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J Biol Chem. Apr 10, 2009; 284(15): 10004–10012.
PMCID: PMC2665055

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

Ni Chen,§ Xueqin Chen,§ Rui Huang,§ Hao Zeng, Jing Gong,§ Wentong Meng,||,1 Yiping Lu, Fang Zhao,** Lin Wang,‡‡ and Qiao Zhou§,2

Abstract

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)3 plays major roles in cellular response to hypoxia as well as in disease processes including carcinogenesis (1-4). Many genes have been identified as HIF-1 targets (3, 4), 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 (3-5), in which it is associated with advanced clinical stage and treatment failure (6). HIF-1α overexpression has been identified in both prostate adenocarcinoma tissue (5, 7) and cell lines (8).

Although acute hypoxia may lead to cell death, prolonged hypoxia results in resistance to apoptosis as well as to radiotherapy and chemotherapy (4, 9, 10), 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 (11) and BIRC5/survivin (12). Although pro-apoptotic molecules BNIP3, NIX (13, 14), and Noxa (15) 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 (16, 17). Despite the correlation of BCL-xL overexpression with HIF-1α in some tumors (18) and the observation that BCL-xL is a key molecule underlying hypoxia-driven cell death resistance (10), 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) (19), NF-κB (20), Ets (21), GATA (22), PAX3 (and the PAX3/FKHR (Forkhead related transcription factor) fusion) (23), and POU (Brn-3a) (24). 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.

EXPERIMENTAL PROCEDURES

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 for RT. PCR primers were designed according to cDNA sequences (GenBank™) as follows: HIF-1α (5′-CCT ATG ACC TGC TTG GTG CTG-3′, 5′-CTG GCT CAT ATC CCA TCA ATT CG-3′, product length 157 bp), BNIP3 (5′-ACC AAC AGG GCT TCT GAA AC-3′, 5′-GAG GGT GGC CGT GCG C-3′, 202 bp), GLUT-1 (5′-GCA AGT CCT TTG AGA TGC TGA TCC-3′, 5′-GCC GAC TCT CTT CCT TCA TCT CC-3′, 402 bp), GAPDH (5′-CCA CCC ATG GCA AAT TCC ATG GCA-3′, 5′-TCA AGA CGG CAG GTC AGG TCC ACC-3′, 597 bp), BCL-xL/BCL-xS (5′-GCA GGC GAC GAG TTT GAA CT-3′, 5′-CTC GGC TGC TGC ATT GTT C-3′, BCL-xL 330bp, BCL-xS, 141 bp), BIK (5′-GAG GTT CTT GGC ATG ACT GAC-3′, 5′-GTG GTG AAA CCG TCC ATG AAA C-3′, 247 bp), BID (5′-GCA GCT CAG GAA CAC CAG C-3′, 5′-GAC ATC ACG GAG CAA GGA CG-3′, 178 bp), BAX (5′-GCT TCA GGG TTT CAT CCA GG-3′, 5′-CCA GTT GAA GTT GCC GTC AG-3′, 244 bp), BIRC5/survivin (5′-GCA GTT TGA AGA ATT AAC CCT TG-3′, 5′-CAC TTT CTC CGC AGT TTC CTC-3′, 121 bp), CIAP1 (5′-TTG TCA ACT TCA GAT ACC ACT GGA G-3′, 5′-CAA GGC AGA TTT AAC CAC AGG TG-3′, 123 bp), CIAP2 (5′-AGG GAA GAG GAG AGA GAA AGA GC-3′, 5′-CGG CAG TTA GTA GAC TAT CCA GG-3′, 133 bp), XIAP (5′-GGG TTC AGT TTC AAG GAC ATT AAG-3′, 5′-CGC CTT AGC TGC TCT TCA GTA C-3′, 182 bp), CASP3 (5′-GTT TGT GTG CTT CTG AGC CAT G-3′, 5′-CCA CTG TCT GTC TCA ATG CCA C-3′, 188 bp), CASP6 (5′-TCA GAC AGA GAA GTT GGA CAC C-3′, 5′-CTG TGA ACT CTA AGG AGG AGC C-3′, 200 bp), CASP7 (5′-CCC ATC AAT GAC ACA GAT GC-3′, 5′-CAC AAA CCA GGA GCC TCT TC-3′, 126 bp), CASP9 (5′-CCA CAC CCA GTG ACA TCT TTG-3′, 5′-ACC GAA ACA GCA TTA GCG AC-3′, 172 bp). β-Actin was used as internal control (5′-CTG GCA CCA CAC CTT CTA CAA TG-3′, 5′-CCT CGT AGA TGG GCA CAG TGT G-3′, 248bp). Standard PCR protocols were used, and products resolved by 2% agarose gel electrophoresis and visualized by staining with the fluorescent dye Goldview™ (SBS, Beijing, China).

