BCL-xL Is a Target Gene Regulated by Hypoxia-inducible Factor-1α

The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays pivotal roles in physiology and pathophysiology. Constitutive or hypoxia-induced HIF-1α overexpression is observed in many types of cancers including prostate adenocarcinoma, in which it is associated with resistance to apoptosis and therapeutic agents. BCL-xL, a hypoxia-responsive, anti-apoptotic protein of the Bcl-2 family, is also overexpressed in prostate carcinoma and many other cancers. Despite this connection, whether BCL-xL expression is directly regulated by HIF-1α is not known. We used prostate cancer PC-3 cell with constitutive high HIF-1α level as a model to address this important question. We first generated prostate cancer PC-3 cells in which HIF-1α was stably knocked-down (HIF-KD) by using small interference RNA. BCL-xL was dramatically decreased in HIF-KD PC-3 cells, in parallel with sensitization to apoptosis with caspase-3 activation as well as decreased cell proliferation. We then demonstrated that HIF-1α directly regulated BCL-xL transcription by binding to a hypoxia-responsive element in the BCL-xL promoter (-865 to -847) by reporter gene assay, chromatin immunoprecipitation, and electrophoretic mobility shift and supershift assays. HIF-1α-dependent BCL-xL overexpression may be an important mechanism by which HIF-1α protects prostate cancer cells from apoptosis and leads to treatment resistance. The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays pivotal roles in physiology and pathophysiology. Constitutive or hypoxia-induced HIF-1α overexpression is observed in many types of cancers including prostate adenocarcinoma, in which it is associated with resistance to apoptosis and therapeutic agents. BCL-xL, a hypoxia-responsive, anti-apoptotic protein of the Bcl-2 family, is also overexpressed in prostate carcinoma and many other cancers. Despite this connection, whether BCL-xL expression is directly regulated by HIF-1α is not known. We used prostate cancer PC-3 cell with constitutive high HIF-1α level as a model to address this important question. We first generated prostate cancer PC-3 cells in which HIF-1α was stably knocked-down (HIF-KD) by using small interference RNA. BCL-xL was dramatically decreased in HIF-KD PC-3 cells, in parallel with sensitization to apoptosis with caspase-3 activation as well as decreased cell proliferation. We then demonstrated that HIF-1α directly regulated BCL-xL transcription by binding to a hypoxia-responsive element in the BCL-xL promoter (-865 to -847) by reporter gene assay, chromatin immunoprecipitation, and electrophoretic mobility shift and supershift assays. HIF-1α-dependent BCL-xL overexpression may be an important mechanism by which HIF-1α protects prostate cancer cells from apoptosis and leads to treatment resistance. The transcription factor hypoxia-inducible factor-1α (HIF-1α) 3The abbreviations used are: HIF-1α, hypoxia-inducible factor 1; BCL-xL, BCL2L1 or BCL2-like 1; BNIP3, BCL2/adenovirus E1B 19-kDa interacting protein 3; CASP, caspase; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT-1, glucose transporter-1; HIF-KD, HIF-1α knock-down PC-3 cells; HRE, hypoxia-responsive element; IAP, inhibitor of apoptosis protein; Mcl-1, myeloid cell factor-1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NIX, BCL2/adenovirus E1B 19-kDa interacting protein 3-like; RNAi, RNA interference; siRNA, small interference RNA; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP digoxigenin nick end labeling; VEGF, vascular endothelial growth factor; PI3K, phosphatidylinositol 3-kinase; RT, reverse transcription; KD, knocked-down; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid; CMV, cytomegalovirus. plays major roles in cellular response to hypoxia as well as in disease processes including carcinogenesis (1Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Crossref PubMed Scopus (1234) Google Scholar, 2Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar, 3Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5502) Google Scholar, 4Harris A.L. Nat. Rev. Cancer. 