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• W1983991527 abstract "Resistance of carcinoma cells to hypoxic stress is of importance to the growth of solid tumors. The mucin 1 (MUC1) oncoprotein is aberrantly overexpressed by most human carcinomas; however, there is no known relationship between MUC1 and the hypoxic stress response. The present work has demonstrated that MUC1 attenuates activation of hypoxia-inducible factor-1α (HIF-1α), a regulator of gene transcription in the response of cells to hypoxic stress. In cells with stable gain and loss of MUC1 function, we have shown that MUC1 up-regulates prolyl hydroxylase 3 (PHD3) expression and promotes HIF-1α degradation. PHD activity is attenuated by increases in reactive oxygen species (ROS) generated in the hypoxic stress response. Our results further demonstrate that MUC1 blocks hypoxia-induced increases in ROS and thereby potentiates PHD-mediated HIF-1α suppression. Importantly, MUC1 also blocks hypoxia-induced apoptosis and necrosis by suppressing accumulation of ROS. These findings indicate that MUC1 attenuates HIF-1α activation in a survival response to hypoxic stress. Resistance of carcinoma cells to hypoxic stress is of importance to the growth of solid tumors. The mucin 1 (MUC1) oncoprotein is aberrantly overexpressed by most human carcinomas; however, there is no known relationship between MUC1 and the hypoxic stress response. The present work has demonstrated that MUC1 attenuates activation of hypoxia-inducible factor-1α (HIF-1α), a regulator of gene transcription in the response of cells to hypoxic stress. In cells with stable gain and loss of MUC1 function, we have shown that MUC1 up-regulates prolyl hydroxylase 3 (PHD3) expression and promotes HIF-1α degradation. PHD activity is attenuated by increases in reactive oxygen species (ROS) generated in the hypoxic stress response. Our results further demonstrate that MUC1 blocks hypoxia-induced increases in ROS and thereby potentiates PHD-mediated HIF-1α suppression. Importantly, MUC1 also blocks hypoxia-induced apoptosis and necrosis by suppressing accumulation of ROS. These findings indicate that MUC1 attenuates HIF-1α activation in a survival response to hypoxic stress. Solid tumors outgrow their blood supply, resulting in regions with decreased oxygen tension (1Vaupel P. Thews O. Kelleher D.K. Hoeckel M. Adv. Exp. Med. Biol. 1998; 454: 591-602Crossref PubMed Scopus (80) Google Scholar, 2Hockel M. Vaupel P. J. Natl. Cancer Inst. 2001; 93: 266-276Crossref PubMed Scopus (2128) Google Scholar). Cancer cells, however, adapt to survive and proliferate in hypoxic environments (3Wouters B.G. Brown J.M. Radiat. Res. 1997; 147: 541-550Crossref PubMed Scopus (272) Google Scholar). The hypoxia-inducible factor 1 (HIF-1) 2The abbreviations used are: HIF-1α, hypoxia-inducible factor 1α; PHD, prolyl hydroxylase; MUC1-C, MUC1 C-terminal subunit; MUC1-CD, MUC1 cytoplasmic domain; siRNA, small interfering RNA; CsiRNA, control siRNA; MUC1siRNA, MUC1-specific siRNA; ROS, reactive oxygen species; E3, ubiquitin-protein isopeptide ligase; CREB, cAMP-response element-binding protein; RT, reverse transcription; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide; DCFH-DA, 5-(and -6-)-carboxy-2′,7′-dichlorohydrofluorescin diacetate (carboxy-H2DCFDA). 2The abbreviations used are: HIF-1α, hypoxia-inducible factor 1α; PHD, prolyl hydroxylase; MUC1-C, MUC1 C-terminal subunit; MUC1-CD, MUC1 cytoplasmic domain; siRNA, small interfering RNA; CsiRNA, control siRNA; MUC1siRNA, MUC1-specific siRNA; ROS, reactive oxygen species; E3, ubiquitin-protein isopeptide ligase; CREB, cAMP-response element-binding protein; RT, reverse transcription; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide; DCFH-DA, 5-(and -6-)-carboxy-2′,7′-dichlorohydrofluorescin diacetate (carboxy-H2DCFDA). mediates adaptive changes in the response to hypoxia by regulating gene transcription. HIF-1 is a heterodimer of the tightly regulated HIF-1α subunit and the constitutively expressed HIF-1β subunit. The stability of HIF-1α is regulated through oxygen-dependent trans-4-hydroxylation of prolines by prolyl hydroxylase domain containing proteins PHD1, PHD2, and PHD3 (4Bruick R.