The Real-time PCR Master Mix containing SYBR Green (ToYoBo) was used for real-time PCR on Light Cycler 2.0 (Roche Diagnostics), and data were recorded and analyzed by the Light Cycler software 4.05. Copy number of target genes (relative to β-actin) was defined by 2-ΔΔCt, where ΔΔCt = ΔCtHIF-KDCtHIF-CON = (CtHIF-KD-target - CtHIF-KD-actin) - (CtHIF-CON-target - CtHIF-CON-actin).

Inhibition of PI3K in PC-3 Cells—PC-3 cells were cultured in 6-well plates in fetal calf serum-free media and treated with 0, 10, 20, and 50 μm PI3K inhibitor LY294002 for 1 h. Cells were collected for Western analysis of HIF-1α, BCL-xL, AKT1/2, and phosphorylated AKT.

Hypoxia Mimetic Treatment of PC-3 Cells—PC-3 cells were cultured in 6-well plates in fetal calf serum-free media and treated with 0, 200, 400 μm CoCl2 for 4 h. Cells were collected for Western analysis of HIF-1α and BCL-xL.

HIF-1α RNA Interference—The expression vector pRNAT-U6.1/Neo (GenScript Corp. Piscataway, NJ) was used to construct HIF-1α siRNA plasmids by inserting siRNA-coding sequences under U6 promoter for siRNA expression. Two (HIF-siRNA1 and -2) expressing vectors were constructed with the following siRNA sequences: HIF-siRNA1, GCCACATCATCACCATATA (nucleotides 1960-1978, NM_001530); HIF-siRNA2, CTAACTGGACACAGTGTGT (nucleotides 379-397). Corresponding control siRNAs with scrambled sequences were also designed and prepared as Scrambled 1, GACCTACAACTACCTATCA, and Scrambled 2, GTGGACACCCGATAAGTTT. These sequences were checked to ensure non-homology with known human mRNA sequences. Co-expression of green fluorescence protein from the plasmid was used for checking transfection efficiency.

PC-3 cells were transfected by using Lipofectamine 2000 (Invitrogen). To obtain HIF knock-down cells (HIF-KD) with stable transfection of HIF-1α-siRNA1 and HIF-1α-siRNA2 (designated as HIF-KD1 and HIF-KD2, respectively) and the control plasmids (RNAi-CON1 and RNAi-CON2, respectively), cells were selected by G418 at 500 μg/ml for 2 weeks (starting at 48 h after transfection) and maintained in growth medium supplemented with G418 (200 μg/ml).

Western Blot Analysis—The primary antibodies used were: HIF-1α (mouse monoclonal, 1:1,500) from Chemicon Inc., Pittsburgh, PA; BCL-xL (rabbit polyclonal,1:1,000) and phosphorylated AKT (rabbit polyclonal, 1:600) from Cell Signaling Technology Inc., Danvers, MA; BNIP3 (mouse monoclonal, 1:3,000) from Sigma; BIRC5/survivin (rabbit polyclonal, 1:1,000) from R and D Systems Inc., Minneapolis, MN; BAX (mouse monoclonal, 1:800), CIAP1 (rabbit polyclonal, 1:800), CIAP2 (rabbit polyclonal, 1:800), CASP3 (rabbit polyclonal, 1:800), CASP9 (rabbit polyclonal, 1:600), and AKT1/2 (goat polyclonal, 1:800) from Santa Cruz Biotechnology, Santa Cruz, CA; GAPDH (mouse monoclonal, 1:10,000) from Kangcheng, Shanghai, China; and β-tubulin (mouse monoclonal, 1:1,000) from Huatesheng, Shenzhen, China. Horseradish peroxidase-labeled secondary antibodies were from Zymed Laboratories Inc.

