Cleavage Inhibitors Of Transforming Growth Factor Beta Type I Receptor And Uses Thereof In Cancer Therapy

Abstract

Disclosed herein are methods to inhibit cleavage of a type I receptor of transforming growth factor beta (T.beta.RI) and reduce cancer cell invasiveness/metastasis, and agents and pharmaceutical compositions for use in these methods. Also disclosed herein are methods for identifying agents that inhibit cleavage of T.beta.RI and methods for diagnosing and/or prognosing cancer based on nuclear localization of a intracellular domain of T.beta.RI, which is a product of T.beta.RI cleavage.

Description

RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional application No. 61/452,549, filed Mar. 14, 2011 under 35 U.S.C. .sctn.119, the entire content of which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Transforming growth factor .beta. (TGF.beta.) is a cytokine that plays an important role during normal embryogenesis due to its multifunctional effects on cellular responses such as proliferation, differentiation, apoptosis, and migration. TGF.beta. has during recent years become recognized as a potent regulator of cellular plasticity, which is a central event during embryogenesis and tumor progression. TGF.beta. signals through its binding to the type II and type I serine/threonine kinase receptors (T.beta.RII and T.beta.RI, respectively), resulting in their hetero-oligomerization, which subsequently activates various intracellular signalling pathways. For example, TGF.beta. activates the kinase activity of T.beta.RI to phosphorylate the latent transcription factors Smad2 and Smad3 in early endosomes, which induces complex formation with Smad4 and nuclear translocation, allowing regulation of target genes. Sorrentino et al., Nat. Cell Biol. 10:1199-1207; 2008. In addition to the Smad pathways, TGF.beta./T.beta.R signaling can also activate non-Smad pathways.

[0003] T.beta.RI has been found to harbour a consensus binding site for the ubiquitin ligase tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), which was initially identified as mediating the activation of NF-.kappa.B by Interleukin-1. Cao et al., 1996. When bound to TGF.beta., TRAF6 activates the TGF.beta. activated kinase 1 (TAK1), which in turn activates p38 mitogen activated protein (MAP) kinase pathway. Yamashita et al., Mol. Cell. 31:918-924; 2008; and Liu et al., Mol. Cell. 35:26-36; 2009. TRAF6 was also known as an E3 ligase, which was reported to interact with T.beta.RI (also known as activin like kinase (ALK) 5) at a highly conserved consensus motif. Sorrentino et al., Nat. Cell Biol. 10(10):1199-1207; 2008. The T.beta.RI-TRAF6 interaction leads to TGF.beta. induced TRAF6 autoubiquitination and Lys63-dependent polyubiqitination of TAK1. The activated TAK1 in turn activates MKK3/6 leading to p38 activation and resulting in apoptosis. Thakur et al., Future Oncol. 5(1):1-3; 2009 and Landstrm et al., Int. J. Biochem Cell Biol. 42(5):585-589; 2010.

[0004] Posttranslational modifications of T.beta.RI, such as monoubiquitination or Lys63-linked polyubiquitination, have emerged as an important mechanism to control the localization or function of this protein, whereas Lys48-linked polyubiquitination of T.beta.RI was originally described to instead target its substrate for proteasomal degradation (Hershko and Ciechanover 1998, and Ikeda and Dikic 2010). TRAF6 is known to induce Lys63-linked polyubiquitination of its substrates, including TAK1. Yamashita et al., 2008.

SUMMARY OF THE INVENTION

[0005] The present disclosure is based on unexpected discoveries that TGF.beta. induces polyubiquitination of its type I receptor (T.beta.RI) via tumor necrosis factor receptor-associated factor 6 (TRAF6), which, in turn, induces cleavage of T.beta.RI by a cleavage enzyme (e.g., tumor necrosis factor alpha converting enzyme or TACE and presenilin 1 or PS1), leading to nuclear translocation of an intracellular domain of T.beta.RI (T.beta.RI ICD) and activation of genes involved in cancer invasion (e.g., Snail), and that nuclear localization of T.beta.RI ICD, which occurs in cancer cells but not in normal cells, is a reliable cancer biomarker.

[0006] Accordingly, one aspect of this disclosure relates to a method for inhibiting cleavage of a T.beta.RI, comprising contacting a cell (e.g., a cancer cell) with a T.beta.RI cleavage inhibitor in an amount sufficient to inhibit cleavage of a T.beta.RI to release a T.beta.RI ICD, thereby blocking the T.beta.RI ICD from translocating to cell nuclei. The contacting step can be performed by administering the T.beta.RI cleavage inhibitor to a subject in need of the treatment (e.g., a human cancer patient).

[0007] In another aspect, disclosed herein is a method for reducing invasiveness of cancer cells in a subject (e.g., a human cancer patient), comprising administering to the subject a pharmaceutical composition comprising at least a T.beta.RI cleavage inhibitor in an amount sufficient to inhibit cleavage of a T.beta.RI to release an ICD of the T.beta.RI, thereby blocking the ICD from translocating to the nuclei of the cancer cells and reducing their invasiveness. In some embodiments, the T.beta.RI cleavage inhibitor is administered to a cancer patient in an amount sufficient to reduce metastasis of a cancer (e.g., prostate cancer, renal carcinoma, bladder carcinoma, breast cancer, lung cancer, and colorectal cancer). In some embodiments, the pharmaceutical composition is free of MMP14 inhibitors.

[0008] In one example, the cleavage of the T.beta.RI occurs between the G and L residues corresponding to G.sub.120 and L.sub.121 in SEQ ID NO: 1 and an amount of the T.beta.RI cleavage inhibitor sufficient to inhibit T.beta.RI cleavage at this site is used in any of the methods described above. In another example, the cleavage of the T.beta.RI occurs between the V and I residues corresponding to V.sub.129 and I.sub.130 in SEQ ID NO: 1 and an amount of the T.beta.RI cleavage inhibitor sufficient to inhibit this cleavage is used in the methods described herein.

[0009] The T.beta.RI cleavage inhibitor can be (i) an antibody (e.g., a full-length antibody or an antigen-binding fragment thereof) that binds to the T.beta.RI and blocks its cleavage; (ii) an inhibitor of TRAF6, e.g., an anti-TRAF6 antibody (full-length or antigen-binding fragment), a TRAF6-specific interfering nucleic acid, a TRAF6 inhibitory peptide, or a small molecule TRAF6 inhibitor; (iii) an inhibitor of TACE, e.g., an anti-TACE antibody (full-length or antigen-binding fragment), a TACE-specific interfering nucleic acid, or a small molecule TACE inhibitor; (iv) an inhibitor of protein kinase C zeta (PKC.zeta.), e.g., an anti-PKC.zeta. antibody (full-length or antigen-binding fragment), a PKC.zeta.-specific interfering nucleic acid, a PKC.zeta. pseudosubstrate, or a small molecule PKC.zeta. inhibitor, (v) an inhibitor of PS1, e.g., an anti-PS1 antibody (full-length or antigen-binding fragment), a PS1-specific interfering nucleic acid, or a small molecule PS1 inhibitor; or (vi) a combination of any of (i) to (v). In some embodiments, the T.beta.RI cleavage inhibitor is not an inhibitor of MMP14.

[0010] The antibody of (i) noted above, which is also within the scope of this disclosure, can be an antibody that binds to the T.beta.RI and blocks cleavage of the T.beta.RI between the G and L residues noted above. In some embodiments, the antibody binds to an antigen epitope (linear or conformational) that encompasses the G residue, the L residue, or both. Alternatively, the antibody binds to the T.beta.RI and blocks its cleavage between the V and I residues also noted above, e.g., binding to an antigen epitope (linear or conformational) that encompasses the V residue, the L residue, or both. In some examples, the antibody binds to an epitope within residues 114-124 in SEQ ID NO:1. Any of the antibodies of (i) as described above can be bispecific antibodies that bind to both T.beta.RI and TACE, MMP14, or PS1.

[0011] The T.beta.RI cleavage inhibitor described herein can inhibit T.beta.RI in various cancer cells, including, but are not limited to, prostate cancer cells, renal carcinoma cells, bladder carcinoma cells, breast cancer cells, lung cancer cells, or colorectal cancer cells. It therefore is effective in reducing cancer cell invasion in patients carrying the various types of cancer cells.

[0012] In another aspect, provided herein is a diagnostic method, comprising: (i) examining presence or absence of an T.beta.RI ICD in the nuclei of cells in a sample, and, (ii) determining whether the sample contains cancer cells by, e.g., contacting the cells in the sample, or a fraction thereof, with an antibody that binds to the ICD. Nuclear localization of the T.beta.RI ICD indicates presence of cancer cells in the sample. In one example, the sample is suspected of containing cancer cells, such as prostate cancer cells, renal carcinoma cells, bladder carcinoma cells, breast cancer cells, lung cancer cells, or colorectal cancer. In another example, the sample is a tissue sample obtained from, e.g., a patient suspected of having or at risk for a cancer, e.g., those listed above.

[0013] The just-described diagnostic method can further comprise a step of assessing the likelihood of cancer invasiveness/metastasis in the subject. Nuclear localization of the ICD indicates that the subject has cancer cells, which are likely to metastasize.

[0014] Further, the present disclosure provides a prognostic method comprising (i) examining presence or absence of a T.beta.RI ICD in the nuclei of cancer cells in a tissue sample of a cancer patient, and (ii) assessing invasiveness of the cancer cells. Nuclear localization of the ICD indicates that the cancer cells are invasive. Accordingly, it is predicted that cancer metastasis is likely to occur in the patient. This method can further comprise a step of determining a treatment choice for the patient based on the result obtained from the assessing step. The prognostic methods described herein is applicable to various cancers, including, but are not limited to, prostate cancer, renal carcinoma, bladder carcinoma, breast cancer, lung cancer, or colorectal cancer.

[0015] The present disclosure also provides a method for identifying a T.beta.RI cleavage inhibitor. The method comprises (i) contacting a cancer cell that expresses a T.beta.RI with a candidate compound in the presence of TGF.beta., which induces T.beta.RI cleavage, (ii) examining presence or absence of an ICD in the nuclear of the cancer cell, and (iii) assessing whether the candidate compound is a T.beta.RI cleavage inhibitor. The candidate compound is identified as a T.beta.RI cleavage inhibitor if (i) the level of ICD nuclear localization in the cell treated with the candidate compound is lower than that in a cell not treated with the candidate compound, or (ii) the level of an extracellular domain of the T.beta.RI in the culture medium of the cell treated with the candidate compound is lower than that in the culture medium of a cell not treated with the candidate compound. In one example, this method is performed in a high-throughput format.

[0016] Also within the scope of this disclosure are (a) pharmaceutical compositions for use in inhibiting T.beta.RI cleavage and thus reducing cancer cell invasiveness, the composition comprising any of the T.beta.RI cleavage inhibitors described herein or a combination thereof, and a pharmaceutically acceptable carrier, and (b) use of the T.beta.RI cleavage inhibitors in manufacturing medicaments for the therapeutical uses described herein. Optionally, the pharmaceutical compositions are free of MMP14 inhibitors.

[0017] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed descriptions of two examples and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings are first described.

[0019] FIG. 1 is a graph showing that TGF.beta. induces nuclear accumulation of the T.beta.RI intracellular domain (T.beta.RI-ICD). Panels a and b: knockdown of T.beta.RI by siRNA in PC-3U cells to demonstrate specificity of antibodies V22 and H100, which is specific to the C-terminal fragment and N-terminal fragment of T.beta.RI, respectively. In panel b, .beta.-actin served as a loading control. RNA was isolated from untreated or siRNA treated cells 24, 48, and 72 h after transfection. The T.beta.RI mRNA was measured by qRT-PCR (mean.+-.s.e.m., n=3 independent experiments). Panel c: Cell lysates of PC-3U cells treated with TGF.beta. as indicated were fractionated to produce the cytoplasmic and nuclear protein fractions, which were subjected to SDS-gel electrophoresis. Gels were immunoblotted and probed with the V22 and H100 antibodies. .beta.-tubulin and lamin A served as loading controls for the cytoplasmic and nuclear fractions, respectively. Panel d: Representative confocal microscopy images of C-terminally GFP-tagged wt T.beta.RI (pT.beta.RI-EGFP) and the pEGFP-N3-vector, which were expressed in PC-3U cells treated with TGF.beta. for 6 h (left). 3-5 independent experiments were performed. Cell nuclei were stained with DAPI. The percent of cells having T.beta.RI nuclear accumulation was quantified (mean.+-.s.d., n=350 cells); scale bar 20 .mu.m (right).

[0020] FIG. 2 is a graph showing that TRAF6 promotes Lys63-dependent polyubiquitination of wt T.beta.RI, but not the T.beta.RI E161A mutant. Panel a: PC-3U cells transiently transfected with C-terminally HA-tagged wt T.beta.RI (HA-ca T.beta.RI), or the T.beta.RI E161A mutant (HA-E161A) were treated with or without TGF.beta.. Ubiquitination of T.beta.RI was examined by an in vivo ubiquitination assay. A fraction of cell lysates was boiled in 1% SDS for 10 min to disrupt non-covalent protein-protein interactions, then diluted (1:10) in buffer. The proteins were immunoprecipitated (IP) with an anti-HA antibody and immunoblotted. Immunoblots (IB) were probed with antibodies specific for Lys63 (K63) linked polyubiquitin. A light-chain specific antibody (IgG L.C.) was used to avoid cross-reaction with the IgG heavy chain. The IP-filter was reblotted with HA to verify equal expression levels of wt T.beta.RI and the E161A mutant. Another portion of the total cell lysates (TCL) was immunoblotted with an anti-HA antibody to detect the T.beta.RI-ICD fragment. Phospho-specific antibodies were used to test whether transcription factors p38 and Smad2 were activated by phosphorylation (p-p38, and p-Smad2, respectively). Panel b: In vivo ubiquitination assays were performed in PC-3U cells transiently transfected with HA-tagged wt ubiquitin and K63 or K48 ubiquitin mutants. Cell lysates were immunoprecipitated with the V22 antibody. K63-dependent polyubiquitination was visualised by probing immunoblots with the P4D1-antisera. An IgG L.C. antibody was used to avoid cross-reaction with the IgG heavy chain. The IP-filter was reblotted with T.beta.RI to verify equal expression of T.beta.RI. The TCL-filter was subjected to immunoblotting with HA-antisera to verify equal expression the wild-type ubiquitin and the mutants. Panel c: PC-3U cells ectopically expressing HA-ca wt T.beta.RI or the E161A mutant were stimulated with TGF.beta. for 30 min and then stained with an anti-HA antibody (red). Staining with DAPI (blue) was used to visualise cell nuclei (scale bar 20 .mu.m).

[0021] FIG. 3 is a graph showing that TACE regulates nuclear accumulation of the T.beta.RI intracellular domain (ICD). Panel a, left portion: PC-3U cells were treated with TPA for 6 h and prepared for immunofluorescence imaging. Endogenous T.beta.RI was visualised using the V22 antibody (red). Cell nuclei were detected by DAPI (blue) staining. Scale bar 20 .mu.m. Panel a, right portion: Quantification of the percent of cells that showed endogenous T.beta.RI in the nucleus (mean.+-.s.e.m., n=3 independent experiment, where N=200-300 cells counted in each group). Panel b: Cell lysates from PC-3U cells transiently transfected with C-terminally HA-tagged T.beta.RI (HA-ca T.beta.RI) were subjected to immunoprecipitation with an anti-HA antibody. The T.beta.RI-ICD was visualised by immunoblotting with the anti-HA antibody. The membrane was then stripped and reblotted with the VPN antiserum, which is specific to the ICD. Panel c, left portion: PC-3U cells were treated with TGF.beta. alone, TPA alone, or both TGF.beta. and TAPI-2 for 6 h. Endogenous T.beta.RI was visualised by immunofluorescence using the V22 antibody (red). DAPI (blue) staining was applied to detect cell nuclei. Scale bar 20 .mu.m. Panel c, right portion: Quantification of the percent of cells that showed endogenous T.beta.RI in the nucleus (mean.+-.s.e.m., n=3 independent experiments, where N=200-300 cells as counted from each group). Panel d: Cell lysates of PC-3U cells were treated with TGF.beta. in the presence or absence of TAPI-2 as indicated. Cell lysates were fractionated into cytoplasmic and nuclear proteins and subjected to SDS-gel electrophoresis. Gels were immunoblotted and probed with the V22 antibody. .beta.-tubulin and lamin A served as loading controls for the cytoplasmic and nuclear fractions, respectively. Panel e: PC-3U cells were treated with or without TGF.beta. for 30 min as indicated. Endogenous TACE and T.beta.RI are visualised by co-immunofluorescence with an anti-TACE antibody (TRITC-labeled, red) and the V22 antibody (FITC-labeled, green). Their colocalization is shown by the yellow color, as shown in merged images. The images on the left are enlarged from the areas enclosed in white boxes in the images to the right. DAPI staining was performed to detect cell nuclei (blue).

[0022] FIG. 4 is a graph showing the Identification of TGF.beta. induced TACE cleavage site in the extracellular domain of T.beta.RI. Panel a: Total cell lysates, derived from PC-3U cells transiently transfected with wt T.beta.RI or the G120I mutant and stimulated with TGF.beta. as indicated, were immunoblotted with an anti-HA antibody to detect the T.beta.RI-ICD fragment (the filter was reprobed with .beta.-actin antibodies to show equal loading of proteins in all lanes). Phospho-specific antibodies were used to examine whether transcription factors p38 and Smad2 were activated by phosphorylation (p-p38, and p-Smad2, respectively). Panel b: PC-3U cells ectopically expressing HA-ca wt T.beta.RI or the G120I mutant were stimulated with TGF.beta. for 30 min and then stained with an anti-HA antibody (red). DAPI (blue) staining was performed to detect cell nuclei. Scale bar 20 .mu.m.

[0023] FIG. 5 is a graph showing that PKC.zeta. promotes nuclear accumulation of T.beta.RI. Panel a: Endogenous T.beta.RI in PC-3U cells treated with TGF.beta. was examined using the V22 antibody via Immunofluorescence in the presence or absence of a PKC.zeta. pseudosubstrate, which inhibits PKC.zeta.. Staining with DAPI was used to visualise cell nuclei (blue). Scale bar 20 .mu.m. Panel b: Cell lysates from PC-3U cells treated with TGF.beta. or TPA were subjected to immunoblotting with an antiserum against pPKC.zeta. to detect phosphorylated PKC.zeta. (p-PKC.zeta.). Total cell lysates from cells transiently transfected with anti-PKC.zeta. siRNA served as a negative control and cells treated with 10% FBS served as a positive control. The filter was reprobed with an anti-PKC.zeta. antibody to show equal loading of proteins in all lanes. Panel c: Cell lysates from wild-type MEFs and TRAF6-/- MEFs were subjected to immunoblotting with antibodies specific for p-PKC.zeta.. The filter was reprobed with antibodies specific to PKC.zeta., TRAF6 and .beta.-actin to show, respectively, specificity, knock down of TRAF6 by siRNA, and equal loading of proteins in all lanes. Panel d: PC-3U cells were transiently transfected with siRNA to silence endogenous PKC.zeta.. The transfected cells were treated with TGF.beta., subjected to cell fractionation followed by SDS-gel electrophoresis. Samples were immunoblotted to investigate the subcellular localization of endogenous full-length (FL) or the intracellular domain (ICD) of T.beta.RI. Lamin A and .beta.-tubulin served as controls for the nuclear and cytoplasmic fractions, respectively. Panels e and f: PC-3U cells were transiently transfected and treated as indicated, in the presence or absence of wt PKC.zeta.. The HA-KDT.beta.RI is a mutant T.beta.RI with abolished kinase activity. SB505124 is a T.beta.RI kinase inhibitor. Cell lysates were subjected to immunoblotting to detect presence of full-length T.beta.RI and T.beta.RI ICD. Panel g: PC-3U cells were treated with TGF.beta. with the PKC.zeta. pseudosubstrate. Endogenous TACE and T.beta.RI were visualised by immunofluorescence with an anti-TACE antibodies (TRITC-labeled, red) and the V22 antibody (FITC-labeled, green). Their co-localization is demonstrated by the yellow colour as shown in the merged images. The images in the top three rows are enlarged from the areas enclosed in white boxes in the images below. Staining with DAPI was used to visualise cell nuclei. Scale bar 20 .mu.m.