2002; 2: 38-47Crossref PubMed Scopus (4420) Google Scholar). Many genes have been identified as HIF-1 targets (3Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5502) Google Scholar, 4Harris A.L. Nat. Rev. Cancer. 2002; 2: 38-47Crossref PubMed Scopus (4420) Google Scholar), including GLUT-1, GAPDH, and VEGF, which are involved in such biological processes as energy metabolism, cell survival, and angiogenesis. Hypoxia inhibits proteasome-dependent degradation of HIF-1α, resulting in HIF-1α stabilization, which dimerizes with HIF-1β and activates target genes by binding to hypoxia responsive element (HRE) within their promoters. Hypoxia and HIF-1α overexpression are implicated in the pathogenesis of many cancers, including prostate carcinoma (3Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5502) Google Scholar, 4Harris A.L. Nat. Rev. Cancer. 2002; 2: 38-47Crossref PubMed Scopus (4420) Google Scholar, 5Zhong H. Semenza G.L. Simons J.W. De Marzo A.M. Cancer Detect. Prev. 2004; 28: 88-93Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), in which it is associated with advanced clinical stage and treatment failure (6Movsas B. Chapman J.D. Hanlon A.L. Horwitz E.M. Greenberg R.E. Stobbe C. Hanks G.E. Pollack A. Urology. 2002; 60: 634-639Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). HIF-1α overexpression has been identified in both prostate adenocarcinoma tissue (5Zhong H. Semenza G.L. Simons J.W. De Marzo A.M. Cancer Detect. Prev. 2004; 28: 88-93Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 7Zhong H. De Marzo A.M. Laughner E. Lim M. Hilton D.A. Zagzag D. Buechler P. Isaacs W.B. Semenza G.L. Simons J.W. Cancer Res. 1999; 59: 5830-5835PubMed Google Scholar) and cell lines (8Zhong H. Agani F. Baccala A.A. Laughner E. Rioseco-Camacho N. Isaacs W.B. Simons J.W. Semenza G.L. Cancer Res. 1998; 58: 5280-5284PubMed Google Scholar). Although acute hypoxia may lead to cell death, prolonged hypoxia results in resistance to apoptosis as well as to radiotherapy and chemotherapy (4Harris A.L. Nat. Rev. Cancer. 2002; 2: 38-47Crossref PubMed Scopus (4420) Google Scholar, 9Graeber T.G. Osmanian C. Jacks T. Housman D.E. Koch C.J. Lowe S.W. Giaccia A.J. Nature. 1996; 379: 88-91Crossref PubMed Scopus (2191) Google Scholar, 10Dong Z. Wang J. J. Biol. Chem. 2004; 279: 9215-9221Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), the mechanism of which is not well understood. Only recently have a few apoptosis regulators been identified as HIF-1α target genes, most notably the anti-apoptotic Mcl-1 (11Piret J.P. Minet E. Cosse J.P. Ninane N. Debacq C. Raes M. Michiels C. J. Biol. Chem. 2005; 280: 9336-9344Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and BIRC5/survivin (12Peng X.H. Karna P. Cao Z. Jiang B.H. Zhou M. Yang L. J. Biol. Chem. 2006; 281: 25903-25914Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Although pro-apoptotic molecules BNIP3, NIX (13Bruick R.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9082-9087Crossref PubMed Scopus (678) Google Scholar, 14Sowter H.M. Ratcliffe P.J. Watson P. Greenberg A.H. Harris A.L. Cancer Res. 2001; 61: 6669-6673PubMed Google Scholar), and Noxa (15Kim J.Y. Ahn H.J. Ryu J.H. Suk K. Park J.H. J. Exp. Med. 2004; 199: 113-124Crossref PubMed Scopus (240) Google Scholar) are also responsive to HIF-1α, hypoxia-induced apoptosis-resistant phenotype eventually predominates. BCL-xL (BCL2-like 1 or BCL2L1), a major anti-apoptotic protein of the Bcl-2 family, is also overexpressed in prostate carcinoma and many other cancers. BCL-xL overexpression is associated with the hormone-refractory phenotype and renders prostate cancer cells apoptosis-resistant, whereas BCL-xL knock-down increases sensitivity to chemotherapeutic agents (16Lebedeva I. Rando R. Ojwang J. Cossum P. Stein C.A. Cancer Res. 2000; 60: 6052-6060PubMed Google Scholar, 17Vilenchik M. Raffo A.J. Benimetskaya L. Shames D. Stein C.A. Cancer Res. 2002; 62: 2175-2183PubMed Google Scholar). Despite the correlation of BCL-xL overexpression with HIF-1α in some tumors (18Park S.Y. Billiar T.R. Seol D.W. Biochem. Biophys. Res. Commun. 