Total proteins resolved by SDS-polyacrylamide (Sigma) gel electrophoresis were electroblotted to polyvinylidene difluoride membrane (Amersham Biosciences), blocked with 5% nonfat milk and 0.1% Tween 20, and incubated with primary and secondary antibodies at room temperature for 2 and 1.5 h, respectively. Signals were detected by exposure to x-ray films after treatment with the SuperSignal enhanced chemiluminescence kit (Pierce).

Ultraviolet (UV) Irradiation Induced Cell Death—Cells in culture plates were briefly exposed to UV irradiation in a UV cross-linker (UVC-500, Hoefer, San Francisco, CA) at 120 mJ/cm2 for 30 s. Cells were then cultured as appropriate for subsequent assays.

Immunocytochemistry—Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and incubated overnight at 4 °C with anti-human Ki67/MIB-1 antibody (mouse monoclonal, 1:100, DakoCytomation, Glostrup, Denmark), anti-human cleaved caspase-3 antibody (rabbit polyclonal, 1:200, Cell Signaling Technology), or anti-human BCL-xL antibody (1:200). Standard labeled streptavidin-biotin protocol was used for staining with 3′-diaminobenzidine as chromogen and hematoxylin as counterstain.

Cell Viability Assay—Cells were cultured in 96-well plates and measured by tetrazolium-based MTT (Sigma) cell proliferation assay. The working concentration of MTT was 1 mg/ml.

Caspase-3 Activity Assay—Cultured cells were lysed with lysis buffer containing 50 mm Hepes (pH 7.4), 100 mm NaCl, 0.1% CHAPS, 1 mm EDTA, 10% glycerol, and 10 mm dithiothreitol. The soluble fraction of the cell lysate was used for colorimetric caspase-3 activity assay using acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-PNA) (Calbiochem) as a substrate on FL600 plate reader (BIO-TEK, Winooski, VT).

Terminal Deoxynucleotidyltransferase-mediated Biotinylated dUTP Nick end-labeling (TUNEL)—TUNEL was performed by using in situ cell death detection kit (Roche Diagnostics). Cells were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton-100, 0.1% sodium citrate, incubated with TUNEL reaction mixture then with alkaline phosphatase-conjugated anti-fluorescein antibody, stained with nitro blue tetrazolium/5-bromo-4-chloro-3-Indolyl phosphate and counterstained with methyl green. Reaction without terminal transferase was used as negative control. The Apoptotic index was represented as number of TUNEL(+) cells/total number of cells (%).

Overexpression of BCL-xL in HIF-KD1 Cells—Full-length cDNA of BCL-xL coding sequence was cloned into TA vector pMD18-T (TaKaRa, Dalian, China) and subcloned into pDsRed vector (Clontech, Palo Alto, CA). The primers used for cloning were: 5′-AGA TCT AAT GTC TCA GAG CAA CCG GGA-3′ and 5′-GTC GAC CGT TTC CGA CTG AAG AGT GAG-3′. HIF-KD1 cells were transfected with BCL-xL expression plasmid (HIF-KD1-xL) or pDsRed control vector (HIF-KD1-DsRed) using Lipofectamine 2000. Transfected HIF-KD1-xL and HIF-KD1-DsRed cells were maintained in RPMI1640.