[0024] FIG. 6 is a graph showing that T.beta.RI promotes expression of Snail and invasion of prostate cancer cells in a TGF.beta.-dependent manner. Panel a: PC-3U cells were starved and treated with TGF.beta. as indicated. Endogenous T.beta.RI and p300 were visualised by immunofluorescence using the V22 antibody (TRITC-labeled, red) and an p300-specific antibody (FITC-labeled, green). Panel b: PC-3U cells were starved and treated with TGF.beta. in the absence or presence of the PKC.zeta. pseudosubstrate (p.s.) as indicated. Endogenous T.beta.RI and PML were shown by immunofluorescence using the V22 antibody (TRITC-labeled, red) and an anti-PML antibody (FITC-labeled, green). Panel c: TGF.beta. induces association between endogenous T.beta.RI and p300. Cell lysates from PC-3U cells treated with TGF.beta. were immunoprecipitated (IP) with the V22 antibody, subjected to SDS-gel electrophoresis, and immunoblotted (IB) with the anti-p300 antibody. Panel d: PC-3U cells were transiently transfected with HA-tagged wt (HA-ca) T.beta.RI or the E161A mutant and treated with TGF.beta. as indicated. Cell lysates were immunoprecipitated with the anti-p300 antibody, subjected to SDS-gel electrophoresis, and immunoblotted with the anti-HA antibody. Panel e: PC-3U cells were transiently transfected with HA-caT.beta.RI or the E161A mutant and treated with TGF.beta. as indicated. Cell lysates were immunoprecipitated with an antiserum against HA, subjected to SDS-gel electrophoresis, and immunoblotted with an antibody specific to acetylated lysine (AcK). Panels f and g: qRT-PCR analysis to quantifying the expression levels of p300, Snail-1, MMP2, PAI1, and Smad7, using mRNAs extracted from PC-3U cells that were transiently transfected with HA-caT.beta.RI or the E161A mutant and stimulated with TGF.beta. for different time periods. Error bars represent the s.e.m. n=3 independent experiments. Panel h: A chromatin immunoprecipitation assay to detect T.beta.RI association with the Snail promoter in PC-3U cells treated with TGF.beta.1. Immunoblots were probed with the V22 antibody against the endogenous T.beta.RI. Error bars represent s.d. (n=3 independent experiments). Panel i: An invasion assay on PC-3U cells transiently transfected with HA-caT.beta.RI or the E161A mutant and treated with TGF.beta. or EGF. Cells were visualised by staining with crystal violet cell stain solution. The chart at right represents mean values for optical density (OD) of invasive cells. Error bars represent s.d. (n=3 independent experiments). Panel j: Immunofluorescence staining to detect cytoskeletal reorganisation of actin (phalloidin probe, red) and subcellular localization of T.beta.RI (green) in TGF.beta.-treated primary prostate epithelial cells (PREC) and PC-3U cells. Staining with DAPI was used to visualise cell nuclei. Scale bar 20 .mu.m. Panel k: Quantification of the percent of cells in panel j that showed endogenous T.beta.RI in the nuclei (mean.+-.s.e.m., n=3 independent experiments, where N=200 cells as counted in each group).

[0025] FIG. 7 is a graph showing that endogenous T.beta.RI is localized in nuclei in human lung and breast carcinoma cells and is associated with tumor invasion. Panel a: Human lung (A549) and breast carcinoma (MDA-MB-231) cells were starved and treated with TGF.beta. in the presence or absence of the PKC.zeta. pseudosubstrate or TAPI-2 as indicated. Endogenous C-terminal T.beta.RI was detected by immunofluorescence using the V22 antibody (TRITC-labeled, red). Panels b and c: Invasion assays on MDA-MB231 and A549 cells treated with TGF.beta. in the absence or presence of TAPI-2 and the PKC.zeta. pseudosubstrate. Cells were visualised by staining with crystal violet cell stain solution. The right chart shows mean values of the optical density (OD) of invasive cells. Error bars represent s.d. (n=3 independent experiments).

[0026] FIG. 8 is a graph showing that endogenous T.beta.RI is localized in nuclei in various types of malignant tumors. Tumor tissues from prostate cancer, renal carcinoma, and bladder cancer were stained with the V22 and H100 antibodies.

[0027] FIG. 9 is a graph showing that TGF.beta. induces expression and activation of PS1, which promotes cleavage of T.beta.RI in human prostate cancer PC-3U cells. Panel a: Cell lysates derived from PC-3U cells were subjected to immunoblotting with an antibody that recognizes the PS1-holoprotein (PS1-FL; 45 kDa) and the PS1--C-terminal fragment (CTF) (18 kDa). Panel b: Cell lysates from PC-3U cells transiently transfected with Myc-PS1 at various amounts (0, 2, 4, and 6 .mu.g) and treated with TGF.beta. were subjected to immunoblotting to detect PS1. Panels c and d: Cell lysates derived from PC-3U cells transiently transfected with a PS1 specific siRNA (siPS1) or a non-targeting control siRNA (siCtrl) and treated with TGF.beta. were subjected to immunoblotting to detect PS1. Non-transfected cells (NT) were used as a blank control in panel c. PS1 mRNA levels, obtained from RT-PCR analyses, are shown in the lower part of panel c. Panel d: Immunoblotting for detecting T.beta.RI, p-p38, p38, p-Smad2 and Smad2 in total cell lysates (TCL) derived from PC-3U cells treated as indicated. Panel e: Cell lysates derived from wild type PS1 (.sup.+/.sup.+) and PS1 deficient (.sup.-/.sup.-) mouse embryonic fibroblast (MEFs) treated with TGF.beta. were subjected to immunoblotting to detect T.beta.RI, p-p38, p38, p-Smad2, and Smad2. Immunoblotting using an anti-p-actin antibody served as an internal control for equal loading of proteins in all lanes in panels d and e.

[0028] FIG. 10 is a graph showing that endogenous PS1 is associated with T.beta.RI. Panel a: PC-3U was treated with TGF.beta. and proteins in cell lysates were immunoprecipitated (IP) with an anti-PS1 antibody and immunoblotted with an anti-T.beta.RI antibody. A light-chain specific antibody (IgG L.C.) was used to avoid cross-reaction with the IgG heavy chain. The reverse IP was also conducted. The corresponding total cell lysates (TCL) were immunoblotted with antibodies specific to T.beta.RI and PS1. Panel b: PC-3U cells were stimulated with TGF.beta. as indicated and the cell lysates were co-immunoprecipitated with the anti-PS1 antibody and immunoblotted with the V22 antibody. An IgG L.C. antibody was used to avoid cross-reaction with the IgG heavy chain. The IP-filter was reblotted with the anti-PS1 antibody to verify the specificity of the PS1 antibody. The TCL-filter was subjected to immunoblotting with antisera against T.beta.RI and PS1. Panel c: PC-3U cells were treated with TGF.beta. as indicated and subjected to immunofluorescence and confocal imaging. Endogenous PS1 and T.beta.RI was visualised using the anti-PS1 antibody (green) and the V22 antibody (red). Staining with DAPI (blue) was used to visualise cell nuclei (blue, bottom panels). Arrow indicates co-localization of the proteins.

[0029] FIG. 11 is a graph showing that TGF.beta. promotes Lys63-dependent polyubiquitination of PS1 in in vivo ubiquitination assays. Panel a: PC-3U cells were treated with TGF.beta.. Ubiquitination of PS1 were examined with an in vivo ubiquitination assay as described in Sorrentino et al., 2008. Panel b: In vivo ubiquitination assays were performed in PC-3U cells transiently transfected with HA-tagged wt ubiquitin and the K63 or K48 mutant. Cell lysates were immunoprecipitated with an anti-PS1 antibody. Polyubiquitination was visualised by probing immunoblots with P4D1-antisera. An IgG L.C. antibody was used to avoid cross-reaction with the IgG heavy chain. Panel c: PC-3U cells were transiently transfected with a control siRNA or an anti-TRAF6 siRNA to silence the endogenous TRAF6. The cells were treated with TGF.beta.. The ubiquitination of PS1 was examined by an in vivo ubiquitination assay as described above.

[0030] FIG. 12 is graph showing that the association between TRAF6 and PS1 is dependent on the catalytic activity of TRAF6. Panel a: PC-3U cells were treated with or without TGF.beta.. Proteins were immunoprecipitated (IP) with an antibody specific to the N-terminus of PS1 and immunoblotted with an antibody specific to TRAF6. A light-chain specific antibody (IgG L.C.) was used to avoid cross-reaction with the IgG heavy chain. The corresponding total cell lysates were immunoblotted with antibodies specific to TRAF6 and PS1. The level of .beta.-Actin served as an internal control for equal loading of proteins. Panel b: PC-3U cells were transiently transfected with Flag-tagged wild type (wt) or C70A mutant TRAF6 and treated with TGF.beta.. Proteins were immunoprecipitated (IP) with an anti-Flag antibody and immunoblotted with an anti-PS1 antibody. A light-chain specific antibody (IgG L.C.) was used to avoid cross-reaction with the IgG heavy chain. The corresponding total cell lysates were immunoblotted with antibodies specific to Flag (TRAF6) and PS1. The level of .beta.-Actin served as an internal control for equal loading of proteins. Panel c: PC-3U cells were transiently transfected with Flag-tagged wild type (wt) or C70A mutant TRAF6 and treated with TGF.beta.. Proteins were immunoprecipitated (IP) with an anti-PS1 antibody and immunoblotted with antibodies specific to Flag. A light-chain specific antibody (IgG L.C.) was used to avoid cross-reaction with the IgG heavy chain. The corresponding total cell lysates were immunoblotted with antibodies specific for Flag (TRAF6) and PS1. The level of .beta.-Actin served as an internal control for equal loading of proteins.

[0031] FIG. 13 is a graph showing that TRAF6 is required for the association between T.beta.RI and PS1. Panel a: PC-3U cells were transiently transfected with a control siRNA (siCtrl) or an anti-TRAF6 siRNA (siTRAF6) to silence the endogenous TRAF6. The cells were treated with TGF.beta.. Proteins were immunoprecipitated (IP) with an anti-T.beta.RI antibody and immunoblotted with an anti-PS1 antibody. A light-chain specific antibody (IgG L.C.) was used to avoid cross-reaction with the IgG heavy chain. The corresponding total cell lysates were immunoblotted with an antibody specific to TRAF6. The level of .beta.-Actin served as an internal control for equal loading of proteins. Panel b: PC-3U cells were transiently transfected with HA-tagged constitutively active (ca) T.beta.RI or the E161A mutant T.beta.RI and treated with TGF.beta.. Proteins were immunoprecipitated (IP) with an anti-PS1 antibody and immunoblotted with antibodies specific to HA. A light-chain specific antibody (IgG L.C.) was used to avoid cross-reaction with the IgG heavy chain. The corresponding total cell lysates were immunoblotted with antibodies specific to HA (T.beta.RI) and PS1. The level of .beta.-Actin served as an internal control for equal loading of proteins.

[0032] FIG. 14 is a graph showing the identification of the PS1 cleavage site in T.beta.RI. Panel a: PC-3U cells were transiently transfected with HA-tagged constitutively active (ca) or the V129A/I130A T.beta.RI mutant and treated with TGF.beta.. Non transfected cells (NT) served as a blank control. A fraction of the proteins was extracted from the total cell lysate and immunoblotted with an anti-HA antibody (T.beta.RI). The level of .beta.-Actin served as an internal control for equal loading of proteins. Another fraction of proteins derived from the total cell lysate was immunoprecipitated (IP) with an anti-PS1 antibody and immunoblotted with an antibody specific to HA to investigate the association of PS1 to the ectopically expressed T.beta.RI. A light-chain specific antibody (IgG L.C.) was used to avoid cross-reaction with the IgG heavy chain. Panel b: 293T cells were transiently transfected with HA-tagged constitutively active (ca) or the V129A/I130A T.beta.RI mutant and treated with TGF.beta.. Total cell lysates (TCL) were subjected to immunoblotting with the anti-HA antibody (T.beta.RI). The importance of TGF.beta.-induced cleavage of T.beta.RI in activating the Smad2 and p38 pathways was examined by immunoblotting of TCL to detect p-Smad2/Smad2 and p-p38/p38. The level of .beta.-Actin served as an internal control for equal loading of proteins. Panel c: PC-3U cells were transiently transfected with HA-tagged constitutively active (ca) or the V129A/I130A T.beta.RI mutant and treated with TGF.beta. as indicated. The cells were then subjected to immuno-fluorescence and confocal imaging. HA-tagged constitutively active (ca) and the mutant T.beta.RI were detected using the anti-HA antibody (red). Staining with DAPI (blue) was used to visualise cell nuclei. Panel d: a chart showing the level of T.beta.RI ICD that binds to T.beta.RI promoter as obtained in a ChIP assays.

[0033] FIG. 15 is a graph showing cleavage of the TGF.beta. type I receptor (T.beta.RI). Panel a: PC-3U cells were transiently transfected with various amounts of C-terminally tagged HA-T.beta.RI (HA-ca T.beta.RI). The full-length (FL) T.beta.RI migrated at a position corresponding to the molecular weight of 53 kDa in SDS-gel electrophoresis and a T.beta.RI intracellular fragment (ICD) migrated at a position corresponding to the molecular weight of 37 kDa (the asterisk indicates a background band). Panel b: Cell lysates from PC-3U cells transiently transfected with HA-ca T.beta.RI and treated with TGF.beta. as indicated and fractionated for produce cytoplasmic and nuclear protein fractions, which were subjected to SDS-gel electrophoresis. Gels were immunoblotted and probed with an anti-HA antibody. .beta.-tubulin and lamin A served as loading controls for the cytoplasmic and nuclear fractions, respectively. Panel c, left portion: The nuclear localization of T.beta.RI was detected with the V22 antibody. PC-3U cells were treated with TGF.beta. for 30 minutes. Control cells (left) were stained with the V22 antibody to visualise endogenous T.beta.RI (green). To demonstrate its specificity, the V22 antibody was incubated with the peptide used for immunization for 1 h at room temperature before adding it to the cells (right; peptide blocking). Panel c, right portion: PC-3U cells ectopically expressing HA-ca T.beta.RI or the N-terminal HA-tagged T.beta.RI were stimulated with TGF.beta. for 30 min and then stained with an anti-HA antibody (red). Staining with DAPI (blue) was used to visualise cell nuclei. Scale bar 20 .mu.m.

[0034] FIG. 16 is a graph showing that TRAF6 promotes TGF.beta.-induced Lys63-linked polyubiquitination and cleavage of T.beta.RI. Panel a: PC-3U cells were transiently transfected with a control siRNA or an anti-TRAF6 siRNA to silence the endogenous TRAF6. The cells were treated with TGF.beta.. The ubiquitination of T.beta.RI was examined by an in vivo ubiquitination assay described in Sorrentino et al., 2008. Panel b: PC3U cells were transiently transfected with the control siRNA or the anti-TRAF6 siRNA and then treated with TGF.beta., as indicated. Cell lysates were immunoblotted and probed with the V22 antibody to detect endogenous T.beta.RI (full length). Panel c: PC-3U cells were transiently transfected with C-terminally HA-tagged wt T.beta.RI, or the T.beta.RI E161A mutant. Cells were treated with TGF.beta., and ubiquitination of T.beta.RI was examined with an in vivo ubiquitination assay. The IP-filter was reblotted with HA to verify equal expression levels of wt and E161A mutant T.beta.RI.

[0035] FIG. 17 is a graph showing that TRAF6 acts with the ubiquitin-conjugating enzyme, Ubc13-Uev1A, to promote the polyubiquitination of T.beta.RI in vitro. A GST-T.beta.RI intracellular domain fusion protein were incubated in the presence or absence of a recombinant glutathione-S-transferase-tagged TRAF6 (GST-TRAF6) fusion protein (approximately 0.1 .mu.g at maximum concentration) in a reaction mixture containing 20 mM Tris, pH 7.4, 50 mM NaCl, 10 mM MgCl.sub.2, 10 mM dithiothreitol, 10 mM ATP, 0.5 .mu.g .mu.l-1 ubiquitin (Sigma), 2 .mu.M ubiquitin aldehyde (BIOMOL), 100 .mu.M MG132 (Sigma), 0.1 .mu.g E1 (human recombinant from Biomol), 0.2 .mu.g E2 Ubc13-Uev1A (Biomol) at 30.degree. C. for 1 h, then subjected to SDS-PAGE. After incubation at 30.degree. C. for 1 h, the reaction products were immunoblotted (IB) and probed with antibodies against T.beta.RI and TRAF6 to examine the polyubiquitination of T.beta.RI.

[0036] FIG. 18 is a graph showing that PKC.zeta. promotes nuclear accumulation of T.beta.RI. Top panel: PC-3U cells were treated with TGF.beta. for 6 h and stained for endogenous T.beta.RI in the presence or absence of various amounts of the PKC.zeta. pseudosubstrate. The subcellular localization of the endogenous T.beta.RI was visualized with the V22 antibody (green, top row). The nuclei were stained with DAPI (blue) and the phalloidin stain (red) indicates the cytoplasm. Bottom panel: Quantification of the number of cells shows that the PKC.zeta. pseudosubstrate (p.s.) blocked the PKC.zeta.-induced translocation of endogenous T.beta.RI into the nuclei (mean.+-.s.e.m., n=3 independent experiments where 200-300 cells were counted).

[0037] FIG. 19 is a graph showing that T.beta.RI promotes invasion of human cancer cells. Panel a: Invasion assay for human prostate cancer LNCaP cells, which were transiently transfected with the HA-tagged wt (HA-ca T.beta.RI; left) or the E161A mutant (HA-E161A; right) and treated with TGF.beta.s indicated. Cells were visualiszed by staining with crystal violet cell stain solution. Panel b: Quantification of invasive LnCaP cells transiently transfected with wt or E161A mutant T.beta.RI and treated with TGF.beta.. Invasive cell density was measured by optical density at 560 nm in the invasion assays, (mean.+-.s.d., n=3 independent experiments). P<0.01 by Student's t test. Panel c: Invasiveness of human breast cancer MB 231 cells in response to TGF.beta. and reduction of cancer cell invasion by TACE and PKC.zeta. inhibitors. Panel d: Invasiveness of human lung cancer A549 cells in response to TGF.beta. and reduction of cancer cell invasion by TACE and PKC.zeta. inhibitors.

[0038] FIG. 20 is a graph showing that the levels of Smads do not influence PKC.zeta.-dependent generation of T.beta.RI-ICD. Panel a: PC3U cells were transiently transfected with a control siRNA and an anti-Smad4 siRNA, pcDNA3, C-terminally HA-tagged T.beta.RI, alone or together with PKC.zeta. as indicated. Cell lysates were immunoblotted and probed with an anti-HA antibody to detect T.beta.RI-ICD. Total cell lysates were also subjected immunoblotting with anti-Smad4 and anti-PKC.zeta. antibodies (full length T.beta.RI=T.beta.RI-FL). One of the filters was stripped and reprobed with an anti-.beta.-actin antibody to serve as a control for equal loading of proteins. Panel b: PC3U cells were transiently transfected with C-terminally HA-tagged T.beta.RI alone or together with Smad2, 3, 4, and PKC.zeta. as indicated. Cell lysates were immunoblotted and probed with the anti-HA antibody to detect T.beta.RI-ICD. Total cell lysates were subjected immunoblotting using antibodies against Smad2, Smad3, Smad4 and PKC.zeta..

