Fibronectin Protects Prostate Cancer Cells from Tumor Necrosis Factor-α-induced Apoptosis via the AKT/Survivin Pathway

Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84→ Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via β1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-α (TNF-α). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-α-induced apoptosis. Evidence is provided that expression of β1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-α-induced apoptosis. In contrast, the β1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-α in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis. Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84→ Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via β1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-α (TNF-α). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-α-induced apoptosis. Evidence is provided that expression of β1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-α-induced apoptosis. In contrast, the β1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-α in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis. It has become increasingly clear that the interactions between extracellular matrix (ECM) 1The abbreviations used are: ECMextracellular matrixIAPinhibitor of apoptosisERKextracellular signal-regulated kinasemAbmonoclonal antibodyTNF-αtumor necrosis factor-αPARPpoly(ADP ribose) polymeraseCHXcycloheximideAct Dactinomycin DHAhemagglutininPI(3,4)P2phosphatidylinositol 3,4-bisphosphatePI(3,4,5)P3phosphatidylinositol 3,4,5-trisphosphateZbenzyloxycarbonylfmkfluoromethyl ketoneBSAbovine serum albuminntnucleotideEGFepidermal growth factorFACSfluorescence-activated cell sorterGFPgreen fluorescent proteindndominant negative. proteins and integrins, their cognate cell surface receptors, mediate cell adhesion but also engender signals that participate in cell survival (1.Frisch S.M. Ruoslahti E. Curr. Opin. Cell Biol. 1997; 9: 701-706Crossref PubMed Scopus (992) Google Scholar). It has been shown that insufficient or inappropriate cell-ECM interactions cause apoptosis, previously designated anoikis (1.Frisch S.M. Ruoslahti E. Curr. Opin. Cell Biol. 1997; 9: 701-706Crossref PubMed Scopus (992) Google Scholar). This mechanism of integrin-dependent cytoprotection is operative against a variety of death-promoting stimuli, acting through the intrinsic (mitochondrial) or extrinsic (death receptor) apoptotic pathway and involving growth factor withdrawal (2.Zhang Z. Vuori K. Reed J.C. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6161-6165Crossref PubMed Scopus (575) Google Scholar, 3.O'Brien V. Frisch S.M. Juliano R.L. Exp. Cell Res. 1996; 224: 208-213Crossref PubMed Scopus (85) Google Scholar, 4.Lee J.W. Juliano R.L. Mol. Biol. Cell. 2000; 11: 1973-1987Crossref PubMed Scopus (143) Google Scholar, 5.Almeida E.A. Ilic D. Han Q. Hauch C.R. Jin F. Kawakatsu H. Schlaepfer D.D. Damsky C.H. J. Cell Biol. 2000; 149: 741-754Crossref PubMed Scopus (337) Google Scholar), exposure to cytotoxic drugs (6.Damiano J.S. Curr. Cancer Drug Targets. 2002; 2: 37-43Crossref PubMed Scopus (91) Google Scholar, 7.Uhm J.H. Dooley N.P. Kyritsis A.P. Rao J.S. Gladson C.L. Clin. Cancer Res. 1999; 5: 1587-1594PubMed Google Scholar), and ligation of death receptors (8.Aoudjit F. Vuori K. J. Cell Biol. 2001; 152: 633-643Crossref PubMed Scopus (254) Google Scholar, 9.Shain K.H. Landowski T.H. Dalton W.S. J. Immunol. 2002; 168: 2544-2553Crossref PubMed Scopus (101) Google Scholar). Critical signaling intermediates involved in integrin-dependent cell survival have been identified, and integrin-mediated adhesion to the ECM stimulates the production of PI(3,4)P2 and PI(3,4,5)P3 (10.Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (940) Google Scholar, 11.King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar), the association of the p85 PI 3-kinase subunit with focal adhesion kinase (12.Chen H.-C. Guan J.-L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (478) Google Scholar) and AKT activation (10.Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (940) Google Scholar, 11.King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar). In turn, active AKT interferes with the apoptotic machinery by phosphorylating thus sequestering the proapoptotic Bcl-2 family protein BAD, by inactivating members of the forkhead family of transcription factors, and by promoting NF-κB-dependent cytoprotection via transcriptional activation of a plethora of downstream target genes (13.Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar). extracellular matrix inhibitor of apoptosis extracellular signal-regulated kinase monoclonal antibody tumor necrosis factor-α poly(ADP ribose) polymerase cycloheximide actinomycin D hemagglutinin phosphatidylinositol 3,4-bisphosphate phosphatidylinositol 3,4,5-trisphosphate benzyloxycarbonyl fluoromethyl ketone bovine serum albumin nucleotide epidermal growth factor fluorescence-activated cell sorter green fluorescent protein dominant negative. The effector molecules involved in integrin-dependent cell survival have not been completely elucidated. Engagement of fibronectin through α5β1 and αvβ1 and of vitronectin through αvβ3 integrins has been associated with up-regulation of anti-apoptotic Bcl-2 and with Bcl-2-mediated protection from apoptosis induced by serum deprivation (2.Zhang Z. Vuori K. Reed J.C. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6161-6165Crossref PubMed Scopus (575) Google Scholar, 14.Matter M.L. Ruoslahti E. J. Biol. Chem. 2001; 276: 27757-27763Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Whether other cytoprotective mechanisms for integrin-dependent cell survival exist in normal or tumor cells has not been determined. In addition to the Bcl-2 family of cell death regulators, a second group of inhibitor of apoptosis (IAP) proteins has been recently identified (15.Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2285) Google Scholar). Survivin, a member of the IAP family of proteins, is expressed during embryonic and fetal development but undetectable in most normal adult tissues (16.Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3025) Google Scholar). However, survivin becomes the fourth most expressed transcript in human cancer (17.Velculescu V.E. Madden S.L. Zhang L. J. C. A. H. K. 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Google Scholar), survivin expression in of the prostate cancer cell lines The highest levels of survivin present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 with the results in survivin expression in non-tumorigenic and LNCaP prostate cancer cells or in normal prostate cells of human prostatic that survivin expressed in as well as in cells and of PC3 cells with the mutant F. G. J. S. Altieri D.C. PubMed Scopus Google Scholar), or survivin antisense cDNA in the of PC3 cells with as shown by and and fragmentation, as by of cells In expression of mutant or antisense cDNA in apoptosis with of cells and of PC3 cells with survivin mutant in a in caspase-3 as by F. G. J. S. Altieri D.C. PubMed Scopus Google Scholar). of PC3 cells with survivin mutant in cleavage of the with the of a of PC3 cells with caspase-3 and cleavage induced by survivin and These findings indicate that of survivin in prostate cancer cells is to cause of the of apoptosis caspase-3 and cleavage of caspase of survivin prostate cancer cell survival. PC3 cells with or the mutant and in growth for by and are of of of apoptosis. and cells the and as in A. cell for caspase-3 by of the substrate are the of of a of the are the as in with the that of PC3 cells with the with or a caspase with antibody to The of and are by of survivin antisense prostate cancer cell survival. PC3 cells with or antisense and in growth for and or for the nuclear of PC3 cells with or antisense by The indicate apoptotic antisense cells in growth for with or by and The of cells with is in of apoptosis. with or antisense the and as in in PC3 but in LNCaP matrix survivin PC3 and LNCaP cells in or with fibronectin and survivin expression by shown in PC3 cell adhesion to fibronectin for in survivin expression levels with cells in and and in PC3 cell adhesion to fibronectin is mediated by β1 integrins, to be expressed levels in cells G. Cancer Res. 1999; Google Scholar, Cancer Res. Clin. PubMed Scopus Google Scholar). In contrast, survivin levels in LNCaP cells not by adhesion to fibronectin and We that fibronectin not survivin levels in PC3 cells in the of a transcription or a protein inhibitor These results that regulation of survivin levels by fibronectin transcriptional and In in expression of anti-apoptotic Bcl-2 or cell adhesion to fibronectin of the and not However, the adhesion of PC3 cells to vitronectin in levels of Bcl-2 by not in with J.H. Dooley N.P. Kyritsis A.P. Rao J.S. Gladson C.L. Clin. Cancer Res. 1999; 5: 1587-1594PubMed Google Scholar, 14.Matter M.L. Ruoslahti E. J. Biol. 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PubMed Scopus Google Scholar) that a for of PC3 cells with in apoptosis as by and with cells with or in cells in expression of the of cells with induced by TNF-α with cells with or The β1A for Cell is well that the cytoplasmic of the β1 subunit is required to modulate cellular as well as to integrin signaling Biol. 1997; 16: PubMed Scopus Google Scholar). β1 cytoplasmic have been identified and and have been shown to cell cell adhesion and interactions with proteins, and and activation of signaling molecules M.E. S. The Scholar). β1C expression cell and is in prostate cancer Biol. 1997; 16: PubMed Scopus Google Scholar). In PC3 the β1C integrin variant is whereas the β1A is and L. the of the β1 integrin in protection against we PC3 cell lines β1C or β1A integrins of the β1 extracellular and transmembrane and human β1C or human β1A the of a These used to between β1 integrin and β1C and β1A integrin in human of the by using to β1 levels of surface expression of β1C and β1A in of the and and not the of β1 in protection from TNF-α-induced apoptosis, we and cell with or of in cells in the of cells with induced by TNF-α with cells with and In contrast, expression of in cells in not able to apoptosis by TNF-α and These that the β1A is necessary to protect cells from TNF-α-induced apoptosis in a via matrix results in activation of AKT and integrin survival signaling (10.Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (940) Google Scholar, 11.King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar, J. Curr. Opin. Cell Biol. PubMed Scopus Google Scholar). This pathway as a cell that may also survivin expression A. D. K. F. Altieri D.C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the of AKT in in survivin we PC3 cells with a for a shown in expression of in but not of completely the ability of fibronectin to survivin expression in PC3 cells as by of AKT not survivin levels The expression of AKT the of AKT as by using a antibody S. H. S. W.S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), thus that the AKT mutant is These results that integrin-mediated of survivin expression through the AKT pathway is responsible for the against apoptosis. In we have shown that integrin ligation by fibronectin stimulates survival signals that apoptosis in prostate PC3 of pathway the of the IAP protein but not Bcl-2 as a critical downstream effector of and the of mechanism We also show that cytoprotection of non-adherent cells by TNF-α the β1A integrin subunit has been that integrins participate in cell survival and of cells from their or of by their is by a of cell death designated anoikis (1.Frisch S.M. Ruoslahti E. Curr. Opin. Cell Biol. 1997; 9: 701-706Crossref PubMed Scopus (992) Google Scholar), survival that integrin ligation to the cellular apoptotic machinery have not been completely elucidated. integrin in PC3 cells with fibronectin associated with up-regulation of survivin expression and protection from apoptosis, whereas interference with survivin using induced apoptosis and the cytoprotective of with a report E. PubMed Scopus Google Scholar), which has shown that integrin by fibronectin results in survivin expression in results demonstrate that integrin signaling as a cell pathway in cancer cells using expression of survivin as a critical downstream with expression in D.C. Mol. Med. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), we that the highest levels of survivin present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 S. G. E. Clin. Cancer Res. 1996; 2: Google Scholar, Cancer Res. Clin. PubMed Scopus Google Scholar). the molecular for the of survivin in cancer has not been a of survivin expression in cells D.C. Mol. Med. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) that may the of S. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and of survivin This mechanism may be with the findings in which survivin expression is in PC3 cells but not in non-tumorigenic LNCaP cells D. S. 1997; PubMed Scopus Google Scholar) and also with the ability of to integrin-dependent survival signals A. S. J. Cell Biol. 1999; PubMed Scopus Google Scholar). have shown that cell adhesion to fibronectin through α5β1 and αvβ1 and to vitronectin through αvβ3 integrins in up-regulation of Bcl-2 in cells (2.Zhang Z. Vuori K. Reed J.C. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6161-6165Crossref PubMed Scopus (575) Google Scholar, 14.Matter M.L. Ruoslahti E. J. Biol. Chem. 2001; 276: 27757-27763Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) and that Bcl-2 anoikis in prostate cancer cells K. J. Scopus Google Scholar), whereas the levels of Bcl-2 in to fibronectin adhesion in cells J.S. Dalton W.S. 1999; PubMed Google Scholar). We have that PC3 cell adhesion to vitronectin the levels of Bcl-2 not we not a in the levels of Bcl-2 in to fibronectin The between results and the be to the cell the survivin expression by fibronectin adhesion to protection from apoptosis, we used a dominant negative survivin that has shown for ability to apoptosis and to cell in vitro and in a of cancer in vivo J. Altieri D.C. J. Clin. 2001; PubMed Scopus Google Scholar, S. V. PubMed Scopus Google Scholar). with of the Ala mutant the cytoprotective of fibronectin against TNF-α-induced apoptosis. the molecular mechanisms of apoptosis induced by survivin have not been completely a critical in survivin levels to the ability of to with survivin and to This is of the apoptotic the of survivin levels by antisense and of cell death F. G. J. S. Altieri D.C. PubMed Scopus Google Scholar, A.P. D. U. Cancer Res. 2000; Google Scholar). It recently that expression of survivin Ala in of cell of apoptosis, and in of thus in with the A. F. D. P. G. E. Mol. Biol. Cell. PubMed Scopus Google Scholar). the that survivin Ala mutant to protect against apoptotic stimuli, which their and the used in adhesion death apoptosis. The of PC3 cells transduced to survivin cytoprotection the of survivin of apoptosis have not been a in the of the intrinsic apoptotic for a of survivin in the death receptor-mediated apoptotic has also been and survivin apoptosis in cells and TNF-α-induced apoptosis in cells H. Altieri D.C. Nat. Med. 2002; PubMed Scopus Google Scholar, J. D. Altieri D. A. Cancer Res. 2002; Google Scholar). may a interference of survivin in the extrinsic apoptotic has been recently that prostate cancer cells death signaling to the of cell death via cleavage and from A. Cancer Res. 2000; Google Scholar). from the is the that expression of the β1A but not of the β1C cytoplasmic integrin variant protection against apoptosis to cells transduced with survivin. These indicate that the β1A cytoplasmic is required for the anti-apoptotic by survivin in non-adherent we have not the expression of and in PC3 is that to the by survivin their expression has been shown to be to and and to and Biol. 1997; 16: PubMed Scopus Google Scholar). indicate that expression of dominant negative AKT integrin-mediated regulation of survivin. In with that have shown that integrin ligation survival signals and protect cells from apoptosis by AKT (6.Damiano J.S. Curr. Cancer Drug Targets. 2002; 2: 37-43Crossref PubMed Scopus (91) Google Scholar), results demonstrate that pathway also apoptotic signals by death ligation and may to the development of apoptosis in prostate cancer findings that survivin may be required to maintain a critical anti-apoptotic threshold in prostate cancer cells and that the ECM is factor that to tumor mechanisms by survivin the molecular mechanisms integrin-mediated survival signals in cancer cells may for cancer We and for and