Reporter Gene Assay for HIF-1α-dependent BCL-xL Promoter Activity—The basic pGL3 luciferase reporter vector (Promega, Madison, WI) was used to construct reporter plasmids with various lengths of the BCL-xL promoter. Four plasmids were constructed in which the BCL-xL promoter spanned -1075 to +617 (relative to the transcription start site) or truncated fragments of which were inserted upstream of the luciferase gene. The reporter constructs were designated as pGL1642 (-1075 to +617), pGL1281 (-664 to +617), pGL828 (-211 to +617), and pGL621 (-4 to +617), respectively. Two additional plasmids with HRE1 and HRE2 site-specific mutation were constructed: pGL828-MUT (-211 to +617, with CGTG at -78 to -75 of HRE1 mutated to TCGG) and pGL1642-MUT (-1075 to +617, with CGTG at -858 to -855 of HRE2 mutated to TCGG). Each reporter construct and the pRL-CMV plasmid (Promega) containing the Renilla luciferase gene as internal control were used in dual reporter gene assay for studying HIF-1α-dependent gene expression. Cells were transfected with plasmids by using Lipofectamine 2000 (Invitrogen). Four hours after transfection, the medium was replaced by fresh medium. Thirty-six hours after transfection, cells were treated with 400 μm CoCl2 for 12 h, and luciferase activity was determined by using Luminometer TD-20/20 (Turner Designs, Sunnyvale, CA).

Chromatin Immunoprecipitation—Cells were lysed, and nuclei were pelleted. The extract was sonicated, and supernatants were collected and treated with sheared salmon sperm DNA (Invitrogen) and protein A/G-Sepharose (Santa Cruz). Immunoprecipitation was performed overnight at 4 °C with 3 μg of HIF-1α monoclonal antibody or the control isotype IgG2b (Lab Vision Corp., Fremont, CA) or no antibody and then with protein A/G-Sepharose and salmon sperm DNA. Precipitates were washed, and extracted with 1% SDS and 0.1 m NaHCO3. Eluates were pooled and heated. DNA fragments were purified and used as template for PCR. The promoter-specific primers used were: BCL-xL, 5′-CGA GCA GTC AGC CAG GTA G-3′ and 5′-GAC GGC GAA GGC TCC TAT TG-3′; VEGF (as positive control), 5′-GTT CCC TGG CAA CAT CTG G-3′ and 5′-GAC ATC AAA GTG AGC GGC AG-3′.

Electrophoretic Mobility Shift Assay (EMSA) and Supershift Assay—The sequences of the two BCL-xL promoter oligonucleotide probes were: BCL-xL-Pro1, 5′-GAGCCAAGGGG-CGTGCAAGAGAGAGG-3′ (-89 to -64), and BCL-xL-Pro2, 5′-CCCTGTGCGTGACAGCCGT-3′ (-865 to -847). The VEGF promoter probe 5′-CAGTGCATACGTGGGCTCCA-3′ (-989 to -970) was used as positive control. Three corresponding probes with HRE mutation were prepared: BCL-xL-Pro1-MUT, 5′-GAGCCAAGGGGTCGGCAAGAGAGAGG-3′, BCL-xL-Pro2-MUT, 5′-CCCTGTGTCGGACAGCCGT-3′, VEGF-Pro-MUT, 5′-CAGTGCATATCGGGGCTCCA-3′. Labeled wild-type probes were prepared by biotinylation (Invitrogen). Unlabeled wild-type and mutant probes were used for competition experiments. For each probe, complementary strands were synthesized, and equimolar concentrations of complementary strands were annealed for use in EMSA.

PC-3 cells were harvested after 6 h of incubation with 400 μm CoCl2. Nuclear extract was prepared, and 10 μg was incubated with 100 pmol of biotinylated probe in a 10-μl reaction mixture (with 0.5 μg of poly[dI-dC]) for 30 min at room temperature. For competition assays, a 50-fold excess of unlabeled wild-type probe or mutant probe was used. For supershift assays, 1.0 μg of monoclonal anti-HIF-1α antibody was added to the reaction mixture and incubated at 4 °C overnight. The mixture was electrophoresed at 4 °C on 6% PAGE for 3 h and transferred to nylon membranes (Roche Diagnostics) by electroblotting. After baking and blocking, horseradish peroxidase-labeled streptavidin (1:1000, Zymed Laboratories Inc.) was added and incubated at room temperature for 2 h. Signals were detected by exposure to x-ray films after treatment with the SuperSignal enhanced chemiluminescence kit (Pierce).

Statistical Analysis—Statistical analysis was performed by using the SPSS 10 software package (Chicago, IL).