[0039] FIG. 21 shows negative controls of TbRI immunohistochemical stainings in prostate cancer tissues, using the V22 and H100 antibodies.

[0040] FIG. 22 shows that TGF.beta. induces expression and activation of PS1 which promotes cleavage of T.beta.RI in human prostate cancer (PC-3U) cells. Panel A: a graph showing levels of PS1-holoprotein (PS1-FL; 45 kDa) and the PS1-C-terminal fragment (CTF) (18 kDa) in cell lysates derived from PC-3U cells by an immunoblotting assay using an antibody that recognizes the PS1-holoprotein. Endogenous expression of PS1-FL was enhanced by TGF.beta. and PS1 CTF (a band of 18 kDa) was observed after TGF.beta. treatment for 0.15 h. Panel B: a graph showing levels of PS-1 and the N-terminal fragment of PS1 (PS1-NTF) in cell lysates from PC-3U cells, which were transiently transfected with Myc-PS1 at various amounts (0, 2, 4, 6 .mu.g) in the presence or absence of TGF.beta., as determined by an immunoblotting assay. TGF.beta. was observed to induce expression of both PS1--FL and PS1--NTF, migrating at 32 kDa. Note also the TGF.beta.-induced increased smear on top of PS1-FL. Panel C: a graph showing levels of PS1, as determined by an immunoblotting assay, in cell lysates derived from PC-3U cells transiently transfected with PS1 specific (siPS1) or non-targeting control siRNA (siCtrl), in the presence or absence of TGF.beta.. Panel D: a chart showing the levels of PS1 mRNA in the PC-3U cells as described above via RT-PCR analyses. Panel E: a graph showing levels of T.beta.RI and PS1 in PC-3U cells treated as indicated above via immunoblotting. Decrease of T.beta.RI-ICD was observed in cells transfected with siPS1. Panel F: a graph showing levels of T.beta.RI (full-length and ICD) and PS1, determined by immunoblotting, in cell lysates derived from wild type PS1 (.sup.+/.sup.+) and PS1 deficient (.sup.-/.sup.-) mouse embryonic fibroblast (MEFs) treated or not treated with TGF.beta.. Panel G: a graph showing the levels of T.beta.RI (full-length and ICD) and PS1, determined by immunoblotting, in cell lysates derived from PC-3U cells transiently transfected with PS1 specific (siPS1) or a non-targeting control siRNA (siCtrl), or re-transfected with Myc-PS1 in the presence or absence of TGF.beta.. Panel H: a graph showing the levels of T.beta.RI (full-length and ICD) and PS1 (Myc-tagged), determined by immunoblotting, in cell lysates derived from wild type PS1 (.sup.+/.sup.+) and PS1 deficient (.sup.-/.sup.-) MEFs, or re-transfected with Myc-PS1, in the presence or absence of TGF.beta.. Immunoblotting for .beta.-actin served as internal control for equal loading of proteins in all lanes in A, B, C, E, F and G.

[0041] FIG. 23 shows Identification of the PS1 cleavage site in T.beta.RI. Panel A: a schematic illustration showing in silico identification of a cleavage site for PS1 in the transmembrane domain of T.beta.RI. Panel B: a graph showing levels of various proteins in PC-3U cells transiently transfected with HA-tagged constitutively (ca) active or TM-mutant T.beta.RI in the presence or absence of TGF.beta., as determined by an HA-specific antibody or antibodies specific to p-Smad2, Smad2, p-p38, p38, and actin. Non-transfected cells (NT) served as control for HA-blots. A fraction of the proteins was extracted as total cell lysates and immunoblotted with antibodies specific for HA (T.beta.RI) (upper panel). Actin served as an internal control for equal loading of proteins. Another fraction of proteins was immunoprecipitated (IP) with anti-PS1 antibodies and immunoblotted with antibodies specific for HA to investigate the association between PS1 and ectopically expressed T.beta.RI. A light-chain specific antibody (IgG L.C.) was used to avoid cross-reaction with the IgG heavy chain. See the bottom panel. Panel C: a photo showing the levels of various proteins in 293T cells transiently transfected and treated as indicated above. Total cell lysates (TCL) were subjected to immunoblotting with HA-specific antibodies specific to detect HA-tagged T.beta.RI (upper panel). TGF.beta.-induced formation of T.beta.RI-ICD was reduced in cells transfected with the TM-T.beta.RI mutant. The importance of TGF.beta.-induced cleavage of T.beta.RI for activation of Smad2 and p38 pathways was examined by immunoblotting of TCL to detect the levels of p-Smad2/Smad2 and p-p38/p38. Actin served as an internal control for equal loading of proteins. Panel D: a photo showing presence of T.beta.RI in PC-3U cells transfected and treated as indicated above. The cells were then subjected to immuno-fluorescence and confocal imaging. HA-tagged constitutively active (ca) and mutant T.beta.RI were visualised with HA-antibodies (red). Staining with DAPI (blue) was used to visualize cell nuclei. Panel E: a chart showing the levels of T.beta.RI in PC-3U cells transiently transfected and treated as indicated above via qRT-PCR analysis. Panel F: a chart showing the levels of promoter-binding T.beta.RI in PC-3U cells treated or not treated with TGF.beta. via a chromatin immunoprecipitation assay, using V22 antibody against the endogenous T.beta.RI. Panel G: a graph showing cell invasion levels of PC-3U cells transfected and treated as indicated above by an invasion assay as described herein. Cells were visualized by staining with crystal violet cell stain solution (left panel). The right panel presents mean values for optical density (OD) of invasive cells. *P<0.05, **P<0.005 and P<0.001(ANOVA).

[0042] FIG. 24 shows suppression of TGF.beta.-induced cancer cell invation by of .gamma.-secretase inhibitors. Panels A and B: photos/chart showing the invasion levels of PC-3U, human lung carcinoma cells (A549), and human breast carcinoma (MDA-MB-231) cells treated with TGF.beta. in the absence or presence of a .gamma.-secretase inhibitor. Cells were visualized by staining with crystal violet cell stain solution. The right part and bottom parts of A and B present mean values for optical density (OD) of invasive cells. Error bars represents mean.+-.s.d. (n=3 independent experiments; *P<0.05, **P<0.005, ANOVA).

[0043] FIG. 25 shows inhibition of cancer cell invasion by antibodies specific to a TACE-cleavage site in T.beta.RI, which encompass. Panel A: a photo showing invasion levels of PC-3U cells in the presence of ALK5 Ab 114-124 or ALK Ab 490-503. Control cells were not treated with antibodies. Cells were visualized by staining with crystal violet cell stain solution. Panel B: a chart presenting mean values for optical density (OD) of invasive cells. Data in the figure are representative of three independent experiments (mean and s.d). *P>0.5 and **P<0.02.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Described herein are methods and compositions for reducing cancer cell invasiveness/cancer metastasis by blocking T.beta.RI cleavage, methods for identifying agents capable of blocking T.beta.RI cleavage, and methods for diagnosing or prognosing cancer based on presence/absence of nuclear localization of T.beta.RI ICD, a product of T.beta.RI cleavage.

(I) INHIBITION OF T.beta.RI CLEAVAGE AND REDUCTION OF CANCER INVASIVENESS/METASTASIS

[0045] It is disclosed herein that cleavage of T.beta.RI in response to TGF.beta. stimulation releases an intracellular domain (ICD) of the T.beta.RI, which subsequently translocates to the nuclei and regulates expression of genes, some of which are essential to cancer cell invasiveness. Accordingly, blocking T.beta.RI cleavage and ICD nuclear translocation would be effective in reducing cancer cell invasion, leading to inhibiting cancer metastasis or lowering the risk of cancer metastasis.

[0046] Thus, one aspect of the present disclosure relates to methods for inhibiting T.beta.RI cleavage and reducing cancer cell invasiveness by either inhibiting protein factors involved in this cleavage process or blocking the cleavage site(s) in T.beta.RI, thereby disrupting the interaction between T.beta.RI and a cleavage enzyme at the corresponding cleavage site.

(a) Inhibition of Proteins Involved in T.beta.RI Cleavage

[0047] It is disclosed herein that a number of proteins, including TRAF6, TACE, PKC.zeta., and PS1, are involved in T.beta.RI ubiquitination or the subsequent cleavage. For example, TACE has recently been shown to cleave T.beta.RI in its extracellular domain, causing a loss of TGF.beta.-induced inhibition of cell proliferation. Huovila et al., Trends Biochem. Sci. 30:413-422; 2005. Thus, one or more of these proteins can be targeted in the methods disclosed herein.

[0048] All of the above-listed proteins are well known in the art. As examples, the Genbank accession numbers/Gene IDs for human TRAF6, TACE, PKC.zeta., and PS1 are listed below:

[0049] TRAF6: Gene ID: 7189, GenBank accession number NM.sub.--145803,

[0050] TACE: Gene ID: 6868, GenBank accession number U69611,

[0051] PKC.zeta.: Gene ID: 5590, GenBank accession number Q05513, and

[0052] PS1: Gene ID: 5663; GenBank accession number AAC97960.

[0053] Inhibitors of the proteins listed above can be used to reduce T.beta.RI cleavage, including, but are not limited to, antibodies specific to the protein targets, interfering nucleic acids that silence expression of these targets, pseudosubstrates, decoy (inhibitory peptides), and small molecule inhibitors.

Antibodies

[0054] Antibodies capable of binding to TRAF6, TACE, PKC.zeta., and PS1 and neutralizing their activities can be used in the methods described herein. Such an antibody can be a full-length antibody or an antigen-binding fragment thereof, e.g., F(ab').sub.2, Fab, or Fv. Such an antibody can be naturally-occurring or genetically engineered, e.g., a humanized antibody, a chimeric antibody, a single-chain antibody, a domain antibody, or a single variable domain (e.g., VH, VL or VHH) or multi-valent or multi-specific constructs made therefrom, or an antibody isolated from an antibody library. A naturally-occurring antibody can be obtained from any suitable species, such as human, rabbit, mouse, guinea pig, and rat, and can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

[0055] Naturally-occurring antibodies against TRAF6, TACE, PKC.zeta., and PS1, either polyclonal or monoclonal, can be prepared by conventional methods, using these proteins, or fragments thereof as antigens. See, e.g., Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. A monoclonal antibody refers to a homogenous antibody population and a polyclonal antibody refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

[0056] To produce the above-mentioned antibodies, the protein targets or fragments thereof can be (optionally) coupled to a carrier protein, such as KLH, mixed with an adjuvant, and injected into a host animal. Antibodies produced in the animal can then be purified by a protein A column and/or by peptide affinity chromatography. Commonly employed host animals include, but are not limited to, rabbits, mice, guinea pigs, and rats. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, CpG, surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Useful human adjuvants include BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0057] Polyclonal antibodies are present in the sera of the immunized subjects. Monoclonal antibodies can be prepared using standard hybridoma technology (see, for example, Kohler et al. (1975) Nature 256, 495; Kohler et al. (1976) Eur. J. Immunol. 6, 511; Kohler et al. (1976) Eur J Immunol 6, 292; and Hammerling et al. (1981) Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y.). In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described in Kohler et al. (1975) Nature 256, 495 and U.S. Pat. No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al. (1983) Immunol Today 4, 72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2026, and the EBV-hybridoma technique (Cole et al. (1983) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the monoclonal antibodies of the invention may be cultivated in vitro or in vivo. The ability to produce high titers of monoclonal antibodies in vivo makes it a particularly useful method of production. After obtaining antibodies specific to the protein targets, their ability to neutralize the activities of these proteins can be determined by routine procedures.

[0058] Fully human antibodies capable of binding to TRAF6, TACE, PKC.zeta., and PS1, such as those expressed in transgenic animals are also features of the invention. See, e.g., Green et al., Nature Genetics 7:13 (1994), and U.S. Pat. Nos. 5,545,806 and 5,569,825. Fully human antibodies can also be identified by screening human antibody libraries following routine procedures.

[0059] Antigen-binding fragments (e.g., F(ab').sub.2, Fab, or Fv) of naturally-occurring antibodies can be generated by known techniques. For example, F(ab').sub.2 fragments can be produced by pepsin digestion of an antibody molecule and Fab fragments can be generated by reducing the disulfide bridges of F(ab').sub.2 fragments.

[0060] The antibodies to be used in the methods disclosed herein can also be a genetically engineered antibody, e.g., a humanized antibody, a chimeric antibody, a single chain antibody (scFv), or a domain antibody (dAb; see Ward, et al., 1989, Nature, 341:544-546).

[0061] A humanized antibody contains a human immunoglobulin (i.e., recipient antibody) in which regions/residues responsible for antigen binding (i.e., the CDRs, particularly the specificity-determining residues therein) are replaced with those from a non-human immunoglobulin (i.e., donor antibody). In some instances, one or more residues inside a frame region of the recipient antibody are also replaced with those from the donor antibody. A humanized antibody may also contain residues from neither the recipient antibody nor the donor antibody. These residues are included to further refine and optimize antibody performance. Antibodies can also be humanized by methods known in the art, e.g., recombinant technology.

[0062] A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Such an antibody can be prepared via routine techniques described in, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.

[0063] A single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a V.sub.H chain and a nucleotide sequence coding for a V.sub.L chain. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage scFv library and scFv clones specific to any of the target proteins disclosed herein can be identified from the library following routine procedures. Positive clones can be subjected to further screening to identify those that inhibit the activity of TRAF6, TACE, PKC.zeta., or PS1.

Short Interfering Nucleic Acids

[0064] Another family of inhibitory agents to be used in the methods disclosed herein are short interfering nucleic acids (e.g., RNAs) that target TRAF6, TACE, PKC.zeta., or PS1. These short interfering nucleic acids are oligonucleotides at least a portion of which is complementary (i.e., completely or partially) to a fragment of the nucleic acid coding for any of the protein targets (either the sense chain or the antisense chain), i.e., capable of forming a double-strand duplex via base-pairing according to the standard Watson-Crick complementarity rules. They suppress expression of these protein targets via RNA silencing, i.e., mediating RNA transcript cleavage/degradation or translational repression of the target messenger RNA (mRNA).

[0065] Short interfering nucleic acids include, but are not limited to short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of silencing the expression of the target genes. These nucleic acid molecules can be prepared by chemical synthesis or expressed from a vector via routine recombinant technology. They can be unmodified or chemically-modified. The use of chemically-modified siNA improves various properties of native siNA molecules through, for example, increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Furthermore, chemical modifications can help the interfering nucleic acids in retaining their RNAi activity. For example, in some cases, siRNAs are modified to alter potency, target affinity, the safety profile and/or the stability to render them resistant or partially resistant to intracellular degradation. Modifications, such as phosphorothioates, for example, can be made to siRNAs to increase resistance to nuclease degradation, binding affinity and/or uptake. In addition, hydrophobization and bioconjugation enhances siRNA delivery and targeting (De Paula et al., RNA. 13(4):431-56, 2007) and siRNAs with ribo-difluorotoluoyl nucleotides maintain gene silencing activity (Xia et al., ASC Chem. Biol. 1(3):176-83, (2006). siRNAs with amide-linked oligoribonucleosides have been generated that are more resistant to S1 nuclease degradation (Iwase R et al. 2006 Nucleic Acids Symp Ser 50: 175-176). In addition, modification of siRNA at the 2'-sugar position and phosphodiester linkage confers improved serum stability without loss of efficacy (Choung et al., Biochem. Biophys. Res. Commun. 342(3):919-26, 2006). In one study, 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA)-containing antisense oligonucleotides compared favourably to phosphorothioate oligonucleotides, 2'-O-methyl-RNA/DNA chimeric oligonucleotides and siRNAs in terms of suppression potency and resistance to degradation (Ferrari N et al. 2006 Ann N Y Acad Sci 1082: 91-102).

[0066] In some embodiments an siNA is an shRNA molecule encoded by and expressed from a genomically integrated transgene or a plasmid-based expression vector. Thus, in some embodiments a molecule capable of inhibiting gene expression is a transgene or plasmid-based expression vector that encodes a small-interfering nucleic acid. Such transgenes and expression vectors can employ either polymerase II or polymerase III promoters to drive expression of these shRNAs and result in functional siRNAs in cells. The former polymerase permits the use of classic protein expression strategies, including inducible and tissue-specific expression systems. In some embodiments, transgenes and expression vectors are controlled by tissue specific promoters. In other embodiments transgenes and expression vectors are controlled by inducible promoters, such as tetracycline inducible expression systems. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, (Paddison et al., Genes Dev, 2002, 16:948-58; McCaffrey et al., Nature, 2002, 418:38-9; McManus et al., RNA 2002, 8:842-50; Yu et al., Proc Natl Acad Sci USA, 2002, 99:6047-52).

Inhibitory Peptides, Small Molecule Inhibitors, and Pseudosubstrates

[0067] Other inhibitors of TRAF6, TACE, PKC.zeta., and PS1, including inhibitory (decoy) peptides, pseudosubstrates, and small molecules, are well known in the art. Below are some examples.

[0068] Inhibitors of TRAF6 include TRAF6 inhibitory peptides (e.g., those provided by IMGENEX, San Diego, Calif. and disclosed in US 20050130896). Such inhibitory peptides can comprise a TRAF6 binding domain in T.beta.RI, which can be identified based on the consensus TRAF6 binding sequences as disclosed in Sorrentino et al., Nat. Cell Biol. 10(10):1199-1207 and in U.S. Patent Application No. 61/093,181, the content of which is herein incorporated by reference in its entirety.

[0069] Inhibitors of TACE include, but are not limited to, TAPI-1, TAPI-2, BMS-561392, DPC-333, Spiro-cyclic b-amino acid derivatives, INCB 3619, GW280264X, TMI-1, and TNF484, DPC-333, Sch-709156, and Doxycycline.

[0070] Inhibitors of PKC.zeta. include, but are not limited to, 2-(6-phenyl-1H-indazol-3-yl)-1H-benzol(d)imidazol, ethyl (5E)-2-acetylimino-5-[1-(hydroxyamino)ethylidene]-4-phenyl-thiophene-3-ca- rboxylate, and myristylated pseudosubstrates (see Example 1 below).

[0071] Inhibitors of PS1 include, but are not limited to, L685,458 (Hass et al., J. Biol. Chem. 280:9313-9319, 2005) and gamma-secretase inhibitors (e.g., LY-411,575, see US20110059114).