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9082-9087Crossref PubMed Scopus (657) Google Scholar, 5Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2696) Google Scholar, 6Yu F. White S.B. Zhao Q. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9630-9635Crossref PubMed Scopus (634) Google Scholar). Hydroxylation of HIF-1α on Pro-402 and Pro-564 in the oxygen-dependent degradation domain promotes binding of the von Hippel-Lindau protein and the formation of an E3 ubiquitin ligase complex that targets HIF-1α for proteosomal degradation (7Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5195) Google Scholar). Conversely, β-hydroxylation of Asn-803 attenuates the HIF-1α transactivation function by decreasing association with the CREB-binding protein/p300 coactivators (8Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Science. 2002; 295: 858-861Crossref PubMed Scopus (1259) Google Scholar). PHD activity is also dependent on ferrous iron (FeII) and is decreased by accumulation of ROS, which converts FeII to FeIII (9Gerald D. Berra E. Frapart Y.M. Chan D.A. Giaccia A.J. Mansuy D. Pouyssegur J. Yaniv M. Mechta-Grigoriou F. Cell. 2004; 118: 781-794Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). The inhibition of PHD activity under hypoxic conditions or by hypoxia-induced ROS thus results in the stabilization of HIF-1α and activation of HIF-1 target genes. HIF-1 induces the expression of diverse genes that play adaptive roles to hypoxia by increasing angiogenesis, invasion, and resistance to apoptosis (7Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5195) Google Scholar). Under more severe or prolonged hypoxic conditions, HIF-1 contributes to an apoptotic or necrotic response by stabilization of p53 (10An W.G. Kanekal M. Simon M.C. Maltepe E. Blagosklonny M.V. Neckers L.M. Nature. 1998; 392: 405-408Crossref PubMed Scopus (647) Google Scholar) and induction of the pro-death Bcl-2 family members BNIP3 and NIX (11Sowter H.M. Ratcliffe P.J. Watson P. Greenberg A.H. Harris A.L. Cancer Res. 2001; 61: 6669-6673PubMed Google Scholar).Mucin 1 (MUC1) is a heterodimeric mucin that is expressed on the apical borders of normal secretory epithelial cells (12Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (498) Google Scholar). MUC1 is translated as a single polypeptide that undergoes autocleavage into two subunits (13Ligtenberg M.J. Kruijshaar L. Buijs F. van Meijer M. Litvinov S.V. Hilkens J. J. Biol. Chem. 1992; 267: 6171-6177Abstract Full Text PDF PubMed Google Scholar, 14Levitin F. Stern O. Weiss M. Gil-Henn C. Ziv R. Prokocimer Z. Smorodinsky N.I. Rubinstein D.B. Wreschner D.H. J. Biol. Chem. 2005; 280: 33374-33386Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 15Macao B. Johansson D.G. Hansson G.C. Hard T. Nat. Struct. Mol. Biol. 2006; 13: 71-76Crossref PubMed Scopus (199) Google Scholar). The >250-kDa MUC1 N-terminal ectodomain consists of variable numbers of heavily glycosylated tandem repeats that extend beyond the glycocalyx (16Siddiqui J. Abe M. Hayes D. Shani E. Yunis E. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2320-2323Crossref PubMed Scopus (280) Google Scholar, 17Gendler S. Taylor-Papadimitriou J. Duhig T. Rothbard J. Burchell J.A. J. Biol. Chem. 1988; 263: 12820-12823Abstract Full Text PDF PubMed Google Scholar). The MUC1 N-terminal subunit is tethered at the cell membrane to the C-terminal subunit (MUC1-C) that consists of a 58-amino-acid extracellular domain, a 28-amino-acid transmembrane domain and a 72-amino-acid cytoplasmic tail (18Merlo G. Siddiqui J. Cropp C. Liscia D.S. Lidereau R. Callahan R. Kufe D. Cancer Res. 1989; 49: 6966-6971PubMed Google Scholar). With transformation and loss of polarity, MUC1 is expressed at high levels over the entire carcinoma cell surface (12Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (498) Google Scholar). MUC1 associates with members of the ErbB family of receptor tyrosine kinases (19Li Y. Ren J. Yu W.