibitor of PHB.

71. The screening method of claim 69, wherein in step (d) the target gene is upregulated or downregulated by a factor of at least 2, preferably at least 4.

72. The screening method of claim 69, wherein the expression of the at least one gene in step (c) is determined by a microarray technique.

73. A screening method for identification of a compound suitable for treatment of a hyperproliferative disorder comprising the steps

(a) providing a cell or/and a transgenic non-human animal capable of expressing at least one gene selected from Table 1 or/and Table 2,

(b) contacting at least one compound with the cell or/and the transgenic non-human animal,

(c) determining the amount or/and the activity of the gene product of the at least one gene selected from Table 1 or/and Table 2, and

(d) identifying a compound which increases the amount or/and the activity of the gene product of the at least one gene of Table 1 or decreases the amount or/and activity of the gene product of the at least one gene of Table 2.

74. The screening method of claim 69, wherein the hyperproliferative disorder is selected from tumours, metastatic tumours, benign tumours, carcinoma, neoplastic carcinoma, gastric carcinoma, larynx carcinoma, neoplastic thyroid cancer, hepatocellular carcinoma, hyperplasia, adenocarcinoma, bladder carcinoma, EGFR overexpressing tumours, Her-2 family overexpressing tumours, Herceptin resistant tumours, B-Raf transformed tumours, and Raf-1 (C-Raf) transformed tumours.

75. The screening method of claim 69, wherein in step (a) the cell is a mammalian cell.

76. The screening method wherein the downregulation of PHB expression or/and the inhibition of PHB activity in step (b) is performed by an inhibitor as defined in claim 39.