(b) Blockage of Cleavage Site in T.beta.RI

[0072] T.beta.RI is a receptor for TGF.beta.. There are two isoforms of this protein in humans. The GenBank accession numbers for these two isoforms are NM.sub.--004612.2 (mRNA) and NP.sub.--004603.1 (protein); and NM.sub.--001130916.1 (mRMA) and NP.sub.--001124388.1 (protein). The amino acid sequences of an exemplary human T.beta.RI is provided below:

TABLE-US-00001 Amino acid sequence of T.beta.RI isoform 1 (SEQ ID NO: 1): 1 meaavaaprp rllllvlaaa aaaaaallpg atalqcfchl ctkdnftcvt dglcfvsvte 61 ttdkvihnsm ciaeidlipr drpfvcapss ktgsvtttyc cnqdhcnkie lpttvksspG 121 LgpvelaaVI agpvcfvcis lmlmvyichn rtvihhrvpn eedpsldrpf isegttlkdl 181 iydmttsgsg sglpllvqrt iartivlqes igkgrfgevw rgkwrgeeva vkifssreer 241 swfreaeiyq tvmlrhenil gfiaadnkdn gtwtqlwlvs dyhehgslfd ylnrytvtve 301 gmiklalsta sglahlhmei vgtqgkpaia hrdlksknil vkkngtccia dlglavrhds 361 atdtidiapn hrvgtkryma pevlddsinm khfesfkrad iyamglvfwe iarrcsiggi 421 hedyqlpyyd lvpsdpsvee mrkvvceqkl rpnipnrwqs cealrvmaki mrecwyanga 481 arltalrikk tlsqlsqqeg ikm

[0073] As disclosed herein, at least two cleavage sites close to the transmembrane domain of T.beta.RI have been identified, i.e., the G.sub.120-L.sub.121 site and the V.sub.129-I.sub.130 site. See the bold-faced and capitalized residues in SEQ ID NO: 1. Cleavage at either site releases an ICD fragment, which can translocate into the nuclei to regulate gene expression. Thus, agents that bind to T.beta.RI and blocks a cleavage sites from being accessible to a cleavage enzyme can used in the methods disclosed herein.

[0074] In some embodiments, the above-noted agents are antibodies that bind to T.beta.RI and block its cleavage. Such antibodies can bind to any antigen epitope of T.beta.RI (linear or conformational) as long as its binding interferes with the association between the cleavage site in T.beta.RI and a cleavage enzyme. Anti-T.beta.RI antibodies described herein can be an antibody of any kind as known in the art, e.g., those described above. In some instances, these antibodies can be full-length antibodies or antigen-binding fragments, e.g., F(ab').sub.2, Fab, or Fv. In other instances, the antibodies can be naturally-occurring antibodies or genetically-engineered antibodies, e.g., a humanized antibody, a chimeric antibody, a single-chain antibody, a domain antibody, or a single variable domain (e.g., VH, VL or VHH) or multi-valent or multi-specific constructs made therefrom, or antibodies isolated from antibody libraries. Methods for preparation of these antibodies are well known in the art, some of which are exemplified above. In one example, such antibodies bind to epitopes in T.beta.RI that encompassing one or both of the residues in the G.sub.120-L.sub.121 and/or the V.sub.129-I.sub.130 site. Such epitopes can be either linear or conformational. The term "antigen epitope," also known as antigen determinant, refers to a specific portion of a macromolecular antigen to which an antigen binds. It can be either conformational or linear. Typically, a linear epitope is made up of about 6 amino acid residues.

[0075] To prepare these antibodies, the T.beta.RI protein, or a fragment thereof (e.g., fragments encompassing one or both residues in the cleavage sites noted above) can be used as an antigen to induce anti-T.beta.RI antibody production in a host system, following the conventional methods as described above. The resultant antibodies are then tested via routine procedures (e.g., see Example 1 below) for identify those that are capable of binding to T.beta.RI and blocking its cleavage. The naturally-occurring antibodies thus obtained can then used to produce genetically engineered antibodies, following the methods also described above.

[0076] In other embodiments, the blocking antibodies used in the methods disclosed herein are bispecific, i.e., containing one portion that binds to T.beta.RI and blocks its cleavage and another portion that binds to a protein involved in T.beta.RI ubiquitination and the subsequent cleavage (e.g., TRAF6, TACE, PS1, and PKC.zeta. zeta). Methods for preparing such bispecific antibodies are well known in the art.

[0077] Any of the antibodies described herein, including those that bind to T.beta.RI and block its cleavage, and those that bind to one or more of TRAF6, TACE, PS1, and PKC.zeta. zeta and block their activity, can be in isolated form. An isolated antibody refers to an antibody substantially free from naturally associated molecules, i.e., the naturally associated molecules constituting at most 20% by dry weight of a preparation containing the antibody. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, and HPLC. The antibodies can be prepared by any methods known in the art, e.g., those described herein.

(c) Formulation of T.beta.RI Cleavage Inhibitors and Uses Thereof.

[0078] One or more of the T.beta.RI cleavage inhibitors disclosed herein can be mixed with carrier, (e.g., a pharmaceutically acceptable carrier) to form a composition (e.g., a pharmaceutical composition), which can be used either in vitro or in vivo. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

[0079] The pharmaceutical compositions mentioned above may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.

[0080] Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); and phosphoric acid and a salt (0.8-2% W/V). Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).

[0081] The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution, but are not so limited. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

[0082] When administered in vivo, the compositions of the present invention can be administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

[0083] The compositions used in the methods disclosed herein are sterile and in a unit of weight or volume suitable for addition to a cell culture or administration to a subject. As used herein, a subject is a human or non-human animal, including non-human primates, mice, rats, cows, pigs, horses, sheep, goats, dogs, cats, etc. Preferably the subject is a human.

[0084] A "subject (e.g., person or patient) having a cancer" is a subject, person or patient that has detectable cancerous cells. The cancer may be a malignant or non-malignant cancer. Cancers or tumors include but are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas. Cancers also include cancer of the blood and larynx.

[0085] A "subject (e.g., person or patient) suspected of having a cancer" as used herein is a subject, person or patient who may show some clinical or other indications that may suggest to an observer that the subject, person or patient may have cancer. The subject, person or patient suspected of having cancer need not have undergone any tests or examinations to confirm the suspicion. It may later be established that the subject, person or patient suspected of having cancer indeed has cancer.

[0086] A "subject (e.g., person or patient) at risk of developing a cancer" as used herein is a subject, person or patient who has a high probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of developing a cancer and subjects exposed to cancer causing agents such as tobacco, asbestos, or other chemical toxins, or a subject who has previously been treated for cancer and is in apparent remission.

[0087] An effective amount of the pharmaceutical compositions described herein can be administered to a subject in need of the treatment (e.g., a human cancer patient) any conventional route, including injection or by gradual infusion over time. An "effective amount" is that amount of a T.beta.RI inhibitor that alone, or together with further doses, produces the desired response, e.g., inhibiting cleavage of T.beta.RI, blocking translocation of a T.beta.RI ICD into the nucleus, and/or reducing cancer cell invasiveness and cancer metastasis. This can be monitored by routine methods known to one of ordinary skill in the art. The amount effective can be the amount of a single agent that produces a desired result or can be the amount of two or more agents in combination. Such amounts can be determined with no more than routine experimentation.

[0088] The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, intrasternal, transdermal and intratumoral. Other modes of administration include mucosal, rectal, vaginal, sublingual, intranasal, intratracheal, inhalation, ocular, and transdermal.

[0089] For oral administration, the T.beta.RI inhibitors can be formulated readily by combining the active inhibitors with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.

[0090] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0091] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0092] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0093] For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0094] The inhibitors, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0095] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0096] Alternatively, the active inhibitors may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0097] The inhibitors may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0098] In addition to the formulations described previously, the inhibitors may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0099] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0100] Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.

[0101] The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

[0102] Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Pat. No. 4,452,775 (Kent); U.S. Pat. No. 4,667,014 (Nestor et al.); and U.S. Pat. No. 4,748,034 and U.S. Pat. No. 5,239,660 (Leonard) and (b) diffusional systems in which an active component permeates at a controlled rate through a polymer, found in U.S. Pat. No. 3,832,253 (Higuchi et al.) and U.S. Pat. No. 3,854,480 (Zaffaroni). In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation.

[0103] Controlled release of the T.beta.RI inhibitors can also be achieved with appropriate excipient materials that are biocompatible and biodegradable. These polymeric materials which effect slow release of the inhibitors disclosed herein may be any suitable polymeric material for generating particles, including, but not limited to, nonbioerodable/non-biodegradable and bioerodable/biodegradable polymers.

[0104] Such polymers have been described in great detail in the prior art. They include, but are not limited to: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly (methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinyl chloride polystyrene, polyvinylpryrrolidone, hyaluronic acid, and chondroitin sulfate.

[0105] Examples of preferred non-biodegradable polymers include ethylene vinyl acetate, poly(meth) acrylic acid, polyamides, copolymers and mixtures thereof.

[0106] Examples of preferred biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide) and poly(lactide-co-caprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. The foregoing materials may be used alone, as physical mixtures (blends), or as co-polymers. The most preferred polymers are polyesters, polyanhydrides, polystyrenes and blends thereof.

(II) DIAGNOSTIC/PROGNOSTIC METHODS

[0107] It is disclosed herein that nuclear accumulation of a T.beta.RI ICD fragment occurs in cancer cells, but not in normal cells. Thus, nuclear accumulation of the ICD fragment is a reliable biomarker in cancer diagnosis. Further, as translocation of the ICD to the nuclei is associated with expression of genes involved in cancer cell invasion and metastasis, nuclear accumulation of the ICD fragment can also serve as a prognosis marker for predicting cancer progression, particularly metastasis.

[0108] Accordingly, provided herein are methods for diagnosing cancer based on presence/absence of a T.beta.RI ICD in nuclei. To perform this method, a cell-containing sample is obtained from, e.g., a subject suspected having cancer or at risk for cancer. Presence/absence of the ICD in the nuclei is then determined via a conventional method. In one example, in situ immunostaining is performed using an antibody that specifically recognizes the ICD. In another example, the cytoplasmic and nuclear protein fractions are prepared from the cells following routine practice (see Examples below) and presence/absence of the ICD in the nuclear fraction is examined via, e.g., Western blot. Nuclear localization of the ICD indicates that the sample contains cancer cell. It also indicates that the cancer cells are invasive.

[0109] When the cell-containing sample is a tissue sample obtained from a cancer patient, nuclear localization of the ICD indicates that cancer invasion/metastasis is likely to occur in that patient.

[0110] The diagnostic/prognostic methods described above are applicable to human subjects having, suspected of having, or at risk for various types of cancer, including, but are not limited to, biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas.

(III) SCREENING FOR T.beta.RI INHIBITORS

[0111] Also provided herein are screening methods (e.g., high-throughput screening methods) for identifying T.beta.RI inhibitors that can be used in the treatment methods disclosed herein. Below is an example.

[0112] Cancer cells expressing T.beta.RI are cultured in the presence of TGF.beta. (which induces T.beta.RI cleavage) and a candidate compound for a suitable period of time. The levels of a T.beta.RI ICD in the nuclei are determined as described above. A reduction in the nuclear level of the ICD in cells treated with the candidate compound as compared to that in cells not treated with the compound indicates that the candidate compound is a T.beta.RI cleavage inhibitor.

[0113] Alternatively, the levels of an extracellular domain of the T.beta.RI in the culture medium after treatment are determined. A reduced level of the extracellular domain in the culture medium of cells treated with the candidate compound as compared to that in the culture medium of untreated cells indicates that the candidate compound is a T.beta.RI cleavage inhibitor.

[0114] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

Example 1

Cleavage of T.beta.RI by TACE Results in Nuclear Translocation of an Intracellular Domain of T.beta.RI and Increased Invasiveness in Cancer Cells

Materials and Methods

(i) Cell Culture

[0115] The human prostate cancer cell line, PC-3U, originating from PC-3, and LNCaP were purchased from ATCC. The cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and L-glutamine..sup.20 At least 12 h before TGF-.beta.1 stimulation, the cells were starved in RPMI-1640 supplemented with 1% FBS and L-glutamine. Wild-type MEF and TRAF6.sup.-/- MEF cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. Normal human primary prostate epithelial cells (PrEC) were purchased from Cambrex Bio Science, Walkersville. The cells were grown according to the manufacturer's recommendation in Prostate Epithelial Cell Basal Medium supplemented with the Clonetics PrEGM bullet kit, which contains bovine pituitary extract (BPE), hydrocortisone, human epithelial growth factor (hEGF), epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, and GA-1000.

[0116] Before TGF-.beta.1 stimulation, the cells were starved for 12-18 h in medium supplemented with 1% FBS. In all assays, the cells were stimulated with 10 ng/ml TGF.beta.1 (R&D System, UK).

(ii) Antibodies and Reagents

[0117] Antibodies or antisera against the following proteins were used: HA (Y-11), ubiquitin (P4D1), T.beta.RI (V22; the specificity of this antibody was previously reported).sup.21,22, TRAF6 (D10), and p300 (NM11). All of the antibodies were purchased from Santa Cruz Biotechnology. P-Smad2 and T.beta.RI (VPN) antisera were generated in rabbits in-house. Antibodies specific to lamin A, .beta.-tubulin, TACE/ADAM17, pPKC.zeta./.lamda. (Thr410/403), PKC.zeta., p-p38, p38, Smad2, and acetyl-Lys were obtained from Cell Signalling. Antibodies against TRAF6 C-term was purchased from ZYMED Laboratories; anti-UbK63 antibodies were from Enzo Life Sciences; UbK48 Clone Apu 207 antibodies were from V.M. Dixit, Genetech; and antibodies against .beta.-actin were from Sigma. The mouse monoclonal TS2/16 antibodies were used to activate or inactivate .beta.1-integrin. Rat anti-human CD29 antiserum was purchased from BD Pharmingen.

[0118] Secondary antibodies were horseradish-peroxidase-linked whole anti-rabbit, -goat, or -mouse IgG antisera purchased from Sigma. In some experiments, either goat anti-mouse IgG, light chain specific, or mouse anti-rabbit IgG, light chain specific antisera, purchased from Jackson ImmunoResearch Laboratories, were used. 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI) fluorescent dye (purchased from Merck) and TRITC-labelled phalloidin (from Sigma) were used to visualize cell nuclei by microscopy. Alexafluor 555 was purchased from Invitrogen.

[0119] Protein-G Sepharose was obtained from GE Healthcare.; LumiLight Western blotting substrate and Pefabloc were from Roche, PageRuler prestained protein ladder was from Fermentas; and TAPI-2 was from BIOMOL Research Laboratories Inc. TAPI-2 was used at concentrations of 10-20 .mu.M. The p38.alpha./.beta. inhibitor (SB203580) was purchased from Calbiochem (it was used at a concentration of 10 .mu.M). PKC.zeta. pseudosubstrate was from TOCRIS Bioscience and used at concentrations of 10-50 .mu.M. The PKC.zeta. activator, phorbol 12-myristate 13-acetate (PMA, referred to as TPA in our paper), and a T.beta.RI inhibitor (SB505124) were purchased from Sigma. The SB505124 inhibitor was used at a concentration of 10 .mu.M and the TPA at a concentration of 100 nM. The general PKC.zeta. inhibitor, GF109203X, was purchased from Sigma. All inhibitors were added to cells 1 h before TGF.beta. stimulation.

(iii) Western Blotting, Ubiquitination Assay and Immunofluorescence Microscopy

[0120] Cells were starved for 12-18 h and then stimulated with TGF.beta. for the indicated time periods, washed twice in ice-cold PBS, and lysed in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris pH 8.0, 0.5% (v/v) deoxycholate, 1% (v/v) NP40, 10% (v/v) glycerol, 1 mM aprotinin, 1 mM Pefabloc and 2 mM sodium orthovanadate). After centrifugation, supernatants were collected and protein concentrations were determined using the bicinchoninic acid (BCA) protein measurement kit (Nordic Biolabs). Equal volumes were subjected to immunoprecipitation. Equal amounts of protein from total cell lysates and immunoprecipitations were subjected to electrophoresis on 6%, 10%, 12%, or 4-12% gradient SDS-polyacrylamide gels (SDS-PAGE), blotted on to polyvinylidine difluoride membranes, and subjected to immunoblotting, as previously described..sup.23 The A495 cell lysate from Santa Cruz was used as a positive control for T.beta.RI. The ubiquitination assays, immunofluorescence assays, and transient transfections were performed as previously described..sup.5,23,24 Photomicrographs were obtained with a Zeiss 510 Meta (Carl Zeiss Microimaging, Inc.) equipped with a digital camera (RET-EXi-F-M-12-C) from Q-imaging.

(iv) Nuclear Fractionation Assays

[0121] Two different protocols for nuclear fractionation were used to evaluate further the finding that T.beta.RI accumulated in the nucleus in response to TGF.beta. stimulation.

[0122] Protocol 1 was used for cell fractionation, as previously described in the experiments shown in FIG. 1d and FIG. 4d..sup.24 Briefly, cells were washed twice with ice-cold PBS and collected in 1 ml ice-cold PBS then centrifuged (500.times.g, 5 min, 4.degree. C.). The cell pellet was resuspended in 500 .mu.l buffer A (10 mM MES pH 6.2, 10 mM NaCl, 1.5 mM MgCl.sub.2, 1 mM EDTA, 5 mM dithiothreitol, 1% Triton X-100, and protease inhibitors), vortexed for 5 s, and centrifuged (3000.times.g, 5 min). The resulting supernatant was collected as the cytoplasmic fraction. The nuclear pellet was washed twice with buffer B (Buffer A without Triton X-100), then resuspended in 100 .mu.l buffer C (25 mM Tris/HCl pH 10.5, 1 mM EDTA, 0.5M NaCl, 5 mM .beta.-mercaptoethanol, and 0.5% Triton X-100), and incubated on ice for 20 min (vortexed every 5 min) to recover the nuclear proteins. The cytoplasmic and nuclear fractions were centrifuged at 12,000.times.g for 30 min and the supernatants were prepared for immunoblotting.

[0123] Protocol 2 was applied in the experiments shown in FIG. 3b. The Nuclear Complex Co-IP kit purchased from Active Motif, performed according to the manufacturer's instructions, was used in Protocol 2. Briefly, cells in a 10-cm dish were washed twice with ice-cold PBS and then scraped and collected in 1 ml ice-cold PBS. After centrifugation at 1500 rpm for 5 min at 4.degree. C., the cell pellet was gently resuspended in 500 .mu.l Hypotonic Buffer and incubated on ice for 15 min. Twenty-five .mu.l of Detergent Solution was added, gently mixed, and centrifuged at 14,000.times.g for 30 s, at 4.degree. C. The nuclear pellet was resuspended in 100 .mu.l Complete Digestion Buffer. After adding 0.5 .mu.l of Enzymatic Shearing Cocktail, the solution was vortexed gently for 2 s then incubated for 10 min at 37.degree. C. Afterwards, 2 .mu.l of 0.5M EDTA was added to the nuclear lysates to stop the reaction. The mixture was vortexed gently and then incubated on ice for 5 min, centrifuged (14,000.times.g, 10 min, 4.degree. C.), and the supernatant was collected for immunoprecipitation.

(v) Invasion Assay

[0124] Invasion assays were performed using the CytoSelect Cell Invasion Assay (Cell Biolabs, Inc., San Diego, Calif.). Briefly, the basement membrane layer of the cell culture inserts were rehydrated in 300 .mu.l serum-free RPMI-1640 and 2.times.10.sup.6 cells were seeded into the upper sections of the chambers in serum-free RPMI-1640 with or without TGF.beta.. The lower wells of the invasion plates were filled with 500 .mu.l RPMI supplemented with 10% FBS. Non-invasive cells were removed from the upper chamber and invasive cells were stained with crystal violet cell stain solution. Invasive cells were photographed with a Leica DMR light microscope. Colorimetric quantification was performed by transferring inserts into 200 .mu.l of extraction solution for 10 min and then, transferring to a 96-well microtiter plate. The OD at 560 nm was determined with a plate reader (Supplementary Information FIG. S5a).