2002; 291: 150-153Crossref PubMed Scopus (70) Google Scholar) and the observation that BCL-xL is a key molecule underlying hypoxia-driven cell death resistance (10Dong Z. Wang J. J. Biol. Chem. 2004; 279: 9215-9221Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), the mechanism by which hypoxia induces BCL-xL expression is unclear, as it has not been elucidated if HIF-1α directly regulates BCL-xL. BCL-xL gene is regulated by several transcription factor families, including STATs (signal transducers and activators of transcription) (19Fujio Y. Kunisada K. Hirota H. Yamauchi-Takihara K. Kishimoto T. J. Clin. Investig. 1997; 99: 2898-2905Crossref PubMed Scopus (178) Google Scholar), NF-κB (20Chen C. Edelstein L.C. Gelinas C. Mol. Cell. Biol. 2000; 20: 2687-2695Crossref PubMed Scopus (700) Google Scholar), Ets (21Sevilla L. Aperlo C. Dulic V. Chambard J.C. Boutonnet C. Pasquier O. Pognonec P. Boulukos K.E. Mol. Cell. Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (92) Google Scholar), GATA (22Gregory T. Yu C. Ma A. Orkin S.H. Blobel G.A. Weiss M.J. Blood. 1999; 94: 87-96Crossref PubMed Google Scholar), PAX3 (and the PAX3/FKHR (Forkhead related transcription factor) fusion) (23Margue C.M. Bernasconi M. Barr F.G. Schafer B.W. Oncogene. 2000; 19: 2921-2929Crossref PubMed Scopus (92) Google Scholar), and POU (Brn-3a) (24Sugars K.L. Budhram-Mahadeo V. Packham G. Latchman D.S. Nucleic Acids Res. 2001; 29: 4530-4540Crossref PubMed Scopus (55) Google Scholar). These regulators, however, are not closely related to hypoxia as HIF-1α. We tested the hypothesis that BCL-xL is under HIF-1α regulation using prostate cancer PC-3 cell as a model, in which HIF-1α level is constitutively high. We show that stable knockdown of HIF-1α by small interference RNA (siRNA) results in a dramatic decrease of BCL-xL with consequent increase in apoptosis, and most importantly, BCL-xL is transcriptionally regulated by HIF-1α. Cells, Tissues, and General Reagents-Human prostate cancer cell lines LNCaP, DU145, and PC-3 were maintained in RPMI1640 with 10% fetal calf serum (Invitrogen). Prostate adenocarcinoma tissue and normal prostate tissue (from prostatectomy specimens of non-prostate diseases) were snap-frozen in accordance with institutional guidelines. Normal prostate epithelial cells were collected by laser capture microdissection with the Leica AS LMD system (Leica Microsystems, Wetzler, Germany). The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 was from Sigma. Tris base, Tween 20, dithiothreitol, and EDTA were from Amresco (Solon, OH). Phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin were from Roche Diagnostics. Reverse Transcription (RT)-PCR and Real-time Quantitative PCR-Total RNA was extracted by using the TRIzol reagent (Invitrogen). Revertra Ace reverse transcriptase (ToYoBo, Osaka, Japan) was used were to as HIF-1α BCL-xL BIRC5/survivin was used as were and by and by with the The Real-time was used and were and by the of target genes to was by of in PC-3 cells were in in fetal calf and with 20, and inhibitor LY294002 1 were collected of HIF-1α, BCL-xL, and Hypoxia of PC-3 cells were in in fetal calf and with were collected of HIF-1α and BCL-xL. HIF-1α RNA expression was used to HIF-1α by under promoter and were with the with were also and as and These were to with of protein from the was used PC-3 cells were by using (Invitrogen). knock-down cells (HIF-KD) with stable of and as and and the and cells were by and maintained in growth with used HIF-1α from BCL-xL and from from BIRC5/survivin from and and from from and from were from by were to with and Tween 20, and with and and were by to treatment with the in were to in a were then as assays. were in and with and with caspase-3 or BCL-xL was used with as and as were in and by cell The of was 1 cells were with CHAPS, 1 10% and The of the cell was used caspase-3 using as a dUTP was by using in cell death were with and in with then with with and with terminal was used as The was as of of cells of BCL-xL in of BCL-xL was and The used and cells were with BCL-xL expression