RESULTS

HIF-1α siRNA Significantly Decreased Expression of HIF-1α and Its Target Genes—HIF-1α mRNA and protein overexpression in prostate cancer cells and primary prostate adenocarcinoma tissues was validated by conventional RT-PCR (Fig. 1A) and Western blot (Fig. 1B) analysis, respectively. In normal prostate epithelium, HIF-1α mRNA and protein were undetectable (Fig. 1, A and B).

FIGURE 1.
HIF-1α siRNA significantly decreased HIF-1α overexpression in prostate cancer cells. HIF-1α mRNA (A) and protein (B) were overexpressed in prostate cancer cells PC-3, LNCaP, and DU-145, as were in primary prostate cancer tissue ...

The two HIF-siRNA constructs showed comparable interference efficiency, with HIF-siRNA1 being more potent (Fig. 1, C, D, and E). In contrast, the control constructs with scrambled sequences had no effect on HIF-1α expression. The G418-selected PC-3 cells with stably transfected HIF-siRNA1 and HIF-siRNA2 (HIF-KD1 and HIF-KD2 cells, respectively) and the control plasmids (RNAi-CON1 and RNAi-CON2 cells, respectively) were largely homogeneous, as shown by the co-expression of green fluorescence protein (Fig. 1F).

HIF-1α mRNA (Fig. 1G) and protein (Fig. 1H) were significantly reduced in the HIF-KD cells, with consequent down-regulation of known HIF-1α target genes BNIP3, GLUT-1, and GAPDH (Fig. 1, G and H). The control constructs had no effect on mRNA and protein expression of HIF-1α or its target genes (Fig. 1, G and H). Quantitative PCR analysis of mRNA of HIF-1α and its target gene GLUT-1 further validated the interference effect of HIF-1α siRNA, as both of which were significantly reduced (not shown).

HIF-1α siRNA Inhibited PC-3 Cell Proliferation—HIF-KD cells also showed significantly reduced cell growth (Fig. 2, A and B). Decreased cell proliferation was further demonstrated by immunocytochemistry of the proliferative antigen Ki67/MIB-1, which showed much lower Ki67-labeling index (50%) in HIF-KD cells than control (90%) (Fig. 2A).

FIGURE 2.
Effects of HIF-1α knock-down on cell proliferation and apoptosis. Cell growth was significantly reduced in HIF-KD cells as compared with RNAi-CON cells, as demonstrated by immunocytochemical staining of the proliferative antigen (Ki67/MIB-1), ...

HIF-KD Cells Were More Sensitive to Ultraviolet Irradiation and Flutamide Treatment—HIF-KD cells showed a significantly higher cell death rate (Fig. 2, C and D) after UV irradiation. TUNEL assays demonstrating the spontaneous apoptotic index in HIF-KD1 and HIF-KD2 cells were 1.0% (±0.2%) and 0.9% (±0.1%), respectively; in RNAi-CON1 and RNAi-CON2 cells they were 0.2% (±0.01%) and 0.2% (±0.01%), respectively (Fig. 2D). Upon UV irradiation, the apoptotic index in HIF-KD1 and HIF-KD2 cells increased significantly to 17.3% (±1.7%) and 12.7% (±1.3%), respectively (Fig. 2D), but in RNAi-CON1 and RNAi-CON2 cells, the apoptotic index only increased slightly to 1.7% (±0.2%) and 1.5% (±0.1%), respectively.

Immunocytochemistry staining of cleaved caspase-3 (as manifestation of caspase-3 activation) showed prominent activation of caspase-3 in HIF-KD cells but not in RNAi-CON cells (Fig. 2E). Colorimetric caspase-3 activity assay further demonstrated higher caspase-3 activity in HIF-KD cells after UV irradiation (Fig. 2F).

HIF-siRNA also sensitized PC-3 cells to the anti-androgen drug flutamide, treatment by which further inhibited growth rate of HIF-KD cells (Fig. 2B). In contrast, RNAi-CON cell growth was exuberant and essentially remained the same with or without flutamide treatment.

HIF-1α siRNA Dramatically Decreased BCL-xL Expression—To elucidate the mechanisms by which HIF-1α siRNA inhibited cell proliferation and promoted apoptosis in PC-3 cells, we examined the effect of HIF-1α siRNA on major apoptosis regulators: the Bcl-2 family, the IAP family, and the caspase family. Most prominently, BCL-xL expression was significantly down-regulated by HIF-1α siRNA (Fig. 3, A-D), whereas the other examined members of the Bcl-2 family showed little change.