-H. Li G. Kuwahara H. Yin L. Carraway K.L. Kufe D. J. Biol. Chem. 2001; 276: 35239-35242Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 20Schroeder J. Thompson M. Gardner M. Gendler S. J. Biol. Chem. 2001; 276: 13057-13064Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 21Li Y. Yu W.-H. Ren J. Huang L. Kharbanda S. Loda M. Kufe D. Mol. Cancer Res. 2003; 1: 765-775PubMed Google Scholar) and integrates ErbB signaling with the Wnt pathway through direct interactions with β-catenin (22Yamamoto M. Bharti A. Li Y. Kufe D. J. Biol. Chem. 1997; 272: 12492-12494Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 23Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (224) Google Scholar, 24Li Y. Kuwahara H. Ren J. Wen G. Kufe D. J. Biol. Chem. 2001; 276: 6061-6064Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 25Huang L. Chen D. Liu D. Yin L. Kharbanda S. Kufe D. Cancer Res. 2005; 65: 10413-10422Crossref PubMed Scopus (191) Google Scholar). Phosphorylation of the MUC1 cytoplasmic domain by glycogen synthase kinase 3β, c-Src, and protein kinase Cδ regulates binding of MUC1 and β-catenin (23Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (224) Google Scholar, 24Li Y. Kuwahara H. Ren J. Wen G. Kufe D. J. Biol. Chem. 2001; 276: 6061-6064Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 26Ren J. Li Y. Kufe D. J. Biol. Chem. 2002; 277: 17616-17622Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Other studies have demonstrated that MUC1-C accumulates in the cytosol of transformed cells and is targeted to the nucleus (21Li Y. Yu W.-H. Ren J. Huang L. Kharbanda S. Loda M. Kufe D. Mol. Cancer Res. 2003; 1: 765-775PubMed Google Scholar, 25Huang L. Chen D. Liu D. Yin L. Kharbanda S. Kufe D. Cancer Res. 2005; 65: 10413-10422Crossref PubMed Scopus (191) Google Scholar, 27Li Y. Liu D. Chen D. Kharbanda S. Kufe D. Oncogene. 2003; 22: 6107-6110Crossref PubMed Scopus (170) Google Scholar, 28Li Y. Chen W. Ren J. Yu W. Li Q. Yoshida K. Kufe D. Cancer Biol. Ther. 2003; 2: 187-193Crossref PubMed Scopus (70) Google Scholar, 29Wei X. Xu H. Kufe D. Cancer Cell. 2005; 7: 167-178Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 30Wei X. Xu H. Kufe D. Mol. Cell. 2006; 21: 295-305Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and mitochondria (31Ren J. Agata N. Chen D. Li Y. Yu W.-H. Huang L. Raina D. Chen W. Kharbanda S. Kufe D. Cancer Cell. 2004; 5: 163-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 32Ren J. Bharti A. Raina D. Chen W. Ahmad R. Kufe D. Oncogene. 2006; 25: 20-31Crossref PubMed Scopus (106) Google Scholar). Importantly, overexpression of MUC1 is sufficient to induce anchorage-independent growth and tumorigenicity (25Huang L. Chen D. Liu D. Yin L. Kharbanda S. Kufe D. Cancer Res. 2005; 65: 10413-10422Crossref PubMed Scopus (191) Google Scholar, 27Li Y. Liu D. Chen D. Kharbanda S. Kufe D. Oncogene. 2003; 22: 6107-6110Crossref PubMed Scopus (170) Google Scholar, 33Huang L. Ren J. Chen D. Li Y. Kharbanda S. Kufe D. Cancer Biol. Ther. 2003; 2: 702-706Crossref PubMed Scopus (3) Google Scholar, 34Schroeder J.A. Masri A.A. Adriance M.C. Tessier J.C. Kotlarczyk K.L. Thompson M.C. Gendler S.J. Oncogene. 