(vi) Plasmids and DNA Transfections

[0125] GFP-ca T.beta.RI was constructed by inserting the full length ca T.beta.RI between the immediate early promoter of CMV and the EGFP coding sequence. The C-terminus of ca T.beta.RI was fused to the N-terminus of the pEGFP-N3 vector. The pcDNA3 and wild-type PKC.zeta. plasmids were purchased from Addgene. HA-caT.beta.RI and HA-T.beta.RI KR (kinase dead (KD) mutant) with HA fused to the C-terminus of T.beta.RI were obtained from P. ten Dijke (University of Leiden, The Netherlands). Expression vectors for C-terminally tagged HA-tagged G120I mutant of caT.beta.RI were generated by PCR and the mutation was confirmed by sequencing. The caT.beta.RI-E161A plasmid with HA fused to the C-terminus of the T.beta.RI mutant construction was described previously..sup.5 HA-caT.beta.RI with HA inserted between amino acid 27 and 28 was obtained from Dr. S. Corvera (University of Massachusetts Medical School, Worcester, USA). 3.times.Plasmids encoding T.beta.RI with the HA epitope inserted between amino acids 27 and 28 was described in Hayes et al., JCB 2002. HA-tagged wild-type ubiquitin and the K48- and K63-only ubiquitin mutants were obtained from Genentech, San Francisco, Calif. Expression vector for GST-T.beta.RI fusion protein, encoding the complete cytoplasmic part (amino acid 148-503) of ca T.beta.RI (T204D) has been described previously as well as the production and purification of the protein.

(vii) siRNA Transfection

[0126] Twenty-one-base pair siRNA duplexes targeting ALK5 (5' AAC AUA UUG CUG CAA CCA GGA 3') and SMART pool siRNA targeting TRAF6 were synthesised as previously described..sup.5 A non-specific control siRNA (5' AAC AGU CGC GUU UGC GAC UGG 3') was synthesized by Dharmacon Research (Lafayette, Colo.). PKC.zeta. siRNA (h2) was obtained from Santa Cruz biotechnology. The siRNAs were transfected into cells using Oligofectamine (Invitrogen), according to the manufacturer's protocol.

(viii) Expression Analysis

[0127] Total RNAs were isolated from cells with the RNeasy Minikit (Qiagen) and double-stranded cDNAs were prepared using the Thermoscript RT-PCR System (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed with the Power SYBR Green PCR Mastermix (Applied Biosystems) and the Stratagene MX3000P. The following primers were used for qRT-PCR:

TABLE-US-00002 T.beta.RI: forward primer (FP) 5'-TGTTGGTACCCAAGGAAAGC-3', reverse primer (RP) 5'-CACTCTGTGGTTTGGAGCAA-3'; p300: FP 5'-GGGACTAACCAATGGTGGTG-3', RP 5'-GTCATTGGGCTTTTGACCAT-3'; SNAIL 1: FP 5'-GAGCATACAGCCCCATCACT-3', RP 5'-GGGTCTGAAAGCTTGGACTG-3'; Smad7: FP 5'-TCCTGCTGTGCAAAGTGTTC-3', RP 5'-TCTGGACAGTCTGCAGTTGG-3'; MMP-2: FP 5'-AGGCCGACATCATGGTACTC-3', RP 5'-GGTCAGTGCTGGAGAAGGTC-3'; PAI 1: FP 5'-CTCTCTCTGCCCTCACCAAC-3', RP 5'-GTGGAGAGGCTCTTGGTCTG-3'.

(viiii) Chromatin Immunoprecipitation (ChIP)

[0128] ChIPs were performed in three or more biological replicates following the protocol was provided by Abcam, Cambridge UK. Briefly, chromatin was precipitated using the V22 rabbit antibody (Santa Cruz). After precipitation, the DNA was amplified with qRT-PCR to analyze ChIP DNA in triplicate. The following primers were used for ChIP:

[0129] Snail1 forward primer 5'-GGACTCAGGGAGACTCATGG-3', reverse primer 5'-GG GTCTACGGAAACCTCTGG-3'.

(x) Histology of Human Tumors

[0130] Tissue microarrays (TMAs) on healthy and malignant tissues were performed using anti-T.beta.RI antiserum (V22 and H100). 432 tumor samples, obtained from human patients having various cancers (20 different cancers, including prostate cancer, renal carcinoma and bladder cancer), were analyzed. These malignant samples were provided by the Human Proteome Atlas (HPA) facility (http://www.proteinatlas.org). Stained TMA sections were scanned by high-resolution scanners (ScanScope XT, Aperio Technologies), separated in individual spot images, and evaluated by experienced pathologists.

(xi) Statistical Analysis

[0131] Statistical analyses were performed with the Student's t test or ANOVA as indicated in Figure Legends. Values are expressed as mean.+-.s.e.m. of three or more independent experiments, unless otherwise indicated. P values of <0.05 were considered statistically significant.

Results

(i) Nuclear Accumulation of an Intracellular Domain (ICD) of T.beta.RI

[0132] Human prostate cancer cells are known to produce TGF.beta.2 in an autocrine manner. To investigate whether T.beta.RI can be proteolytically cleaved in human prostate cancer cells, a C-terminally haemagglutinin-tagged, constitutively active (ca) T.beta.RI (HA-ca T.beta.RI) was expressed in PC-3U cells (a human prostate cancer cell line). Notably, in addition to the full length receptor, an anti-HA antiserum recognized a T.beta.RI fragment having the estimated size of an intracellular domain of T.beta.RI. See FIG. 15, panel a. Next, subcellular localization of endogenous T.beta.RI in PC-3U cells was investigated by immunofluorescence and confocal microscopy, using an anti-C-terminal T.beta.RI antibody (antibody V22) or an antibody specific to the extracellular domain of T.beta.RI (antibody H100). As shown in FIG. 15, panel c, an enhanced nuclear accumulation of a C-terminal ICD of the T.beta.RI, recognized by the V22 antibody, was observed after the cells were stimulated by TGF.beta.. In contrast, immunofluorescence staining revealed that full length T.beta.RI, containing the N-terminal extracellular domain recognizable by the H-100 antibody, was located on the cell membrane of TGF.beta.-stimulated PC-3U cells. FIG. 15, panel c. To assure the specificity of the V22 bodies and H100-antibodies, siRNA was used to knock-down endogenous T.beta.RI expression in PC-3U cells. See FIG. 1, panel a. qRT-PCR was performed to determine the mRNA levels of T.beta.RI and the results thus obtained demonstrated that they were significantly decreases. FIG. 1, panel b.

[0133] To investigate whether nuclear translocation of the ICD fragment is TGF.beta.-dependent, a nuclear fractionation assay of cell lysates from TGF.beta.-treated PC-3U cells were performed. When nuclear extracts were immunoblotted with the V22 antibody, a T.beta.RI fragment of approximately 34 kDa was revealed. FIG. 1, panel c. This fragment was not recognized by the H100 antibody, indicating that this fragment is a C-terminal fragment lacking the N-terminal extracellular domain. The full-length T.beta.RI was detected in the cytoplasmic fractions of cells stimulated by TGF.beta. for 30 minutes, using both the V22 and H100 antibodies. FIG. 1, panel c. By contrast, the 34 kD fragment recognizable by the V-22 antibody was observed only in the nuclear fraction. The levels of T.beta.RI was much lower in nuclear before TGF.beta. stimulation.

[0134] To further investigate the TGF.beta.-dependency of nuclear accumulation of T.beta.RI, a fusion protein, in which GFP is linked to the C-terminus of T.beta.RI, was expressed in PC-3U cells. After being stimulated with TGF.beta. for 6 h, fluorescent signal released from GFP was observed in the nuclei of the PC-3U cells that expressed the fusion protein, but not in PC-3U cells expressing GFP. FIG. 1, panel d. The nuclear localization of ectopically expressed C-terminally HA-tagged ca T.beta.RI in PC-3U cells was also examined. The results indicated a TGF.beta.-induced nuclear accumulation of the HA-tagged ca T.beta.RI-ICD. FIG. 15, panel c. This observation is consistent with the nuclear accumulation of endogenous T.beta.RI-ICD as shown in FIG. 1, panel c. An N-terminally HA-tagged T.beta.RI (see 4) was also expressed in PC-3U cells and the sub-cellular localization of the HA-tagged protein was examined using an anti-HA antiserum. No positive signal was observed in nuclei of PC-3U cells either treated or not treated with TGF.beta., indicating that full-length T.beta.RI or its N-terminal fragment does not enter the nucleus. FIG. 15, panel c.

[0135] Taken together, the results obtained from this study indicate that an ICD of T.beta.RI enters into the nuclei in PC-3U cells in response to TGF.beta. stimulation.

(ii) Nuclear Accumulation of T.beta.RI-ICD is Dependent on TRAF6

[0136] A consensus TRAF6 binding site for in T.beta.RI was identified recently..sup.5 Ligand-induced oligomerisation of the TGF.beta. receptor complex results in receptor kinase-independent activation of TRAF6. This, in turn, results in Lys63-polyubiquitin-dependent activation of TGF.beta.-activated kinase-1 (TAK1)), which leads to activation of p38 MAPK in PC-3U cells. In contrast, activation of the canonical Smad pathway does not require TRAF6..sup.5

[0137] To examine whether TRAF6 is involved in the cleavage and nuclear accumulation of T.beta.RI, a HA-tagged constitutively active T.beta.RI (HA-caT.beta.RI) or a HA-tagged caE161A T.beta.RI mutant (HA-ca E161A), was expressed in PC-3U cells. HA-ca E161A does not bind to TRAF6 but is still capable of activating the canonical Smad signalling pathway..sup.5 As shown in FIG. 2, panel a, stimulation of PC-3U cells with TGF.beta. resulted in Lys63-linked polyubiquitination of HA-caT.beta.RI, but not HA-ca E161A. Immunoblotting of total cell lysates with HA antiserum showed that HA-caT.beta.RI was cleaved, producing an ICD fragment. FIG. 2, panel a. Moreover, HA-ca E161A failed to activate the p38 MAPK pathway but could activated Smad2. FIG. 2, panel a. In addition, a knock-down of endogenous TRAF6 in PC-3U cells with an anti-TRAF6 siRNA also resulted in loss of both Lys63-linked polyubiquitination in T.beta.RI and the formation of the T.beta.RI ICD in response to TGF.beta. stimulation. FIG. 16, panels a and b. Immunoblotting with antibodies specific to either Lys63 or Lys48 polyubiquitin indicated that, 30 min after TGF.beta. stimulation, wt T.beta.RI contained Lys63 polyubiquitination, but not Lys48 polyubiquitination, in PC-3U cells. FIG. 2, panel a and FIG. 16, panels a and c.

[0138] The pattern of T.beta.RI polyubiquitination was further examined in PC-3U cells that overexpress HA-tagged wt or mutant ubiquitin, in which all lysine residues were mutated except for Lys63 or Lys48 (K63 or K48). TGF.beta. induced polyubiquitination of T.beta.RI in cells transiently expressed the K63 ubiquitin mutant. However, in cells expressing the K48 ubiquitin mutant, the level of TGF.beta.-induced ubiquitination was reduced. FIG. 2, panel b. In addition, TRAF6 was shown to induce the polyubiquitination of T.beta.RI in an in vitro ubiquitination assay, providing firm evidence that T.beta.RI is a substrate for TRAF6. FIG. 17.

[0139] The level of TGF.beta.-induced nuclear accumulation was higher in PC-3U cells expressing HA-caT.beta.RI than in cells expressing HA-ca E161A as analyzed by immunofluorescence stainings. FIG. 2, panel c. This indicates that TRAF6 causes Lys63-dependent polyubiquitination of T.beta.RI in a TGF.beta.-dependent manner, which, in turn, results in generation of an intracellular fragment of T.beta.RI and its nuclear accumulation.

(iii) T.beta.RI is Cleaved by TACE

[0140] When activated, TNF-alpha converting enzyme (TACE) (also known as ADAM17) and ADAM 10, both of which are metalloproteases, cleave certain receptors and adhesion proteins at sites just outside cell membranes. TACE was recently shown to cleave T.beta.RI in an ERK MAP-kinase dependent manner, leading to desensitisation of TGF.beta. signalling. TACE is often overexpressed in tumours and is activated by protein kinase C (PKC)..sup.8

[0141] The role of TACE in T.beta.RI cleavage and nuclear accumulation was investigated to further characterise the underlying molecular mechanisms. Treatment of human PC-3U cells with tetradecanoylphorbol acetate (TPA) to activate PKC.zeta. led to nuclear accumulation of endogenous T.beta.RI ICD, which was detected by immunofluorescence. FIG. 3, panel a. Moreover, when HA-caT.beta.RI-expressing PC-3U cells were treated with TGF.beta. or TPA, the ICD accumulated in the nuclear fraction. FIG. 3, panel b. Pretreatment of cells with TNF-.alpha. protease inhibitor (TAPI)-2, which inhibits TACE, led to a reduction in nuclear translocation of T.beta.RI ICD in response to TGF.beta. stimulation. FIG. 3, panels c and d. TACE was recently shown to associate with overexpression of T.beta.RI in cells that expressed human epidermal growth factor receptor 2..sup.9 In a co-immunofluorescence assay, association between endogenous T.beta.RI and TACE was observed in PC-3U cells stimulated with TGF.beta.. FIG. 3, panel e. These data indicate that activation of TACE, either by TPA treatment or TGF.beta. stimulation, leads to nuclear accumulation of the T.beta.RI ICD.

(iv) Determination of the TACE Cleavage Site in T.beta.RI

[0142] Treatment of PC-3U cells with TAPI-2 partly prevented nuclear translocation of T.beta.RI. This suggests a potential cleavage site in proximity to the transmembrane domain of T.beta.RI. Previous studies with peptide substrates of TNF-a have shown that TACE has a strong preference for cleavage at the Ala-Val sequence and cannot cleave a TNF-a-based peptide when Ala is substituted with Ile at the P1 position. Jin et al., Anal Biochem 302, 269-275, 2002. However, no possible Ala-Val cleavage site was found in the extra-cellular domain of T.beta.RI. TACE has also been shown to have a strong preference for the Gly-Leu sequence and that replacement of this sequence with Gly-Ile blocks cleavage. Chow et al., JBC, 2008. Two Gly-Leu sequences were found in T.beta.RI, Gly.sub.52-Leu.sub.53 and Gly.sub.120-Leu.sub.121. The putative cleavage site is Gly.sub.120-Leu.sub.121, which is in close proximity to the transmembrane domain and would therefore lead to a cleavage product consisting of the complete intracellular domain, including its transmembrane part. To verify that this is the actual cleavage site, Gly120 was mutated to Ile to product a G120I mutant. This mutation did not change the subcellular localization of T.beta.RI in untreated PC-3U cells as confirmed by confocal imaging. FIG. 4, panel b. However, its TGF-induced nuclear accumulation was prevented, when compared with wt T.beta.RI. FIG. 4, panel b. The G120I mutant preserves the kinase activity as judged by its similar capacity to phosphorylate Smad2, when compared to the wt T.beta.RI. FIG. 4, panel a. These data demonstrate that TACE cleaves T.beta.RI at the G.sub.120-L.sub.121 site.

(v) PKC.zeta. is Needed for TACE-Induced Cleavage of T.beta.RI

[0143] PKC.zeta. is the only member in the PKC-family known to form a multiprotein-complex with TRAF6..sup.12 Its involvement in T.beta.RI cleavage and nuclear translocation was investigated. Inhibition of PKC.zeta. with the a PKC.zeta. pseudosubstrate completely prevented T.beta.RI nuclear accumulation in a dose-dependent manner. FIG. 5, panel a and FIG. 18. It is well accepted that TPA can activate classical PKC.zeta. family members but not atypical PKC.zeta. isoforms, like PKC.zeta...sup.13-14 However, it was found in this study that PKC.zeta. was activated by both TGF.beta. and TPA. FIG. 5, panel b. TGF.beta. stimulation did not lead to activation of PKC.zeta. in TRAF6.sup.-/--knock-out mouse embryonic fibroblasts (KO MEFs; see FIG. 5, panel c), indicating that TRAF6 is important for TGF.beta.-induced activation of PKC.zeta.. In a nuclear fractionation assay, siRNA-mediated silencing of PKC.zeta. was associated with a significant loss of TGF.beta.-induced nuclear accumulation of T.beta.RI ICD. FIG. 5, panel d. In addition, ectopic expression of wt PKC.zeta. promoted expression and cleavage of the caT.beta.RI or the kinase dead (KD) T.beta.RI mutant. FIG. 5, panel e. Treatment of cells with the PKC.zeta. inhibitor partially reduced the formation of the T.beta.RI ICD, but treatment with the T.beta.RI kinase inhibitor, SB505124, had no major effect. FIG. 18 and FIG. 5, panel f.

[0144] As shown in FIG. 5, panel g, co-localization of endogenous T.beta.RI and TACE was induced by TGF.beta. and the co-localization was inhibited by the PKC.zeta. pseudosubstrate. This indicates that PKC.zeta. activity was necessary for proper localisation of T.beta.RI to a subcellular compartment, where it could be cleaved by TACE. In conclusion, TGF.beta. caused activation of PKC.zeta. in a TRAF6-dependent manner, which was important for the proteolytic cleavage of T.beta.RI by TACE, leading to nuclear accumulation of T.beta.RI ICD.

(vi) T.beta.RI ICD Regulates Transcription

[0145] The observation that the T.beta.RI ICD entered the nuclei of TGF.beta.-treated PC-3U cells suggested that it might participate in gene regulation. Whether nuclear localization of T.beta.RI ICD is associated expression of p300, a well-known transcription regulator, was examined. TGF.beta. treatment of PC-3U cells led to the co-localisation of T.beta.RI and p300 in nuclear speckles, which were indentified by co-immunofluorescence as PML nuclear bodies..sup.15 FIG. 6, panels a and b. The PKC.zeta. pseudosubstrate prevented the co-localisation of T.beta.RI with PML nuclear bodies. FIG. 6, panel b. In co-immunoprecipitation assays, the acetyltransferase p300 was found to associate with T.beta.RI ICD in vivo (FIG. 6, panels c and d) and acetylation of the T.beta.RI ICD was verified using an antibody specific for acetyl-lysine. FIG. 6, panel e. In contrast, in cells expressing the E161A mutant, a lower level of T.beta.RI ICD was found to be associated with endogenous p300 and less acetylated ICD was detected. FIG. 6, panels d and e. As compared with cells expressing wt T.beta.RI, the level of p300 was lower in cells expressing the E161A mutant, which is unable to bind TRAF6. FIG. 6, panel d. In an qRT-PCR assay, an increased level of p300 mRNA was observed in TGF.beta.-induced cells that express wt T.beta.RI, but not in cells that express the E161A mutant. FIG. 6, panel f. These data indicate that TGF.beta. induces p300 expression in a TRAF6-dependent manner.

[0146] Next, qRT-PCR was performed to analyze the effect of T.beta.RI ICD on known TGF.beta. target genes, including Snail and MMP2. Expression levels of these two genes (determined by their mRNA levels) were found to be induced by wt T.beta.RI in a degree much greater than the E161A mutant. FIG. 6, panel g. In contrast, genes encoding plasminogen activator inhibitor-1 (PAI1) and the antagonist, Smad7, were induced by both wt T.beta.RI and the E161A mutant. FIG. 6, panel g. A chromatin immunoprecipitation (ChIP) assay revealed binding of T.beta.RI or T.beta.RI ICD to the endogenous Snail promoter in a TGF.beta.-dependent manner in PC-3U cells. FIG. 6, panel h. The expression of Snail and MMP2 genes is linked to tumour invasiveness..sup.16 Therefore, PC-3U cells that expressed wt T.beta.RI or the E161A mutant were subjected to an invasion assay. TGF.beta.-induced invasion occurred in PC-3U cells expressing wt T.beta.RI but not in cells expressing the E161A mutant. By contrast, epidermal growth factor (EGF) stimulated invasion in both cells expressing wt T.beta.RI and cells expressing the E161A mutant. FIG. 6, panel i. Similar results were observed when the experiment was repeated with human prostate cancer cell line LNCaP. More specifically, TGF.beta. promoted invasion of LNCaP cells that expressed wt T.beta.RI, but not LNCaP cells that express the E161A mutant. FIG. 19, panels a and b. Taken together, the data obtained from this study indicate that the T.beta.RI ICD associated with p300 in nuclear PML bodies in a PKC.zeta.-dependent manner, and is acetylated by p300. Moreover, Snail and MMP2 were induced by T.beta.RI ICD, which correlates with increased cell invasiveness.