FIGURE 3.
Effects of HIF-1α knock-down on members of Bcl-2 family, IAP family, and caspase family. Expression of BCL-xL was significantly reduced after HIF-1α knock-down, as shown by conventional RT-PCR (A), Western blot analysis (B), real-time ...

BCL-xS, which is encoded by the same gene locus, also showed a decrease in expression upon HIF-1α siRNA. As the base level of BCL-xS was extremely low, the effect appeared less dramatic than BCL-xL (Fig. 3A).

As expected, HIF-1α siRNA also induced a significant decrease of survivin, a documented HIF-1α target (Fig. 3, A and B). Other members of the IAP family, including CIAP1, CIAP2, and XIAP, were not down-regulated. Moreover, HIF-1α siRNA had no effect on expression level of the examined caspases (Fig. 3, A and B).

Overexpression of BCL-xL in HIF-KD Cells Promoted Cell Growth and Inhibited Cell Death—To further demonstrate the importance of HIF-induced BCL-xL expression in apoptosis resistance, BCL-xL was artificially overexpressed in HIF-KD1 cells (HIF-KD1-xL, Fig. 4, A-E), which counteracted the effects of HIF-1α siRNA, resulting in enhanced cell growth (Fig. 4C) and inhibition of UV irradiation-induced cell death and caspase 3 activity (Fig. 4, D and E).

FIGURE 4.
Artificial overexpression of BCL-xL in HIF-KD cells counteracted the effects of HIF-1α knock-down. HIF-KD1 cells (A, left panel, with green fluorescence protein expression) transfected with BCL-xL expression vector (HIF-KD1-xL cells) resulted ...

Inhibition of the PI3K/Akt Pathway Resulted in Down-regulation of Both HIF-1α and BCL-xL—The down-regulation of BCL-xL by HIF-1α siRNA was dramatic given the constitutive high BCL-xL mRNA (Fig. 5A) and protein (Fig. 5B) level in prostate cancer cell lines. Because PI3K/Akt is a major signaling pathway that controls HIF-1α level, we tested if PI3K inhibitor LY294002 could lead to decrease of BCL-xL. As shown in Fig. 5C, both HIF-1α and BCL-xL were simultaneously reduced upon PI3K/Akt inhibition in a dose-dependent manner.

FIGURE 5.
BCL-xL overexpression in prostate cancer cells could be inhibited by the PI3K inhibitor LY294002 but boosted by the hypoxia mimetic CoCl2. BCL-xL mRNA (A) and protein (B) were overexpressed in prostate cancer cells PC-3, LNCaP, and DU-145 and in primary ...

Hypoxia Mimetic CoCl2 Boosted Concomitant BCL-xL and HIF-1α Expression in PC-3 Cells—Treatment of PC-3 cells with the hypoxia mimetic CoCl2 resulted in additional increase of BCL-xL and HIF-1α simultaneously, further supporting the potential dependence of BCL-xL on HIF-1α in response to hypoxia (Fig. 5D).

Potential HREs on BCL-xL Promoter—The above experiments provided important clues to relationship between HIF-1α and BCL-xL. Because BCL-xL is a gene regulated by NF-κB, which could potentially be activated by PI3K/Akt signaling, reduction of BCL-xL expression by PI3K/Akt inhibition might be the effect of either HIF-1α or NF-κB inhibition or both. This prompted us to investigate whether BCL-xL was directly regulated by HIF-1α.

Potential HRE was searched in the human BCL-xL promoter region. Four short HRE consensus motifs were identified within the ~1000-bp region preceding the transcriptional start site. Two of them, starting at positions -78 and -858 (Fig. 6A), fit the extended consensus and were highly conserved across species (Fig. 6B).

FIGURE 6.
HRE in BCL-xL promoter; binding to and transcriptional regulation by HIF-1α. Two potential HRE sites (Site 1 and Site 2, starting at position -78 and -858 of human BCL-xL promoter, respectively) identified by sequence analysis (A) were conserved ...