2004; 23: 5739-5747Crossref PubMed Scopus (123) Google Scholar). Overexpression of MUC1 also suppresses H2O2-induced increases in ROS levels and confers resistance to the induction of apoptosis by oxidative stress (35Yin L. Kufe D. J. Biol. Chem. 2003; 278: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 36Yin L. Huang L. Kufe D. J. Biol. Chem. 2004; 279: 45721-45727Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).The findings that HIF-1α is stabilized by ROS (9Gerald D. Berra E. Frapart Y.M. Chan D.A. Giaccia A.J. Mansuy D. Pouyssegur J. Yaniv M. Mechta-Grigoriou F. Cell. 2004; 118: 781-794Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 37Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11715-11720Crossref PubMed Scopus (1571) Google Scholar) and that MUC1 blocks accumulation of ROS (35Yin L. Kufe D. J. Biol. Chem. 2003; 278: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 36Yin L. Huang L. Kufe D. J. Biol. Chem. 2004; 279: 45721-45727Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) prompted us to investigate whether MUC1 regulates the HIF-1α pathway. The results demonstrate that MUC1 attenuates hypoxia-induced activation of HIF-1α by up-regulating PHD3 and suppressing increases in ROS. The results also demonstrate that MUC1-dependent suppression of ROS blocks hypoxia-induced apoptosis and necrosis.MATERIALS AND METHODSCell Culture—Human HCT116/vector, HCT116/MUC1, HCT116/MUC1-CD, and HCT116/MUC1(Y46F) colon cancer cell clones (24Li Y. Kuwahara H. Ren J. Wen G. Kufe D. J. Biol. Chem. 2001; 276: 6061-6064Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 31Ren J. Agata N. Chen D. Li Y. Yu W.-H. Huang L. Raina D. Chen W. Kharbanda S. Kufe D. Cancer Cell. 2004; 5: 163-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) were grown in Dulbecco's modified Eagle's medium (high glucose; Cellgro, Inc., Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine. Human ZR-75-1 breast cancer cell clones expressing an empty vector, a control siRNA, or a MUC1siRNA that targets the sequence 5′-AAGTTCAGTGCCCAGCTCTAC-3′ (31Ren J. Agata N. Chen D. Li Y. Yu W.-H. Huang L. Raina D. Chen W. Kharbanda S. Kufe D. Cancer Cell. 2004; 5: 163-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 36Yin L. Huang L. Kufe D. J. Biol. Chem. 2004; 279: 45721-45727Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum with antibiotics and l-glutamine. Cells were seeded, grown for 24 h, and then placed in a modular incubator chamber (Billups-Rothenberg, Inc., Del Mar, CA) flushed with a gas mixture containing 1% O2, 5% CO2, and the balance N2. Cells were also treated with 25μm MG132 (Calbiochem), 600μm CoCl2 (Sigma-Aldrich), or 500 units/ml catalase (Sigma).Immunoblot Analysis—Cells were lysed as described previously (35Yin L. Kufe D. J. Biol. Chem. 2003; 278: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and analyzed by immunoblotting with anti-HIF-1α (BD Biosciences), anti-PHD1, anti-PHD3 (Bethyl Laboratories, Montgomery, TX), anti-PHD2 (Novus Biologicals, Littleton, CO), anti-β-actin (Sigma), and anti-MUC1-C (Ab5; NeoMarkers Inc., Fremont, CA). Antigen-antibody complexes were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences).Reverse Transcription (RT)-PCR–Total cellular RNA was extracted in TRIzol dissolved in RNase-free water and incubated for 10 min at 55 °C. HIF-1α (5′-CTCAAAGTCGGACAGCCTCA-3′ and 5′-CCCTGCAGTAGGTTTCTGCT-3′)- and PHD3 (5′-TGAACAATTTCCAGATGTTC-3′ and 5′-TCAAATTGTTCAAGATGCAC-3′)-specific primers were designed to amplify a 460-bp fragment. Primers for β-actin were used as a control (38Ben-Ezra J. Johnson D.A. Rossi J. Cook N. Wu A. J. Histochem. Cytochem. 1991; 39: 351-354Crossref PubMed Scopus (314) Google Scholar). The RNA was reverse-transcribed and amplified using SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen). Amplified fragments were analyzed by electrophoresis in 2% agarose gels.Measurement of ROS Levels–Cells were incubated with 5 μm DCFH-DA (Molecular Probes) for 20 min at 37 °C to assess H2O2-mediated oxidation to the fluorescent compound 2′,7′-dichlorofluorescin. Fluorescence of oxidized 2′,7′-dichlorofluorescin was measured at an excitation wavelength of 480 nm and an emission wavelength of 525 nm by flow cytometry (BD Biosciences).Silencing of MUC1 and PHD3–Cells were seeded at 3 × 105 cells/60-mm well. After 24 h, the cells were transfected with control siRNA, MUC1siRNA, or PHD3siRNA pools (siGENOME SMART pool