(vii) Nuclear Accumulation of T.beta.RI is Found in Malignant but not Normal Prostate Cells

[0147] Immunofluorescence analysis and confocal microscopy were performed to investigate the subcellular localization of endogenous T.beta.RI in primary human prostate epithelial cells (PrEC) cells and in PC-3U cells, using the V22 antibody that is specific to the C-terminal fragment of T.beta.RI. Results thus obtained show that, after TGF.beta. stimulation, nuclear accumulation of T.beta.RI or its ICD took place in the malignant PC-3U cells, but not in the normal PrEC cells. FIG. 6, panels j and k.

(viii) TGF.beta.-Induced Invasion of Human Breast and Lung Carcinoma Cells is Associated with Nuclear Accumulation of T.beta.RI and is Promoted by TACE and PKC.zeta.

[0148] Human breast carcinoma cell line MDA-MB-231 and the human lung carcinoma cell line A549 were used in this study to explore whether the TGF-.beta. induced T.beta.RI nuclear translocation is associated with cancer cell invasiveness. As shown in an immunofluorescence and confocal microscopy assay, TGF.beta. promoted nuclear accumulation of the endogenous T.beta.RI ICD in both MDA-MB-231 and A549 cells. FIG. 7, panes a-d; and FIG. 5S, panels c and d. The T.beta.RI ICD nuclear accumulation was PKC.zeta.- and TACE-dependent. FIG. 6, panel a.

[0149] Next, whether TACE and PKC.zeta. are involved in TGF.beta.-induced invasiveness of these cancer cell lines was investigated. As shown in FIG. 5S, panels c and d, treatment of MDA MB-231 and A549 cells with TACE and PKC.zeta. inhibitors significantly reduced TGF.beta.-induced invasion.

(viiii) Nuclear Accumulation of T.beta.RI in Human Tumors In Vivo

[0150] To explore whether nuclear accumulation of the T.beta.RI ICD occurs in human tumors, immunohistochemistry was performed to investigate the expression and localization of T.beta.RI in a panel of prostate and renal cell carcinomas and bladder tumors. Nuclear accumulation of T.beta.RI ICD was observed in all of the 19 investigated prostate carcinoma tissues, in 19 out of the 24 renal cell carcinomas tissue samples, and in 21 out of the 23 bladder tumors samples, using the V22 antibody. Staining with the H100 antibody, which recognizes the extracellular part of T.beta.RI, only showed cytoplasm localization. FIG. 8. These data indicate that nuclear accumulation of T.beta.RI or its ICD occurs in human tumours.

Discussion

[0151] It has been shown herein that, in response to TGF.beta. stimulation, T.beta.RI undergoes cleavage by TACE in cancer cells and that an ICD segment of T.beta.RI is translocated to the nuclei of the cancer cells to interact with transcriptional regulator p300 in nuclear PML bodies. It has also been shown herein that nuclear accumulation of the T.beta.RI ICD is dependent on TRAF6, TACE, and PKC.zeta.. Overexpression of Smad2, 3 or 4 in PC-3U cells did not influence PKC.zeta.-induced generation of T.beta.RI ICD. FIG. 20, panel a. Knock down of Smad4 by siRNA also did not affect the PKC.zeta.-induced generation of T.beta.RI ICD. FIG. 20, panel b. Moreover, in MDA-MB468 cells, which are Smad4-deficient, the T.beta.RI ICD formed in cells treated with TGF.beta.. Since cells expressing the E161A mutant, which cannot bind to TRAF, still exhibits Smad2 activation but did not produce the T.beta.RI ICD, it indicates that the Smad pathway can operate independently of T.beta.RI ICD formation, which correlates with activation of the TRAF6, TAK1, and p38 MAPK pathways. FIG. 2, panel a.

[0152] Integrins are essential adhesion receptors localized on the surfaces of all metazoan cells and are involved in cell migration and extracellular matrix assembly..sup.17 Integrins forms membrane-spanning heterodimers, which are critical for embryonic development, tissue repair, and immune responses. Anthis N J, Campbell I D, 2011. TGF.beta.-induced activation of the p38 MAPK pathway has been demonstrated to be related to integrin signaling and implicated in epithelial-mesenchymal transition in certain cell lines.sup.18. TS2 and CD29 antibodies, which promote and inhibit integrin activation, respectively, were used to investigate a possible relationship between integrin signaling and TGF.beta.-induced generation of T.beta.RI ICD..sup.19 No major effects on TGF.beta.-induced formation of the T.beta.RI ICD was observed, when integrins were activated or inhibited. FIG. 21.

[0153] Interestingly, results from this study show that the T.beta.RI ICD associates with p300 and the Snail promoter, thereby regulating expression of a subset of genes, including Snail, MMP2, and p300, all of which correlate with increased cell invasiveness.

[0154] Nuclear accumulation of T.beta.RI was observed in prostate, breast, and lung cancer cells and also in several cancer tissues, but not in primary prostate epithelial cells. Thus the pathway elucidated in this report occurs in different kinds of human tumors and is observed in human tumours, indicating that it contributes to tumour progression. Taken together, the data disclosed herein demonstrate that the cleavage and nuclear accumulation of T.beta.RI is part of a tumour promoting TGF.beta. signalling pathway.

References for Example 1

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Protein kinase Cepsilon is linked to 12-O-tetradecanoylphorbol-13-acetate-induced tumor necrosis factor-alpha ectodomain shedding and the development of metastatic squamous cell carcinoma in protein kinase Cepsilon transgenic mice. Cancer Res. 63, 6547-6555 (2003). [0169] 15. R. Bernardi, A. Papa, P. P. Pandolfi. Regulation of apoptosis by PML and the PML-NBs. Oncogene 27, 6299-6312 (2008). [0170] 16. J. P. Thiery, H. Acloque, R. Y. Huang, M. A. Nieto. Epithelial-mesenchymal transitions in development and disease. Cell 139, 871-890 (2009). [0171] 17. Anthis N J, Campbell I D. The tail of integrin activation. Trends Biochem Sci. 2011 Jan. 6. [0172] 18. Bhowmick N A, Zent R, Ghiassi M, McDonnell M, Moses H L Integrin beta 1 signaling is necessary for transforming growth factor-beta activation of p38MAPK and epithelial plasticity. J Biol. Chem. 276:46707-46713 (2001). [0173] 19. Byron A, Humphries J D, Askari J A, Craig S E, Mould A P, Humphries M J. Anti-integrin monoclonal antibodies. J Cell Sci. 2009 Nov. 15; 122(Pt 22):4009-11. [0174] 20. P. Franzen, H. Ichijo, K. Miyazono. Different signals mediate transforming growth factor-beta 1-induced growth inhibition and extracellular matrix production in prostatic carcinoma cells. Exp. Cell Res. 207, 1-7 (1993). [0175] 21. Castan ares, C., Redondo-Horcajo, M., Magan-Marchal, N., ten Dijke, P., Lamas, S. and Rodriguez-Pascual, F. Signaling by ALK5 mediates TGF-beta-induced ET-1 expression in endothelial cells: a role for migration and proliferation. J. Cell Sci. 120, 1256-1266 (2007). [0176] 22. Medici D, Shore E M, Lounev V Y, Kaplan F S, Kalluri R, Olsen B R. Conversion of vascular endothelial cells into multipotent stem-like cells. Nat. Med. 2010 December; 16(12):1400-6. Epub 2010 Nov. 21 [0177] 23. S. Edlund, S. Bu, N. Schuster, P. Aspenstrom, R. Heuchel, N. E. Heldin, P. ten Dijke, C. H. Heldin, M. Landstrim. Transforming growth factor-.beta.1 (TGF-.beta.)-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-activated kinase 1 and mitogen-activated protein kinase kinase 3. Mol. Biol. Cell 2, 529-544 (2003). [0178] 24. S. Edlund, S Y Lee, S. Grimsby, S. Zhang, P. Aspenstrom, C. H. Heldin, M. Landstrim. Interaction between Smad7 and -catenin: importance for transforming growth factor beta-induced apoptosis. Mol. Cell. Biol. 4, 1475-1488 (2005). [0179] 25. Yakymovych I, Engstrim U, Grimsby S, Heldin C H, Souchelnytskyi S. Inhibition of transforming growth factor-beta signaling by low molecular weight compounds interfering with ATP- or substrate-binding sites of the TGF beta type I receptor kinase. Biochemistry. 2002 Sep. 10; 41(36):11000-7 [0180] 26. Hayes S, Chawla A, Corvera S. TGF beta receptor internalization into EEA1-enriched early endosomes: role in signaling to Smad2. J. Cell Biol. 2002 Sep. 30; 158(7):1239-49.

Example 2

Cleavage of T.beta.RI by PS1 Results in Nuclear Translocation of an Intracellular Domain of T.beta.RI

MATERIALS AND METHODS

(i) Cell Culture

[0181] Human prostate cancer cell line PC-3U, derived from PC-3 cells (Frazen et al, 1993) and LnCap cell line were used in this study. PC-3U cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% penicillin/streptomycin (Pest). Cells were incubated at 37.degree. C. with 5% CO.sub.2. Transient transfection of PC-3U cells was performed using Fugene6 (Roche) following the instructions provided by the manufacturer. Cells were starved for at least 12 hours (12 h) in RPMI 1640 medium supplemented with 1% FBS, 1% glutamine and 1% penicillin/streptomycin. Later the cells were stimulated with 10 ng/ml of TGF.beta.1.

[0182] Wild type and presenilin-1 knock out (PS1.sup.-/-) mouse embryo fibroblasts (MEFs) and 293T cells were also used in this study. MEF and 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were cultivated at 37.degree. C. with 5% CO.sub.2. After being starved for at least 12 h in the DEME medium supplemented with 0.5% FBS, the cells were stimulated with 10 ng/ml of TGF.beta.1.

(ii) Antibodies and Other Reagents

[0183] TGF.beta.1 was obtained from R&D systems.

[0184] Antibodies against rabbit phospho-p38, mouse p38, phospho Smad2, rabbit Smad2, rabbit lamin-A, rabbit .beta.-tubulin, rabbit K48 monoclonal antibody were obtained from Cell signaling. Monoclonal anti-.beta.-actin antibody (1:1000) and mouse anti-Flag M2 monoclonal antibody were from Sigma. A rabbit anti-TRAF6 (C-term) antibody was obtained from Zymed laboratories. Rat anti presenilin-1 monoclonal antibody (N-term) was from Millipore. A mouse monoclonal antibody specific to polyubiquitinated Lys63 was from Enzo life sciences. Rabbit anti-HA antibody, rabbit anti-TGF.beta. receptor I, C-terminal fragment (V22 antibody), and mouse anti-ubiquitin (P4D1) were from Santa Cruz Biotechnology.

[0185] Secondary HRP conjugated anti-mouse, anti-rabbit, anti-rat IgG antibodies were from GE Healthcare. Antibodies specific to light chains of rabbit and mouse IgG were from Jackson laboratory. Secondary donkey anti-rabbit antibody (Alexa fluor 555 labeled), goat anti-rabbit antibody (Alexa fluor 488 labeled), donkey anti-mouse antibody (Alexa fluor labeled), and goat anti-rat antibody (Alexa fluor labeled) were from Invitrogen. Pefabloc was from Roche.

(iii) Immunoblotting and In Vivo Protein Interaction Analysis

[0186] After being treated with TGF.beta. for the indicated time periods, cells were washed once with ice-cold PBS and were lysed in ice-cold RIPA lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% Triton X 100, 10% (v/v) glycerol, 1 mM aprotinin, 1 mM Pefabloc). Protein concentration in the resultant lysate was measured using the BCA protein assay kit. Equal amount of proteins were used for immunoprecipitations and subjected to electrophoresis on 10%, 12%, 4-12% SDS-polyacrylamide gels, or 7% Tris acetate gels (Invitrogen). Afterwards, the proteins were transferred from the gel to a nitrocellulose membrane using the iBlot machine (Invitrogen).

(iv) siRNA

[0187] On-TARGETplus SMARTpool siRNA targeting human Presenilin-1 (PSEN-1) and GENOME non-targeting siRNA #1 were obtained from Dharmacon Research. PC-3U cells were transfected with specific or control siRNA using oligofectamine reagent (Invitrogen) following the protocol provided by the manufacturer.

(v) Plasmids and DNA Transfections

[0188] The caT.beta.RI-E161A plasmid construction was described in Sorrentino et al., 2008..sup.4 A construct for expressing constitutively active T.beta.RI fused with the HA tag at the C-terminal (HA-caT.beta.RI) was from P. ten Dijke (University of Leiden, The Netherlands). Amino acid substitutions were introduced into the HA-caT.beta.RI at positions V.sub.129 and I.sub.130, resulting in the T.beta.RI V129A/I130A mutant. A plasmid encoding T.beta.RI with the HA epitope inserted between amino acids 27 and 28 was described in Hayes et al., JCB 2002. HA-tagged wild-type T.beta.RI, K48 mutant, and K63 mutant were from Genentech, San Francisco, Calif. A plasmid expressing myc-tagged PS1 was from Prof. Bart De Strooper.

(vi) Immunofluorescence and Confocal Microscopy

[0189] PC-3U cells, grown on coverslips, were starved in 1% FCS for 24 h and stimulated with TGF.beta.1 for the indicated time periods. Cells were washed once in PBS and then fixed with 4% formaldehyde and then permeabilised with 2% Triton X 100 and later blocked in 5% BSA. Incubations with V22 antibody and antibodies against TRAF6, HA, and PS1 were performed for 1 hr. The cells were then incubated with Alexa 555-labeled or Alexa 488-labeled secondary antibodies. Afterwards, the coverslips were mounted in DAPI according to manufacturer's recommendations. The slides were analyzed with a Zeiss LSM 510 confocal microscope with 63.times. lens (numerical aperture 1.4). The specificity of primary TRAF6 or T.beta.RI antibodies or secondary antibodies was tested and no background staining was observed.

(vii) Nuclear Cytoplasmic Fractionation Assay

[0190] After being treated with TGF.beta. for the indicated time periods, the cells were washed two times with ice-cold PBS and then lysed in ice-cold PBS. The cells were suspended in PBS and briefly centrifuged. After centrifugation, cell pellets were collected and incubated for 5 min in ice-cold buffer 1 (20 mM Tris HCl pH 7.0, 10 mM KCl, 2 mM MgCl.sub.2, 0.5% NP40, 1 mM aprotinin, 1 mM Pefabloc). Afterwards, the cells were sheared mechanically with a syringe and needle and then centrifuged, the supernatant collected (the supernatant is the cytoplasmic protein fraction). The remaining pellet was washed three times with buffer 1, re-suspended in Buffer 2 (Buffer 1+0.5M NaCl), and centrifuged. The supernatant fraction was collected, which is the nuclear protein fraction.

(viii) Ubiquitination Assay

[0191] Ubiquitination assays for various proteins were performed following the protocol described in [9].

(viiii) RNA Isolation and RT-PCR

[0192] Total RNAs were isolated from cells using an RNeasy mini kit (Qiagen), according to the manufacturer instructions. Two microgram of total RNAs were used for complementary DNA (cDNA) synthesis using the Thermoscript RT PCR system (Invitrogen), following the manufacturer's instructions.

(x) Quantitative Real Time PCR (qRT PCR)

[0193] Two microgram of cDNAs, as prepared following the method described in (viiii) above were analyzed in duplicates by real-time PCR (RT-PCR) using the Stratagene system, with SYBR green (Applied Biosystems) to detect the PCR products. Specific primers for TRAF6, PS1 and T.beta.RI were synthesized or purchased from Sigma Aldrich. GAPDH was used as an internal control.

Results

(i) TGF.beta. Regulates PS1 Expression

[0194] Presenilins are synthesized as holoproteins, which are endoproteolysed to produce NTF/CTF heterodimers. In order to examine whether TGF.beta. regulates expression of PS1, PC-3U cells were stimulated with TGF.beta. for the indicated time periods. Western Blot analyses of lysates derived from the TGF.beta.1-treated cells was performed using an antibody that recognizes the PS1-holoprotein (45 kDa) or the PS1-C-terminal fragment (CTF) (18 kDa). Endogenous expression of PS1 holoprotein was enhanced upon TGF.beta. stimulation and a protein band having a molecular weight of 18 kDa, corresponding to PS1 CTF, was observed after TGF.beta.1 treatment for 0.15 hrs. FIG. 9, panel a; and FIG. 22, panel A. Moreover, overexpression of Myc-PS1 in PC-3U cells at increasing concentrations (0, 2, 4, 6 .mu.g) and treatment of cells with or without TGF.beta.1 revealed a prominent band of 32 kDa that corresponds to the NTF of PS1. Interestingly, TGF.beta.1 treatment for 0.5 hrs, enhanced the expression of both full-length PS1 FL and PS1 NTF. FIG. 9, panel b; and FIG. 22, Panel B.

[0195] Next, siRNA silencing assay was perform to inhibit PS1 expression in PC-3U cells. Briefly, the cells were transiently transfected with a PS1-targeting siRNA (siPS1) or a non-targeting control siRNA (siCtrl). Non-transfected cells (NT) were used as a blank control. After transfection, the cells were treated with TGF.beta.1 for indicated time periods. As shown in FIG. 9, panel c; and FIG. 22, panel C. After being treated with TGF.beta.1 for 0.5 hr or 6 hrs, the cells transfected with the PS1-targeting siRNA showed significantly lower level of PS1 NTF expression as compared to the blank control and to the cells transfected with the control siRNA. Furthermore, RT-PCR analyses of PS1 mRNA expression indicate that TGF.beta. regulates the expression of PS1 as observed in the cells transfected with the control siRNA but not in the cells transfected with the PS1-targeting siRNA. FIG. 9, panel c; and FIG. 22, panel D. These data indicate that TGF.beta. regulates the expressions of PS1 holoprotein, PS1 NTF, and PS1 CTF.

(ii) PS1 Cleaves at an Intramembrane Region of T.beta.RI

[0196] PS1 functions as the catalytic core of the .gamma.-secretase complex and regulates the intracellular cleavage of various receptors. To examine whether PS1 regulates cleavage of T.beta.RI in PC-3U cells, siCtrl or siPS1 siRNA was transfected to the cells, which was then stimulated with TGF.beta.1 for 0.5 hr or 6 hrs. Production of a T.beta.RI intracellular domain (ICD, 34 kDa) was observed in the PC-3U cells transfected with the siPS1 siRNA but not in the cells transfected with the control siRNA, indicating that silencing of PS1 expression inhibited the generation of the T.beta.RI ICD fragment. FIG. 9, panel d; and FIG. 22, panel E. However, the expression of T.beta.RI full length (T.beta.RI-FL; 53 kDa) was not affected by silencing of PS1 expression. FIG. 9, panel d; and FIG. 22, panel E. Immunoblotting with specific antisera against p-Smad2, Smad2, phospho-p38, and p38 showed that inhibition of PS1 expression did not affect the level of these proteins, indicating that that cleavage of T.beta.RI does not affect the canonical Smad and p38 signaling. FIG. 9, panel d. Similar results were obtained from mouse embryonic PS1.sup.+/+ and PS1.sup.-/- fibroblasts (MEFs) by immunoblotting assays. FIG. 9, panel e; and FIG. 22, pane F.