or using and cells were maintained in HIF-1α-dependent BCL-xL reporter was used to reporter with of the BCL-xL were in which the BCL-xL promoter to to the transcription or of which were of the The reporter were as to to to and to with and were to with to of to and to with to of to reporter and the the gene as were used in reporter gene HIF-1α-dependent gene were with by using (Invitrogen). the was by cells were with and was by using were and were The was and were collected and with and protein was with of HIF-1α or the or and then with protein and were and extracted with and were and were and used as The used BCL-xL, and and and of the BCL-xL promoter to and (-865 to The promoter to was used as with were were by (Invitrogen). and were used were and of were in PC-3 cells were of with was and was with of in a of a of or was supershift of was to the and The was and to by and was and were by to treatment with the was by using the HIF-1α of HIF-1α and and protein overexpression in prostate cancer cells and prostate adenocarcinoma was by and normal prostate HIF-1α and protein were and The interference with and the with HIF-1α The PC-3 cells with stably and and cells, and the and cells, were as by the of protein HIF-1α and protein were in the HIF-KD cells, with consequent of HIF-1α target genes BNIP3, GLUT-1, and and The and protein expression of HIF-1α or target genes and Quantitative of of HIF-1α and target gene the interference of HIF-1α siRNA, as both of which were HIF-1α PC-3 cells also cell growth and cell was demonstrated by of the which in HIF-KD cells HIF-KD to and cells a cell death and the in and cells were and in and cells were and the in and cells to and in and cells, the to and of caspase-3 of caspase-3 activation of caspase-3 in HIF-KD cells not in cells caspase-3 demonstrated caspase-3 in HIF-KD cells also PC-3 cells to the treatment by which growth of HIF-KD cells cell growth was and the with or HIF-1α BCL-xL the by which HIF-1α cell and apoptosis in PC-3 cells, the of HIF-1α major apoptosis the Bcl-2 family, the family, and the BCL-xL expression was by HIF-1α whereas the other of the Bcl-2 which is by the gene also a decrease in expression HIF-1α the level of was the dramatic BCL-xL HIF-1α also a decrease of a HIF-1α target and of the family, including and were not HIF-1α expression level of the and of BCL-xL in HIF-KD and the of BCL-xL expression in apoptosis BCL-xL was overexpressed in cells which the of HIF-1α siRNA, resulting in cell growth and of cell death and and of the in of HIF-1α and of BCL-xL by HIF-1α was dramatic the constitutive high BCL-xL and protein level in prostate cancer cell is a major that HIF-1α tested if inhibitor LY294002 lead to decrease of BCL-xL. in both HIF-1α and BCL-xL were in a Hypoxia BCL-xL and HIF-1α in PC-3 of PC-3 cells with the hypoxia in increase of BCL-xL and HIF-1α the of BCL-xL HIF-1α in response to hypoxia BCL-xL important to HIF-1α and BCL-xL. BCL-xL is a gene regulated by which be by of BCL-xL expression by be the of HIF-1α or NF-κB or to whether BCL-xL was directly regulated by HIF-1α. was in the BCL-xL promoter were identified within the the of and the and were of reporter were in The to both and NF-κB binding whereas to by The to and to were with of the and PC-3 cells were with of the with as The hypoxia was used to hypoxia and to HIF-1α as a of hypoxia-induced gene transcription in the reporter gene The of the The were dramatic with the whereas of the in a of These that the reporter gene transcription was under of BCL-xL promoter HRE, which to the hypoxia HIF-1α with BCL-xL show HIF-1α to BCL-xL first used chromatin of PC-3 cells by the chromatin by as a to to of BCL-xL promoter was and by was not or was used the and HIF-1α to the of binding of HIF-1α to the in BCL-xL with of the to and (-865 to with were also and were as and HIF-1α binding from the gene promoter was used as with a assays. The that the 1 and not mobility shift with from PC-3 The shift be in the with not with the HIF-1α was in the binding a supershift was observed as well with not mobility shift and supershift were also with the and with and supershift These results demonstrated HIF-1α binding to the in the to of BCL-xL We generated prostate cancer PC-3 cells in which HIF-1α was stably knocked-down by using which in a decrease of the anti-apoptotic molecule BCL-xL. We then that HIF-1α directly regulated BCL-xL gene These to HIF-1α-dependent BCL-xL overexpression as an important mechanism by which HIF-1α protects prostate cancer cells from apoptosis and leads to treatment Hypoxia is in tumors (4Harris A.L. Nat. Rev. Cancer. 