reagents; Dharmacon RNA Technologies) for 72 h.Analysis of Mitochondrial Transmembrane Potential—Cells were incubated in 5 ng/ml DiOC6(3) (Molecular Probes) in phosphate-buffered saline for 30 min at 37 °C and then monitored by flow cytometry.Assays of Apoptosis and Necrosis—Sub-G1 DNA content was assessed by staining ethanol-fixed and citrate buffer-permeabilized cells with propidium iodide and monitoring by flow cytometry (BD Biosciences) as described previously (36Yin L. Huang L. Kufe D. J. Biol. Chem. 2004; 279: 45721-45727Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). For assessment of necrosis, cells were incubated in 1 μg/ml propidium iodide/phosphate-buffered saline for 5 min at room temperature and then monitored by flow cytometry as described previously (36Yin L. Huang L. Kufe D. J. Biol. Chem. 2004; 279: 45721-45727Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).RESULTSMUC1 Attenuates Activation of HIF-1α in the Response of HCT116 Cells to Hypoxia—HCT116 colon cancer cells are null for MUC1 expression (31Ren J. Agata N. Chen D. Li Y. Yu W.-H. Huang L. Raina D. Chen W. Kharbanda S. Kufe D. Cancer Cell. 2004; 5: 163-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). To determine whether MUC1 affects the response to hypoxia, we analyzed HIF-1α levels in HCT116 cells expressing an empty vector or exogenous MUC1. Exposure of HCT116/vector cells to hypoxic conditions was associated with increases in HIF-1α that were maximal at 6 h and remained elevated for 24 h (Fig. 1A). By contrast, hypoxia-induced increases in HIF-1α were attenuated in HCT116 cellss that stably overexpress MUC1 (Fig. 1A). Similar results were obtained with separately isolated HCT116/vector and HCT116/MUC1 clones (supplemental Fig. S1A), indicating that clonal selection is not responsible for the attenuation of HIF-1α activation. To define the region of MUC1 that regulates HIF-1α levels, we performed studies on HCT116 cells stably overexpressing the MUC1 cytoplasmic domain (MUC1-CD) (33Huang L. Ren J. Chen D. Li Y. Kharbanda S. Kufe D. Cancer Biol. Ther. 2003; 2: 702-706Crossref PubMed Scopus (3) Google Scholar). Exposure of separately isolated HCT116/MUC1-CD clones to hypoxic conditions demonstrated that MUC1-CD is sufficient to attenuate HIF-1α activation (Fig. 1B and supplemental Fig. S1B). Members of the c-Src family of non-receptor tyrosine kinases are activated by ROS (39Nakashima I. Kato M. Akhand A.A. Suzuki H. Takeda K. Hossain K. Kawamoto Y. Antioxid. Redox Signal. 2002; 4: 517-531Crossref PubMed Scopus (114) Google Scholar) and phosphorylate the MUC1 cytoplasmic domain on Tyr-46 (24Li Y. Kuwahara H. Ren J. Wen G. Kufe D. J. Biol. Chem. 2001; 276: 6061-6064Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 28Li Y. Chen W. Ren J. Yu W. Li Q. Yoshida K. Kufe D. Cancer Biol. Ther. 2003; 2: 187-193Crossref PubMed Scopus (70) Google Scholar). In this regard, stable overexpression of MUC1 with a mutation at Tyr-46 (Y46F) in the cytoplasmic domain (31Ren J. Agata N. Chen D. Li Y. Yu W.-H. Huang L. Raina D. Chen W. Kharbanda S. Kufe D. Cancer Cell. 2004; 5: 163-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) reversed (although not completely) the suppressive effects of MUC1 on HIF-1α activation (Fig. 1C). HIF-1α is destabilized under normoxic conditions by O2-dependent proteosomal degradation (40Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3825) Google Scholar, 41Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4360) Google Scholar, 42Yu W.H. Woessner Jr., J.F. McNeish J.D. Stamenkovic I. Genes Dev. 2002; 16: 307-323Crossref PubMed Scopus (379) Google Scholar). To define the level at which MUC1 regulates HIF-1α expression, RT-PCR was performed to assess HIF-1α gene transcription. MUC1 overexpression was associated with little effect on HIF-1α mRNA levels under normoxic and hypoxic conditions (supplemental Fig. S1C), indicating that MUC1 disrupts HIF-1α stabilization in the