[0197] PC-3U cells were transiently transfected with PS1 specific siRNA (siPS1), a non-targeting control siRNA (siCtrl), or Myc-PS1 in the presence or absence of TGF.beta.. Cell lysates derived from the transfected cells were subjected to immunoblotting for PS1. The results shown in FIG. 22, panel G indicate that siPS1 significantly reduced the level of PS1 in the cancer cells.

[0198] PS1.sup.+/+ and PS1.sup.-/- MEF cells and PS1.sup.-/- MEF cells transfected with Myc-PS1 were treated with or without TGF.beta.; cell lysates were subjected to immunoblotting for T.beta.RI and Myc(PS1). Little T.beta.RI-ICD was observed in cell lysates from PS1.sup.-/- MEF cells. FIG. 22, panel H.

(iii) PS1 Interacts with T.beta.RI

[0199] First, subconfluent PC-3U cells were starved and stimulated with TGF.beta.1 for various time periods as indicated in FIG. 10, panel a. Proteins in the treated cells were immunoprecipitated using an antibody specific to the NTF of PS1 and then immunoblotted using antibody V22 (recognizing the C-terminal fragment of T.beta.RI). As shown in FIG. 10, panel a, both endogenous PS1 and T.beta.RI were precipitated by the anti-PS1 antibody, indicating that these two proteins form a protein complex. Interaction between PS1 and T.beta.RI was confirmed in an co-immunoprecipitation using antibody V22 for immunoprecipitation and the anti-PS1 antibody for immunoblotting. FIG. 10, panel a.

[0200] In a further immunoprecipitation assay, a PS1 antibody that recognizes the PS1 holoprotein was used for immunoprecipitation and antibody V22 was used for immunoblotting. As shown in FIG. 10, panel b, PS1 and T.beta.RI were coimmuprecipitated from cell lysates of both untreated PC-3U cells and PC-3U cells stimulated TGF.beta.1 for various time periods as shown in FIG. 10, pane b.

[0201] Co-immunofluorescence and confocal imaging assay was performed to examine subcellular localization of PS1 and T.beta.RI in PC-3U cells treated or not treated with TGF.beta.1. As shown in FIG. 10, panel c, TGF.beta. stimulation enhanced co-localization of PS1 and T.beta.RI in the cells.

[0202] Taken together, the data shown above demonstrate that PS1 interacts with T.beta.RI in vivo.

(iii) TGF.beta. Promotes Lys63-Linked Polyubiquitination of PS1 N-Term

[0203] An in vivo ubiquitination assay was performed in PC-3U cells treated with TGF.beta. at various time points (i.e., 0, 0.25, 0.5, 1, and 2 hr after TGF.beta.1 treatment) so as to investigate the mechanism of TGF.beta. mediated activation of PS1. Cell lysates from the treated cells were heated in the presence of 1% SDS to disrupt non-covalent protein-protein interactions and then diluted with a lysis buffer (1:10). PS1 NTF was immunoprecipitated and analyzed by immunoblotting using an antiserum specific to K63-linked ubiquitins in T.beta.RI. The results indicate that PS1 NTF showed K63 ubiquitination 0.15 hr after TGF.beta.1 treatment. FIG. 11, panel a. No K48 ubiquitination of PS1 NTF was observed using an antibody specific to K48-linked ubiquitins. The same results were observed in 293T cells.

[0204] To confirm the finding that PS1 is ubiquitinated in K63-dependent manner, PC-3U cells were transfected with either wild type ubiquitin or the K63 or K48 ubiquitin mutants described in Example 1 above. As shown in FIG. 11, panel b. TGF.beta. induced a K63-linked polyubiquitination in PS1, which resulted in PS1 activation.

(iv) TGF.beta. Promotes TRAF6 Dependent K63 Ubiquitination of PS1 N-Term

[0205] Previous reports suggest that PS1 interacts with TRAF6 in a nerve growth factor (NGF) dependent manner, leading to enhanced autoubiquitination of TRAF6. RNA silencing analysis was performed to investigate whether TRAF6 is the E3 ligase that ubiquitinates PS1. PC-3U cells were transfected with a siRNA targeting TRAF6 (siTRAF6). After being treated with TGF-.beta.1, the transfected cells were subjected to an in vivo ubiquitination assays. PS1 NTF was immunoprecipitated and analyzed using an antiserum specific to K63 linked ubiquitin. The result indicates that PS1 underwent K63 linked ubiquitination in cells transfected with siCtrl and treated with TGF.beta.1 for 0.30 hr. The K63 linked ubiquitination was not observed in cells transfected with siTRAF6. These results demonstrate that TRAF6 is the E3 ligase involved in the ubiquitination of PS1-FL and PS1 NTF.

(v) PS1 Interacts with TRAF6 at the RING Domain

[0206] In line with the previous finding that NGF mediates TRAF6 and PS1 interaction [23], PC-3U cells were stimulated with TGF.beta.1 and co-immunoprecipitation experiments were performed by immunoprecipitating with an anti-PS1 NTF antibody and immunoblotted with an anti-TRAF6 antisera. As shown in FIG. 12, panel a, endogenous PS1 interacts with TRAF6, the interaction being enhanced by TGF.beta.. To examine the binding domain in TRAF6 for interaction with PS1, PC3-U cells were transfected with wild-type TRAF6 (Flag tagged) or with TRAF6 C70A mutant, which is deficient in the E3-ligase activity. Cell lysates where subjected to immunoprecipitation with the anti-PS1 NTF antibody and immunoblotted with an anti-Flag antibody to detect association between TRAF6 and PS1 NTF. As shown in FIG. 12, panel b, only wild-type TRAF6 interacted with endogenous PS1, while the TRAF6 C70A mutant did not, indicating that the E3 ligase activity of TRAF6 is required for its interaction and ubiquitination of PS1. Moreover, the interaction between TRAF6 and PS1 was enhanced by TGF.beta. stimulation. Deletion of the highly conserved RING domain at the N-Terminus of TRAF6 resulted in inhibition of the interaction.

(vi) TRAF6 Mediates Cleavage of T.beta.RI Through PS1

[0207] The expression of TRAF6 was silenced by the siTRAF6 siRNA in PC-3U cells to explore whether TRAF6 acts a central mediator in facilitating the interaction between T.beta.RI and PS1. Proteins obtained from the cells transfected with the siRNA were subjected to co-immunoprecipitation to examine whether endogenous T.beta.RI interacts with PS1 when TRAF6 was knocked out. No association of T.beta.RI and PS1 was observed in the TRAF6-knocked out cells, indicating that TRAF6 is required for the interaction between T.beta.RI and PS1. FIG. 13, panel a.

[0208] PC-3U cells were transfected with either the wild-type T.beta.RI or the T.beta.RI E161A mutant, which does not bind to TRAF6. Sorrentino et al., 2008. Cell lysates from the transfected cells were subjected to co-immunoprecipitation to examine association between T.beta.RI and PS1 in the transfected cells. As shown in FIG. 13, panel b, the wild-type T.beta.RI, but not the E161A mutant, interacts with PS1.

[0209] In sum, the data disclosed above indicates that TRAF6 is a crucial factor in T.beta.RI/PS1 interaction. It plays a major role in recruiting PS1 to interact with T.beta.RI, leading to the cleavage of T.beta.RI and generation of a T.beta.RI ICD.

(vii) Mutation in T.beta.RI Transmembrane Region Inhibits the Cleavage by PS1

[0210] FIG. 23, panel A shows in silico identification of a cleavage site for PS1 in the transmembrane domain of T.beta.RI. It appears that the V.sub.129-I.sub.130 in the transmembrane domain of T.beta.RI constitute a possible consensus motif for PS1 cleavage. To verify this, a TM-V129A/I130A double mutant of T.beta.RI was constructed. Constructs expressing caT.beta.RI and the TM-V129A/I130A T.beta.RI mutant were introduced into PC-3U cells and production of T.beta.RI ICD was examined before and after TGF.beta. stimulation. Production of T.beta.RI ICD was observed only in the TGF-treated cells transfected with caT.beta.RI construct, indicating that mutations at the V.sub.129-I.sub.130 motif suppressed cleavage of T.beta.RI. FIG. 14, panel a; and FIG. 23, panel B.

[0211] Co-immunoprecipitation analysis was performed to examine the interaction between PS1 with caT.beta.RI and the TM-V129A/I130A T.beta.RI mutant. As shown in FIG. 14, panel b, endogenous PS1 interacts with both caT.beta.RI and the mutant.

[0212] Finally, the effect of the TM-V129A/I130A T.beta.RI mutant on the TGF.beta. mediated Smad- and non-Smad (p38) signaling was examined. Neither of these pathways was found to be affected by the cleavage of the T.beta.RI. FIG. 23, panel B. Similar results were observed in 293T cells. FIG. 14, panel b; and FIG. 23, panel C.

[0213] Immunofluorescence analysis was performed to examine whether the TM-V129A/I130A T.beta.RI mutant, which is not cleaved by PS1, affects the nuclear translocation of T.beta.RI ICD. The T.beta.RI KD mutant described in Example 1 above and the TM-V129A/I130A T.beta.RI mutant, both being HA-tagged, were transiently transfected into PC-3U cells, which were then treated with TGF.beta.1 for different time periods (see FIG. 14, panel c). The subcellular localization of both mutants were observed by anti-HA antisera staining and confocal microscopy. As shown in FIG. 14, panel c, and FIG. 23, panel D, nuclear localization of T.beta.RI was only observed in cells transfected with the T.beta.RI KD mutant (after being treated by TGF.beta.1 for 0.5 or 6 hrs) but not in the cells transfected with the TM-V129A/I130A T.beta.RI mutant. Moreover TGF.beta.1 treatment enhanced T.beta.RI ICD nuclear translocation, whereas this did not occur in the cells transfected with the TM-V129A/I130A T.beta.RI mutant.

[0214] In order to further substantiate the finding that the TM-V129A/I130A T.beta.RI mutant does not translocate to the nuclei, nuclear-cytoplasmic fractionation assays were performed. Data from these assays indicate that only the KD mutant translocated to the nuclei, whereas the TM-V129A/I130A T.beta.RI mutant did not.

[0215] qRT-PCR analysis was performed to determine the expression levels of T.beta.RI using mRNAs extracted from PC-3U cells, which were transiently transfected with siCtrl and siPS-1 and treated with TGF.beta. at various dosages. siPS-1 significantly reduced the level of T.beta.RI as shown in FIG. 23, panel E. Next, chromatin immunoprecipitation assay was performed to detect binding of T.beta.RI to its own promoter, using V22 antibody against the endogenous T.beta.RI in PC-3U cells treated or not with TGF.beta.. As shown in FIG. 23, panel F, TGF.beta. significantly increased the level of promoter-bound T.beta.RI. Further, PC-3U cells transiently transfected and treated as indicated in FIG. 23, panel G were subjected to an invasion assay as described above. A much higher level of cancer cell invasion was observed in the presence of TGF.beta., which was associated with a higher level of cleaved T.beta.RI. FIG. 23, panel G.

[0216] Taken together, the data discussed above demonstrate that mutation of the VI motif to AA inhibits the cleavage of T.beta.RI by PS1, thereby inhibiting T.beta.RI ICD nuclear translocation.

(viii) T.beta.RI Promotes its Own Expression

[0217] ChIP assays were performed to investigate whether generation of T.beta.RI ICD and its nuclear translocation promote T.beta.RI expression. The result thus obtained shows that T.beta.RI ICD associates with the promoter of the T.beta.RI gene, thereby up-regulating its expression. FIG. 14, panel d.

Discussion

[0218] Presenilin1 (PS1) is a polytransmembrane protein that plays an integral role as catalytic subunit of the .gamma.-secretase complex [12,13]. The .gamma.-secretase complex cleave various transmembrane receptors, including like Notch receptor, N/E-Cadherin, IL-1, the neurotrophin receptor p75. [14] The inactive PS1 holoprotein (42-43 kDa) undergoes endoproteolytic cleavage by an unknown presenilinase to generate an N-terminal (NTF, 27-28 kDa) and a C-terminal fragment (CTF, 16-17 kDa). [15] PS1 is localized in the endoplamic reticulum (ER), golgi apparatus, ER/Golgi intermediate compartments, endosomes, lysosomes, phagosomes, plasma membrane, and mitochondria. [16-18] The structure of PS1 has been highly debated according to different studies. The recent model proposes PS1 to exist as a nine transmembrane (TM) protein with the cytosolic N-terminal region spanning the first six hydrophobic regions (I-VI TM), a cytosolic loop domain (VII TM) and the C-terminal region spanning three TMDs (VIII-IX TM) localized in the lumen/extracellular space. [19,20] The association of PS1 with nicastrin, presenilin enhancer 2 (pen-2), and the anterior pharynx defective1 (aph-1) proteins, leads to an active .gamma.-secretase complex. [21] Previous reports suggest that PS1 has a conserved TRAF6 binding motif and its association with TRAF6 leads to enhanced TRAF6 autoubiquitination and thereby promotes ubiquitination of p75 neurotrophin receptor (p75.sup.NTR). Powell et al, 2009.

[0219] As disclosed herein, TRAF6 ubiquitinates T.beta.RI in Lys63-dependent manner, leading to its cleavage at the ectodomain region by TACE. This ectodomain shedding leads to generation of an ICD, which translocates to the nuclei and binds to the transcriptional co-activator p300, thereby regulating the expression of certain TGF.beta. target genes such as Snail-1. It was known in the art that such genes are involved in cancer cell invasion. It is further disclosed herein that TGF.beta. regulates PS1 expression, leading to the activation of PS1 by K63 linked polyubiquitination. The activated PS1 in turn leads to cleavage of the T.beta.RI at the transmembrane region in a TGF.beta.-dependent manner, thereby generating an ICD (34 kDa). This ICD fragment was found to accumulate in nuclei, leading to upregulation of T.beta.RI expression. TRAF6 is the E3 ligase that ubiquitinates PS1 in a Lys63-dependent manner. In addition, TRAF6 is required for the interaction between T.beta.RI and PS1.

[0220] As disclosed above, T.beta.RI co-immunoprecipitated with PS1 and this interaction is constitutive and that TGF.beta.1 treatment enhanced the interaction. T.beta.RI was found to interact with PS1 holoprotein and also with PS1-NTF. These data suggest that T.beta.RI interaction with PS1 is a constitutive interaction. Moreover, overexpression of HA-T.beta.RI and Myc-PS1 and analyses of their subcellular localization by confocal imaging revealed that T.beta.RI and PS1 in deed co-localize and that TGF.beta.1 treatment enhances their co-localization.

[0221] TRAF6 was initially identified for its role as an adaptor protein to activate NF-.kappa.B signaling by IL-1[24]. TRAF6 is classified as an E3 ligase as it interacts with the E2 conjugating enzyme (E2) Msm2 and thereby mediates K63 ubiquitination of various proteins. The carboxyl TRAF-domain plays an important role in mediating interaction with various proteins or polyubiquitination of various proteins.

[0222] Previously, it was reported that NGF mediates TRAF6 interaction with PS1 and PS1 has a consensus TRAF6-binding motif which leads to enhanced ubiquitination of TRAF6 upon NGF mediated stimulation of p75.sup.NTR. [23] The disclosure herein that PS1 interacts with TRAF6 at its RING domain is crucial as it leads to ubiquitination of PS1 in a TGF.beta.-dependent manner. In line with the previous report, the disclosure herein shows that there is constitutive interaction between endogenous PS1 and TRAF6, while TGF-.beta. stimulation enhances the interaction at. Transient transfection of wt TRAF6 and the E3-ligase deficient C70A mutant and co-immunoprecipitation experiments revealed that PS1 interacts with TRAF6 at the RING domain, as the interaction was not observed with the E3 ligase deficient C70A mutant. Moreover, the results herein also demonstrate that TRAF6 ubiquitinates PS1 in a K63 dependent manner as in vivo ubiquitination assays with an anti-PS1 NTF antibody revealed that PS1 ubiquitination was observed only in siCtrl but not in siTRAF6. This indicates that TRAF6 is the E3 ligase that ubiquitinates PS1. Moreover, a time course of in vivo ubiquitination assays revealed that PS1 NTF is ubiquitinated upon TGF.beta.1 treatment for 0.15 hrs in a K63 dependent manner, while no significant sign of K48 ubiquitination was observed. This result was confirmed in cells transfected with wild type ubiquitin and the K63 and K48 mutants. Together, the data disclosed herein indicate that PS1 NTF is ubiquitinated in a K63 dependent manner by TRAF6, which is mediated by TGF.beta..

[0223] It was known that TRAF6 interacts with T.beta.RI at a highly conserved motif, leading to TGF.beta. mediated autoubiquitination of TRAF6 and subsequent activation of the TAK1-p38 pathway, which leads to apoptosis. [9] Moreover, the TRAF proteins act as adaptor proteins in transducing signals to various signaling partners. Suppression of TRAF6 expression via RNA silencing inhibited the endogenous interaction of T.beta.RI and PS1, indicating that TRAF6 is required for their interaction. Moreover, the T.beta.RI E161A mutant, which lacks the ability to bind to TRAF6, completely abolished the interaction between T.beta.RI and PS1. In sum, the data disclosed herein show that TRAF6 plays a central role in regulating the interaction between T.beta.RI and PS1 and thereby promoting cleavage of the T.beta.RI.

[0224] In conclusion, the results disclosed in Example 2 demonstrate that (a) the T.beta.RI is cleaved in its transmembrane domain by PS1, (b) TRAF6 plays a major role in the activation of PS1 by K63 linked polyubiquitination and mediates the cleavage of the T.beta.RI by recruiting PS1, (c) the V129A/I130A mutant inhibits the cleavage of the T.beta.RI, and (d) the T.beta.RI ICD generated by the cleavage event translocates to the nuclei and binds to the T.beta.RI promoter and regulates it expression.

References for Example 2

[0225] 1. Massague, J., TGFbeta in Cancer. Cell, 2008. 134(2): p. 215-30. [0226] 2. Heldin, C. H., M. Landstrom, and A. Moustakas, Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition. Curr Opin Cell Biol, 2009. 21(2): p. 166-76. [0227] 3. Wrana, J. L., et al., Mechanism of activation of the TGF-beta receptor. Nature, 1994. 370(6488): p. 341-7. [0228] 4. Souchelnytskyi, S., et al., Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling. J Biol Chem, 1997. 272(44): p. 28107-15. [0229] 5. Abdollah, S., et al., TbetaRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling. J Biol Chem, 1997. 272(44): p. 27678-85. [0230] 6. Macias-Silva, M., et al., MADR2 is a substrate of the TGFbeta receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell, 1996. 87(7): p. 1215-24. [0231] 7. Tsukazaki, T., et al., SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor. Cell, 1998. 95(6): p. 779-91. [0232] 8. Nakao, A., et al., TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J, 1997. 16(17): p. 5353-62. [0233] 9. Sorrentino, A., et al., The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner. Nat Cell Biol, 2008. 10(10): p. 1199-207. [0234] 10. Thakur, N., et al., TGF-beta uses the E3-ligase TRAF6 to turn on the kinase TAK1 to kill prostate cancer cells. Future Oncol, 2009. 5(1): p. 1-3. [0235] 11. Landstrom, M., The TAK1-TRAF6 signalling pathway. Int J Biochem Cell Biol, 2010. 42(5): p. 585-9. [0236] 12. Bergmans, B. A. and B. De Strooper, gamma-secretases: from cell biology to therapeutic strategies. Lancet Neurol, 2010. 9(2): p. 215-26. [0237] 13. De Strooper, B., et al., Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature, 1998. 391(6665): p. 387-90. [0238] 14. McCarthy, J. V., C. Twomey, and P. Wujek, Presenilin-dependent regulated intramembrane proteolysis and gamma-secretase activity. Cell Mol Life Sci, 2009. 66(9): p. 1534-55. [0239] 15. Thinakaran, G., et al., Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo. Neuron, 1996. 17(1): p. 181-90. [0240] 16. Vetrivel, K. S., et al., Pathological and physiological functions of presenilins. Mol Neurodegener, 2006. 1: p. 4. [0241] 17. Brunkan, A. L. and A. M. Goate, Presenilin function and gamma-secretase activity. J Neurochem, 2005. 93(4): p. 769-92. [0242] 18. De Strooper, B., et al., Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins. J Biol Chem, 1997. 272(6): p. 3590-8. [0243] 19. Laudon, H., et al., A nine-transmembrane domain topology for presenilin 1. J Biol Chem, 2005. 280(42): p. 35352-60. [0244] 20. Spasic, D., et al., Presenilin-1 maintains a nine-transmembrane topology throughout the secretory pathway. J Biol Chem, 2006. 281(36): p. 26569-77. [0245] 21. Wakabayashi, T. and B. De Strooper, Presenilins: members of the gamma-secretase quartets, but part-time soloists too. Physiology (Bethesda), 2008. 23: p. 194-204. [0246] 22. Liu, C., et al., TACE-mediated ectodomain shedding of the type I TGF-beta receptor downregulates TGF-beta signaling. Mol Cell, 2009. 35(1): p. 26-36. [0247] 23. Powell, J. C., et al., Association between Presenilin-1 and TRAF6 modulates regulated intramembrane proteolysis of the p75NTR neurotrophin receptor. J Neurochem, 2009. 108(1): p. 216-30. [0248] 24. Cao, Z., et al., TRAF6 is a signal transducer for interleukin-1. Nature, 1996. 383(6599): p. 443-6.