2002; 2: 38-47Crossref PubMed Scopus (4420) Google Scholar), including prostate carcinoma (3Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5502) Google Scholar), in which the of hypoxia is with clinical stage and treatment failure (6Movsas B. Chapman J.D. Hanlon A.L. Horwitz E.M. Greenberg R.E. Stobbe C. Hanks G.E. Pollack A. Urology. 2002; 60: 634-639Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). increase in HIF-1α plays roles in and of many cancers HIF-1α-dependent activation of genes that cancer cell survival, and angiogenesis. of HIF-1α and target genes has been observed in a of tumors of the D. H. Laughner E. Simons J.W. Semenza G.L. Cancer. 2000; PubMed Scopus Google Scholar), and the Y. J. C. R. Y. R. T. T. M. N. S. J. 2005; PubMed Scopus Google Scholar), and A. E. H. K. F. Harris A.L. J. Cancer. 2001; PubMed Scopus Google Scholar) as well as prostate (5Zhong H. Semenza G.L. Simons J.W. De Marzo A.M. Cancer Detect. Prev. 2004; 28: 88-93Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 7Zhong H. De Marzo A.M. Laughner E. Lim M. Hilton D.A. Zagzag D. Buechler P. Isaacs W.B. Semenza G.L. Simons J.W. Cancer Res. 1999; 59: 5830-5835PubMed Google Scholar). of HIF-1α has been observed in prostate cancer tissue and cell lines (5Zhong H. Semenza G.L. Simons J.W. De Marzo A.M. Cancer Detect. Prev. 2004; 28: 88-93Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 7Zhong H. De Marzo A.M. Laughner E. Lim M. Hilton D.A. Zagzag D. Buechler P. Isaacs W.B. Semenza G.L. Simons J.W. Cancer Res. 1999; 59: 5830-5835PubMed Google Scholar, H. Agani F. Baccala A.A. Laughner E. Rioseco-Camacho N. Isaacs W.B. Simons J.W. Semenza G.L. Cancer Res. 1998; 58: 5280-5284PubMed Google Scholar). of HIF-1α be an in prostate as high prostate a HIF-1α level (5Zhong H. Semenza G.L. Simons J.W. De Marzo A.M. Cancer Detect. Prev. 2004; 28: 88-93Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). is HIF-1α overexpression is prostate cancer cells have constitutively high HIF-1α which be by hypoxia (8Zhong H. Agani F. Baccala A.A. Laughner E. Rioseco-Camacho N. Isaacs W.B. 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Cancer. 2004; PubMed Scopus Google Scholar), as the end of a Although BCL-xL has been to be a key molecule involved in hypoxia-induced resistance to cell death (10Dong Z. Wang J. J. Biol. Chem. 2004; 279: 9215-9221Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and BCL-xL overexpression has been associated with HIF-1α in tumors such as cell cancer (18Park S.Y. Billiar T.R. Seol D.W. Biochem. Biophys. Res. Commun. 2002; 291: 150-153Crossref PubMed Scopus (70) Google Scholar), the mechanism by which hypoxia induces BCL-xL and the HIF-1α and BCL-xL has not been known. the first that HIF-1α directly regulates BCL-xL transcription by interacting with in the BCL-xL has been that in the PC-3 BCL-xL is the Bcl-2 (16Lebedeva I. Rando R. Ojwang J. Cossum P. Stein C.A. Cancer Res. 2000; 60: 6052-6060PubMed Google Scholar). that HIF-1α-dependent overexpression of BCL-xL in PC-3 cells is of the major by which prostate cancer cells, cells, apoptosis and the has been identified as a target of HIF-1α (12Peng X.H. Karna P. Cao Z. Jiang B.H. Zhou M. Yang L. J. Biol. Chem. 2006; 281: 25903-25914Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). in many cancers, is to be involved in the regulation of both apoptosis and cell HIF-1α overexpression constitutive or may by key of major gene cell death and that the of cell death by BCL-xL and and of cell by of of HIF-1α-dependent BCL-xL expression may a BCL-xL We of the and in the of the of