response to hypoxia. In concert with these results, attenuation of HIF-1α degradation with the proteosome inhibitor MG132 was associated with hypoxia-induced HIF-1α levels in HCT116/MUC1 and HCT116/MUC1-CD cells that were comparable with those in HCT116/vector cells (Fig. 1D). These findings indicate that MUC1 blocks hypoxia-induced stabilization of HIF-1α in HCT116 cells.Silencing Endogenous MUC1 Increases HIF-1α Activation in the Hypoxic Stress Response—To assess the effects of silencing endogenous MUC1 on HIF-1α, we studied ZR-75-1 cells expressing an empty vector, a control siRNA (CsiRNA) or a MUC1siRNA. As shown previously (31Ren J. Agata N. Chen D. Li Y. Yu W.-H. Huang L. Raina D. Chen W. Kharbanda S. Kufe D. Cancer Cell. 2004; 5: 163-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), the MUC1siRNA stably down-regulates endogenous MUC1 expression (Fig. 2A). Silencing MUC1 in ZR-75-1 cells was associated with substantially higher HIF-1α levels in response to hypoxia (Fig. 2A). The effects of down-regulating MUC1 were similar in two separately isolated ZR-75-1/MUC1siRNA clones (Fig. 2B). RT-PCR demonstrated that silencing MUC1 is associated with increases in HIF-1α mRNA levels under normoxic and hypoxic conditions (Fig. 2C). MG132 treatment of hypoxic ZR-75-1/CsiRNA and ZR-75-1/vector cells that overexpress endogenous MUC1 was associated with an increase in HIF-1α levels (Fig. 2D). However, the effects of MG132 were substantially more pronounced in the ZR-75-1/MUC1siRNA cells (Fig. 2D). These findings indicate that MUC1 attenuates HIF-1α gene transcription and hypoxia-induced stabilization of HIF-1α in ZR-75-1 cells. Whereas MUC1 conferred destabilization of HIF-1α in both HCT116 and ZR-75-1 cells, our subsequent studies focused on signals responsible for HIF-1α degradation.FIGURE 2Silencing MUC1 in ZR-75-1 cells increases HIF-1α activation. A, ZR-75-1/vector and ZR-75-1/MUC1siRNA cells were exposed to hypoxia for the indicated times. Lysates were immunoblotted (IB) with anti-HIF-1α, anti-MUC1-C, and anti-β-actin. B and C, the indicated ZR-75-1 cells were cultured under normoxic (N) or hypoxic (H) conditions for 24 h. Lysates were immunoblotted with the indicated antibodies (B). Total cellular RNA was amplified with HIF-1α- and β-actin-specific primers (C). The intensities of the signals were determined by densitometric scanning and are expressed as the relative signal intensity (RSI) compared with that obtained with normoxic ZR-75-1/CsiRNA cells. D, the indicated ZR-75-1 cells were exposed to hypoxia in the absence (–) and presence (+) of 25 μm MG132 for 8 h. Lysates were immunoblotted with anti-HIF-1α and anti-β-actin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MUC1 Regulates PHD Expression in HCT116 Cells—The constitutive degradation of HIF-1α under normoxic conditions is dependent on the abundance of PHD1, PHD2, and PHD3 (5Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2696) Google Scholar, 6Yu F. White S.B. Zhao Q. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9630-9635Crossref PubMed Scopus (634) Google Scholar, 43Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2087) Google Scholar, 44Appelhoff R.J. Tian Y.M. Raval R.R. Turley H. Harris A.L. Pugh C.W. Ratcliffe P.J. Gleadle J.M. J. Biol. Chem. 2004; 279: 38458-38465Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar). In this context, down-regulation of PHDs during hypoxia is important for stabilization of HIF-1α (5Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2696) Google Scholar, 40Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3825) Google Scholar, 41Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4360) Google Scholar, 45Nakayama K. Frew I.J. Hagensen M. Skals M. Habelhah H. Bhoumik A. Kadoya T. Erdjument-Bromage H. Tempst P. Frappell P.B. Bowtell D.D. Ronai Z. Cell. 2004; 117: 941-952Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Immunoblot analysis of HCT116 cell