Example 3

Inhibition of Cancer Cell Invasion Using .gamma.-Secretase Inhibitors and Anti-T.beta.RI Antibodies

[0249] The cancer cell invasion assay described in Example 1 above was performed to examine the ability of .gamma.-secretase inhibitors in suppressing cancel cell invasion. Prostate cancers cells PC-3U, human lung carcinoma cells A549, and human breast carcinoma cells MDA-MB-231 were cultured following routine procedures, treated with TGF.beta. in the presence of absence of L-685,458, a .gamma.-secretase inhibitor. Cells were visualized by staining with a crystal violet cell staining solution. As shown in FIG. 24, the .gamma.-secretase inhibitor significantly suppressed TGF.beta.-induced cancel cell invasion.

[0250] Next, polyclonal antibodies specific to amino acid residues 114-124 in the extracellular domain of T.beta.RI (SEQ ID NO: 1) and polyclonal antibodies specific to amino acid residues 490-503 were generated in rabbit, following routine methods.

[0251] The ability of the above-noted polyclonal antibodies for inhibiting cancer cell invasion was examined by the invasion assay described in Example 1 above. Prostate cancer cells (PC-3U cells) were incubated in the presence of polyclonal antibodies specific to the 114-124 epitope (ALK5 Ab 114-124) or polyclonal antibodies specific to the 490-503 epitope (ALK5 Ab 490-503), or in the absence of antibodies. Cells were visualized by staining with crystal violet cell stain solution. As shown in FIG. 25, panels A and B, ALK5 Ab 114-124 significantly suppressed cancer invasion, while ALK5 Ab 490-503 did not exihibit this inhibitory effect.

[0252] Monoclonal antibodies are generated via standard hybridoma technology, using various epitopes of T.beta.RI, such as epitope 114-124 and epitope 490-503. The inhibitory effects of these antibodies on cancer cell invasion are tested by the same invasion assay, using various cancer cell lines, e.g., breast cancer cells, lung cancer cells, prostate cancer cells, and colorectal cancer cells.

[0253] After the antibodies' inhibitory effects are confirmed, these antibodies are further analyzed in an animal cancer model such as a rat (Dunning) or mouse (TRAMP) prostate cancer model as described in Johansson et al., 2011, BJU Int. 107(11):1818-1824 to confirm their activity in suppressing cancer cell invasion in vivo.

[0254] The monoclonal antibodies noted above can be modified to produce humanized antibodies by replacing the framework regions in non-human antibodies with the framework regions from a human antibody and retaining regions/residues responsible for antigen binding (e.g., the complementarity determining regions, particularly the specificity-determining residues therein). Methods to identify regions/residues in the heavy and light chains of an antibody are well known in the art. See, e.g., Almagro, J. Mol. Recognit. 17:132-143 (2004); and Chothia et al., J. Mol. Biol. 227:799-817 (1987).

Other Embodiments

[0255] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0256] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Sequence CWU 1

1

211503PRTH. sapiens 1Met Glu Ala Ala Val Ala Ala Pro Arg Pro Arg Leu Leu Leu Leu Val 1 5 10 15 Leu Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Leu Pro Gly Ala Thr 20 25 30 Ala Leu Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys 35 40 45 Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys 50 55 60 Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg 65 70 75 80 Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr 85 90 95 Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro 100 105 110 Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Leu Ala Ala 115 120 125 Val Ile Ala Gly Pro Val Cys Phe Val Cys Ile Ser Leu Met Leu Met 130 135 140 Val Tyr Ile Cys His Asn Arg Thr Val Ile His His Arg Val Pro Asn 145 150 155 160 Glu Glu Asp Pro Ser Leu Asp Arg Pro Phe Ile Ser Glu Gly Thr Thr 165 170 175 Leu Lys Asp Leu Ile Tyr Asp Met Thr Thr Ser Gly Ser Gly Ser Gly 180 185 190 Leu Pro Leu Leu Val Gln Arg Thr Ile Ala Arg Thr Ile Val Leu Gln 195 200 205 Glu Ser Ile Gly Lys Gly Arg Phe Gly Glu Val Trp Arg Gly Lys Trp 210 215 220 Arg Gly Glu Glu Val Ala Val Lys Ile Phe Ser Ser Arg Glu Glu Arg 225 230 235 240 Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr Val Met Leu Arg His 245 250 255 Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Asn Lys Asp Asn Gly Thr 260 265 270 Trp Thr Gln Leu Trp Leu Val Ser Asp Tyr His Glu His Gly Ser Leu 275 280 285 Phe Asp Tyr Leu Asn Arg Tyr Thr Val Thr Val Glu Gly Met Ile Lys 290 295 300 Leu Ala Leu Ser Thr Ala Ser Gly Leu Ala His Leu His Met Glu Ile 305 310 315 320 Val Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser 325 330 335 Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu 340 345 350 Gly Leu Ala Val Arg His Asp Ser Ala Thr Asp Thr Ile Asp Ile Ala 355 360 365 Pro Asn His Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu 370 375 380 Asp Asp Ser Ile Asn Met Lys His Phe Glu Ser Phe Lys Arg Ala Asp 385 390 395 400 Ile Tyr Ala Met Gly Leu Val Phe Trp Glu Ile Ala Arg Arg Cys Ser 405 410 415 Ile Gly Gly Ile His Glu Asp Tyr Gln Leu Pro Tyr Tyr Asp Leu Val 420 425 430 Pro Ser Asp Pro Ser Val Glu Glu Met Arg Lys Val Val Cys Glu Gln 435 440 445 Lys Leu Arg Pro Asn Ile Pro Asn Arg Trp Gln Ser Cys Glu Ala Leu 450 455 460 Arg Val Met Ala Lys Ile Met Arg Glu Cys Trp Tyr Ala Asn Gly Ala 465 470 475 480 Ala Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu Ser Gln Leu Ser 485 490 495 Gln Gln Glu Gly Ile Lys Met 500 221RNAArtificial SequencesiRNA targeting ALK5 2aacauauugc ugcaaccagg a 21321RNAArtificial SequenceNon-specific control siRNA 3aacagucgcg uuugcgacug g 21420DNAArtificial SequenceTbetaRI forward primer 4tgttggtacc caaggaaagc 20520DNAArtificial SequenceTbetaRI reverse primer 5cactctgtgg tttggagcaa 20620DNAArtificial Sequencep300 forward primer 6gggactaacc aatggtggtg 20720DNAArtificial Sequencep300 reverse primer 7gtcattgggc ttttgaccat 20820DNAArtificial SequenceSNAIL 1 forward primer 8gagcatacag ccccatcact 20920DNAArtificial SequenceSNAIL 1 reverse primer 9gggtctgaaa gcttggactg 201020DNAArtificial SequenceSmad7 forward primer 10tcctgctgtg caaagtgttc 201120DNAArtificial SequenceSmad7 reverse primer 11tctggacagt ctgcagttgg 201220DNAArtificial SequenceMMP-2 forward primer 12aggccgacat catggtactc 201320DNAArtificial SequenceMMP-2 reverse primer 13ggtcagtgct ggagaaggtc 201420DNAArtificial SequencePAI 1 forward primer 14ctctctctgc cctcaccaac 201520DNAArtificial SequencePAI 1 reverse primer 15gtggagaggc tcttggtctg 201620DNAArtificial SequenceSNAIL1 forward primer 16ggactcaggg agactcatgg 201720DNAArtificial SequenceSNAIL1 reverse primer 17gggtctacgg aaacctctgg 201824PRTH. sapiens 18Leu Gly Pro Val Glu Leu Ala Ala Val Ile Ala Gly Pro Val Cys Phe 1 5 10 15 Val Cys Ile Ser Leu Met Leu Met 20 1924PRTR. norvegicus 19Leu Gly Pro Val Glu Leu Ala Ala Val Ile Ala Gly Pro Val Cys Phe 1 5 10 15 Val Cys Ile Ser Leu Met Leu Met 20 2024PRTB. taurus 20Leu Gly Pro Val Glu Leu Ala Ala Val Ile Ala Gly Pro Val Cys Phe 1 5 10 15 Val Cys Ile Ala Leu Met Leu Met 20 2124PRTG. gallus 21Leu Gly Pro Val Glu Leu Ala Ala Val Ile Ala Gly Pro Val Cys Phe 1 5 10 15 Val Cys Ile Ser Leu Met Leu Ile 20

Claims

1. An isolated antibody that binds to a type I receptor of transforming growth factor beta (T.beta.RI) and blocks its cleavage.

2. (canceled)

3. The isolated antibody of claim 1, wherein the antibody binds to a T.beta.RI antigen epitope that (i) encompasses the G residue, the L residue, or both, that correspond to G.sub.120 and L.sub.121, respectively, in SEQ ID NO: 1; or (ii) (ii) encompasses the V residue, the I residue, or both that correspond to V.sub.129 and I.sub.130, respectively, in SEQ ID NO:1.

4-6. (canceled)

7. The isolated antibody of 1, wherein the antibody binds to an epitope within residues 114-124 in SEQ ID NO:1.

8. (canceled)

9. The isolated antibody of claim 1, wherein the antibody is a humanized antibody or a human antibody.

10. (canceled)

11. The isolated antibody of claim 1, wherein the antibody is a bi-specific antibody that: (i) binds to both the T.beta.RI and TACE, (ii) binds to both the T.beta.RI and PS1, or (iii) binds to both the T.beta.RI and MMP14.

12-16. (canceled)

17. A method for inhibiting cleavage of a type I receptor of transforming growth factor beta (T.beta.RI), the method comprising contacting a cell with a T.beta.RI cleavage inhibitor in an amount sufficient to inhibit cleavage of a T.beta.RI to release an intracellular domain (ICD) of the T.beta.RI, thereby blocking the ICD from translocating to the nucleus of the cell.

18. The method of claim 17, wherein the amount of the T.beta.RI cleavage inhibitor is sufficient to inhibit cleavage of the T.beta.RI between the G and L residues therein that correspond to G.sub.120 and L.sub.121, respectively, in SEQ ID NO: 1, or sufficient to inhibit cleavage of the T.beta.RI between the V and I residues therein that correspond to V.sub.129 and I.sub.130, respectively, in SEQ ID NO:1.

19. (canceled)

20. The method of claim 17, wherein the T.beta.RI cleavage inhibitor is: (i) an antibody that binds to the T.beta.RI and blocks its cleavage, (ii) an inhibitor of tumor necrosis factor receptor-associated factor 6 (TRAF6), (iii) an inhibitor of tumor necrosis factor-alpha converting enzyme (TACE), (iv) an inhibitor of protein kinase C zeta (PKC.zeta.), (v) an inhibitor of Presenilin 1 (PS1), or (vi) a combination of any of (i) to (v).

21. The method of claim 20, wherein the T.beta.RI cleavage inhibitor is the antibody set forth in (i), which binds to an antigen epitope that encompasses the G residue, the L residue, or both in the T.beta.RI that correspond to G.sub.120 and L.sub.121, respectively, in SEQ ID NO:1; or which binds to an antigen epitope of the T.beta.RI that encompasses the V residue, the I residue, or both in the T.beta.RI that correspond to V.sub.129 and I.sub.130, respectively, in SEQ ID NO:1.

22-26. (canceled)

27. The method of claim 21, wherein the antigen epitope is within residues 114-124 in SEQ ID NO:1.

28. (canceled)

29. The method of claim 21, wherein the antibody is a bispecific antibody that: (i) binds to both T.beta.RI and TACE, (ii) binds to both the T.beta.RI and PS1, or (iii) binds to both the T.beta.RI and MMP14.

30-31. (canceled)

32. The method of claim 20, wherein: (a) the T.beta.RI cleavage inhibitor is (ii) and wherein the TRAF6 inhibitor is an anti-TRAF6 antibody, a TRAF6-specific interfering nucleic acid, a TRAF6 inhibitory peptide, or a small molecule; (b) the T.beta.RI cleavage inhibitor is (iii) and wherein the TACE inhibitor is an anti-TACE antibody, a TACE-specific interfering nucleic acid, or a small molecule; (c) the T.beta.RI cleavage inhibitor is (iv) and wherein the PKC.zeta. inhibitor is an anti-PKC.zeta. antibody, a PKC.zeta.-specific interfering nucleic acid, a PKC.zeta. pseudosubstrate, or a small molecule; or (d) the T.beta.RI cleavage inhibitor is (v) and wherein the PS1 inhibitor is an anti-PS1 antibody, a PS1-specific interfering nucleic acid, or a small molecule.

33. (canceled)

34. The method of claim 32, wherein the TACE inhibitor is a small molecule selected from the group consisting of TAPI-2, BMS-561392, TAPI-I, DPC-333, Spiro-cyclic b-amino acid derivatives, INCB 3619, GW280264X, TMI-1, and TNF484, DPC-333, Sch-709156, and Doxycycline.

35-37. (canceled)

38. The method of claim 17, wherein the cell is a cancer cell.

39. (canceled)

40. The method of claim 38, wherein the contacting step is performed by administering the T.beta.RI cleavage inhibitor to a patient carrying the cancer cell.

41-65. (canceled)

66. A method for reducing invasiveness of cancer cells in a subject, comprising administering to a subject carrying cancer cells a pharmaceutical composition comprising a T.beta.RI cleavage inhibitor in an amount sufficient to inhibit cleavage of a T.beta.RI to release an intracellular domain (ICD) of the T.beta.RI, thereby blocking the ICD from translocating to the nuclei of the cancer cells and reducing their invasiveness.

67. The method of claim 66, wherein the amount of the T.beta.RI cleavage inhibitor is sufficient to inhibit cleavage of the T.beta.RI between the G and L residues therein that correspond to G.sub.120 and L.sub.121, respectively, in SEQ ID NO: 1 or sufficient to inhibit cleavage of the T.beta.RI between the V and I residues therein that correspond to V.sub.129 and I.sub.130, respectively, in SEQ ID NO:1.

68. (canceled)

69. The method of claim 66, wherein the T.beta.RI cleavage inhibitor is: (i) an antibody that binds to the T.beta.RI and blocks the cleavage of the T.beta.RI, (ii) an inhibitor of tumor necrosis factor receptor-associated factor 6 (TRAF6), (iii) an inhibitor of tumor necrosis factor-alpha converting enzyme (TACE), (iv) an inhibitor of Protein Kinase C zeta (PKC.zeta.), (v) an inhibitor of Presenilin 1(PS1), or (vi) a combination of any of (i) to (v).

70. The method of claim 69, wherein the T.beta.RI cleavage inhibitor is the antibody set forth in (i), which binds to an antigen epitope of the T.beta.RI that encompasses the G residue, the L residue, or both in the T.beta.RI that correspond to G.sub.120 and L.sub.121, respectively in SEQ ID NO:1; or which binds to an antigen epitope of the T.beta.RI that encompasses the V residue, the I residue, or both in the T.beta.RI that correspond to V.sub.129 and I.sub.130, respectively, in SEQ ID NO: 1.

71-75. (canceled)

76. The method of claim 70, wherein the antigen epitope is within residues 114-124 in SEQ ID NO: 1.

77. (canceled)

78. The method of claim 70, wherein the antibody is a bispecific antibody that: (iv) binds to both T.beta.RI and TACE, (v) binds to both the T.beta.RI and PS1, or (vi) binds to both the T.beta.RI and MMP14.

79-80. (canceled)

81. The method of claim 69, wherein; (a) the T.beta.RI cleavage inhibitor is (ii) and wherein the TRAF6 inhibitor is an anti-TRAF6 antibody, a TRAF6-specific interfering nucleic acid, a TRAF6 inhibitory peptide, or a small molecule; (b) the T.beta.RI cleavage inhibitor is (iii) and wherein the TACE inhibitor is an anti-TACE antibody, a TACE-specific interfering nucleic acid, or a small molecule; (c) the T.beta.RI cleavage inhibitor is (iv) and wherein the PKC.zeta. inhibitor is an anti-PKC.zeta. antibody, a PKC.zeta.-specific interfering nucleic acid, a PKC.zeta. pseudosubstrate, or a small molecule; or (d) the T.beta.RI cleavage inhibitor is (v) and wherein the PS1 inhibitor is an anti-PS1 antibody, a PS1-specific interfering nucleic acid, or a small molecule.

82. (canceled)

83. The method of claim 81, wherein the TACE inhibitor is a small molecule selected from TAPI-2, BMS-561392, TAPI-1, DPC-333, Spiro-cyclic b-amino acid derivatives, INCB 3619, GW280264X, TMI-1, and TNF484, DPC-333, Sch-709156, and Doxycycline.

84-88. (canceled)

89. The method of claim 87, wherein the amount of the T.beta.RI cleavage inhibitor is sufficient to reduce cancer metastasis in a human cancer patient.

90. A method, comprising: examining presence or absence of an intracellular domain (ICD) of a type I receptor of transforming growth factor beta (T.beta.RI) in the nuclei of cells in a sample, and determining whether the sample contains cancer cells, wherein nuclear localization of the ICD indicates presence of cancer cells in the sample.

91-96. (canceled)

97. A method, comprising: examining presence or absence of an intracellular domain (ICD) of a type I receptor of transforming growth factor beta (T.beta.RI) in the nuclei of cancer cells in a tissue sample of cancer patient, and assessing invasiveness of the cancer cells, wherein nuclear localization of the ICD indicates that the cancer cells are invasive.

98-101. (canceled)

102. A method for identifying a T.beta.RI cleavage inhibitor, comprising: contacting a cancer cell that expresses a T.beta.RI with a candidate compound in the presence of TGF.beta., wherein TGF.beta. induces T.beta.RI cleavage, examining presence or absence of an intracellular domain (ICD) of the T.beta.RI in the nuclear of the cancer cell, and assessing whether the candidate compound is a T.beta.RI cleavage inhibitor, wherein the candidate compound is identified as a T.beta.RI cleavage inhibitor if (i) the level of ICD nuclear localization in the cell treated with the candidate compound is lower than that in a cell not treated with the candidate compound, or (ii) the level of an extracellular domain of the T.beta.RI in the culture medium of the cell treated with the candidate compound is higher than that in the culture medium of a cell not treated with the candidate compound.

103. (canceled)