Reporter Gene Assay of Putative HRE Activity—The reporter constructs were shown in Fig. 6C. The pGL1642 (-1075 to +617) contained both HREs (HRE2 and HRE1) flanking two NF-κB binding sites, whereas pGL1281 (-664 to +617) differed by lacking HRE2. The pGL828 (-211 to +617) contained HRE1 only, and pGL621 (-4 to +617) contained no HRE. Two constructs were prepared with site-specific mutation of the respective HREs: pGL828-MUT and pGL1642-MUT.

PC-3 cells were transiently transfected with one of the six constructs, with PRL-CMV co-transfection as internal control. The hypoxia mimetic CoCl2 was used to simulate hypoxia and to further boost HIF-1α as a means of showing hypoxia-induced gene transcription in the reporter gene assay.

The experiment showed significantly higher luciferase activity of the HRE-bearing constructs (Fig. 6C) than base line (pGL3). The effects were more dramatic with the HRE2-bearing construct upon CoCl2 treatment, whereas mutation of the HRE (notably HRE2) core sequence resulted in a significant reduction of transcriptional activity. These experiments indicated that the reporter gene transcription was under control of BCL-xL promoter containing HRE, particularly HRE2, which responded to the hypoxia mimetic.

Chromatin Immunoprecipitation Assay Displayed HIF-1α Interaction with BCL-xL Promoter—To show HIF-1α physically bind to BCL-xL promoter, we first used chromatin immunoprecipitation assay of PC-3 cells treated by CoCl2. Using the chromatin fraction pulled down by anti-HIF-1α antibody as template, a PCR fragment corresponding to -1025 to -821 (containing HRE2) of BCL-xL promoter was detected (Fig. 6D) and verified by sequencing. This fragment was not detected when isotype control IgG2b or no antibody was used for the pulldown assay (Fig. 6D).

EMSA and Super Shift Assay Demonstrated HIF-1α Binding to the Promoter Region of BCL-xL—To further confirm binding of HIF-1α to the putative HRE in human BCL-xL promoter, we performed EMSA with two oligonucleotide probes, each containing one of the extended HRE consensus sequence, designated BCL-xL-Pro1 (-89 to -64) and BCL-xL-Pro2 (-865 to -847), respectively. Two probes with HRE core sequence mutations were also prepared for competition experiments and were designated as BCL-xL-Pro1-MUT and BCL-xL-Pro2-MUT, respectively. A known HIF-1α binding oligonucleotide derived from the VEGF receptor gene promoter (VEGF-Pro) was used as positive control (Fig. 6E) together with a corresponding HRE mutation probe, VEGF-Pro-MUT, for competition assays.

The experiments showed that the labeled BCL-xL-Pro2 (Fig. 6E, lanes 1 and 2), but not BCL-xL-Pro1 (not shown), caused gel mobility shift when incubated with nuclear proteins from PC-3 cells. The shift could be suppressed in the competition experiments with excess unlabeled wild-type probe BCL-xL-Pro2 (Fig. 6E, lane 3) but not with the HRE-mutated probe BCL-xL-Pro2-MUT (Fig. 6E, lane 4). When HIF-1α monoclonal antibody was included in the binding reaction, a supershift band was observed as well with BCL-xL-Pro2 (Fig. 6E, lane 5) but not BCL-xL-Pro1 (not shown). Gel mobility shift and supershift were also shown with the VEGF-Pro positive control (Fig. 6E, lanes 6 and 7) together with competition and supershift assays (Fig. 6E, lanes 8-10). These results further demonstrated HIF-1α binding to the HRE2 in the -865 to -847 region of BCL-xL promoter.

DISCUSSION

We generated prostate cancer PC-3 cells in which HIF-1α was stably knocked-down by using siRNAs, which resulted in a significant decrease of the anti-apoptotic molecule BCL-xL. We then showed that HIF-1α directly regulated BCL-xL gene transcription. These novel findings point 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 failure.