lysates demonstrated that MUC1 overexpression has little if any effect on levels of the 43-kDa PHD1 protein (Fig. 3A). However, MUC1 overexpression was associated with down-regulation of the 46-kDa PHD2 (Fig. 3A). MUC1 overexpression was also associated with increases in levels of the 27-kDa PHD3 protein (Fig. 3A). In addition, hypoxia had little effect on the MUC1-induced increases in PHD3 (Fig. 3A and supplemental Fig. S2A). Similar results were obtained with the separately isolated HCT116/vector and HCT116/MUC1 cells (supplemental Fig. S2A). Increases in PHD3 levels were also found in HCT116 cells overexpressing MUC1-CD (Fig. 3B and supplemental Fig. S2B). By contrast, there was no apparent effect on PHD3 expression in the HCT116/MUC1(Y46F) cells (Fig. 3C). To determine whether MUC1 attenuates HIF-1α activation by up-regulating PHD3, we treated the HCT116 cells with CoCl2, an inhibitor of PHD activity (46Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell Biol. 2004; 5: 343-354Crossref PubMed Scopus (1580) Google Scholar). The results show that CoCl2-induced HIF-1α activation is similar in the absence and presence of MUC1 or MUC1-CD (Fig. 3D). To further assess whether PHD3 is involved in the regulation of HIF-1α in HCT116/MUC1 cells, we transiently down-regulated PHD3 expression with a PHD3 siRNA pool (Fig. 3E). Silencing PHD3 was associated with increases in HIF-1α (Fig. 3E). These findings indicate that MUC1 up-regulates PHD3 and thereby decreases HIF-1α in HCT116 cells.FIGURE 3MUC1 up-regulates PHD3 expression in HCT116 cells. A and B, HCT116/vector, HCT116/MUC1, and HCT116/MUC1-CD cells were cultured under hypoxic conditions for the indicated times. Lysates were immunoblotted (IB) with antibodies against PHD1–3. NS, nonspecific band. C, the indicated HCT116 cells were cultured under normoxic (N) or hypoxic (H) conditions for 24 h. Lysates were immunoblotted with the indicated antibodies. D, the indicated HCT116 cells were incubated in the absence (–) and presence (+) of 600 μm CoCl2 for 24 h. Lysates were immunoblotted with the indicated antibodies. E, HCT116/MUC1 cells were transfected with control CsiRNA or PHD3 siRNA pools. After 72 h, the cells were cultured under hypoxic conditions for 6 h. Lysates were immunoblotted with the indicated antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Silencing MUC1 in ZR-75-1 Cells Decreases PHD3 Expression—Analysis of PHD levels in ZR-75-1 cells demonstrated that silencing of MUC1 has little effect on PHD1 or PHD2 levels (Fig. 4A). By contrast, silencing MUC1 was associated with a substantial down-regulation of PHD3 (Fig. 4A). A role for MUC1 in up-regulating PHD3 expression was confirmed in the ZR-75-1/CsiRNA cells and both ZR-75-1/MUC1siRNA clones (Fig. 4B). As a control, silencing MUC1 with a MUC1 siRNA pool was also associated with decreases in PHD3 and increases in HIF-1α levels (Fig. 4C), indicating that the results observed are not due to off-target effects of the MUC1siRNA. Analysis of PHD3 mRNA levels by RT-PCR further indicated that the down-regulation of PHD3 protein associated with silencing MUC1 is conferred by a post-transcriptional mechanism (supplemental Fig. S3). MUC1-mediated attenuation of HIF-1α activation was reversed in large part by CoCl2 (Fig. 4D), consistent with regulation by a PHD-dependent mechanism. Moreover, silencing PHD3 was associated with increases in HIF-1α levels (Fig. 4E). These findings in ZR-75-1 cells indicate that MUC1 increases PHD3 levels and that this response contributes to the attenuation of hypoxia-induced HIF-1α activation.FIGURE 4Silencing MUC1 in ZR-75-1 cells decreases PHD3 levels. A, ZR-75-1/vector and ZR-75-1/MUC1siRNA-A cells" @default.
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• W1983991527 title "Mucin 1 Oncoprotein Blocks Hypoxia-inducible Factor 1α Activation in a Survival Response to Hypoxia" @default.
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