Hypoxia is common in solid tumors (4), including prostate carcinoma (3), in which the extent of hypoxia is correlated with clinical stage and treatment failure (6). A hypoxia-mediated increase in HIF-1α plays critical roles in tumorigenesis and progression of many cancers through HIF-1α-dependent activation of genes that promote cancer cell survival, proliferation, spreading, and angiogenesis. Overexpression of HIF-1α and its target genes has been observed in a variety of solid tumors, for example, tumors of the brain (25), kidney and the urinary tract (26), and lung (27) as well as prostate (5, 7).

High levels of HIF-1α has been observed in prostate cancer tissue and cell lines (5, 7, 8). Up-regulation of HIF-1α might be an early event in prostate carcinogenesis, as high grade prostate intraepithelial neoplasia showed higher a HIF-1α level than benign epithelium (5). It is noteworthy that, although HIF-1α overexpression is often hypoxia-dependent, prostate cancer cells have constitutively high HIF-1α level, which could be further increased by hypoxia (8). HIF-1α gene amplification (28) and P582S polymorphism or mutation in the oxygen-dependent domain (29) might contribute to overexpression of HIF-1α at normoxic conditions.

Hypoxia and HIF-1α overexpression contribute to resistance to radiotherapy and chemotherapy (4). For example, the multi-drug resistance 1 gene has been observed to be hypoxia-responsive and is regulated by HIF-1α (30). Treatment of LNCaP cells with the androgen receptor antagonist Casodex results in up-regulation of a subset of hypoxia-related genes, including membrane metallo-endopeptidase and cyclin G2, which might be involved in development of the androgen-independent phenotype (31).

Knock-down of HIF-1α by siRNA or antisense techniques inhibits cell growth, proliferation, or migration, and promotes apoptosis. The effects have been observed in human respiratory epithelium (32) and umbilical vascular endothelial cells (33) as well as in a variety of tumors, including glioma (34), non-small cell lung cancer (35), hepatocellular carcinoma (11), pancreatic cancer (36), pituitary adenoma (37), squamous cell carcinoma (38), and prostate cancer (39).

Of more clinical interest is that silencing of HIF-1α gene results in sensitization of cancer cells to therapeutic agents. For example, HIF-1α knock-down increases sensitivity to 5-fluorouracil, doxorubicin, and gemcitabine in pancreatic cancer cell (36). Our finding that HIF-1α knock-down renders the androgen-independent PC-3 cells more sensitive to UV or flutamide treatment also supports that HIF-1α is a potential therapeutic target in androgen-independent prostate cancer.

The effects of such inhibition are mainly mediated by down-regulation of HIF transcriptional targets involved in diverse biological processes as cell proliferation, metabolism, and angiogenesis. These targets include, for example, phosphoglycerate kinase (35), GLUT-1 (34), chemokine receptors CXCR1 and CXCR2 (39) or CRCX4 (40), and VEGF (35).

Promotion of apoptosis is a recurrent theme of HIF-1α knock-down (11, 32-40). However, the underlying mechanisms have been less clear. Caspases could be increased or activated upon HIF-1α siRNA, but it most probably reflects the activation of caspase-dependent pathways rather than transactivation. It was only recently that two anti-apoptotic genes, BIRC5/survivin (12) and Mcl-1 (11, 32), were identified as HIF-1α targets. Despite responsiveness to HIF-1α by pro-apoptotic genes BNIP3, NIX, and Noxa (13-15), most experiments have shown that knock-down of HIF-1α promotes cell death and inhibits cell proliferation (32-35, 37, 38), apparently as the end result of a complex regulatory circuit.

Although BCL-xL has been found to be a key molecule involved in hypoxia-induced resistance to cell death (10) and BCL-xL overexpression has been associated with increased HIF-1α in tumors such as non-small cell lung cancer (18), the mechanism by which hypoxia induces BCL-xL up-regulation and the relationship between HIF-1α and BCL-xL has not been known.

Our study, thus, provides the first evidence that HIF-1α directly regulates BCL-xL transcription by interacting with HRE in the BCL-xL promoter. It has been shown that in the androgen-independent PC-3 cell, BCL-xL is more responsible for apoptosis-resistance than the prototypic Bcl-2 (16). Our data, therefore, indicate that HIF-1α-dependent overexpression of BCL-xL in PC-3 cells is one of the major mechanisms by which prostate cancer cells, particularly androgen-independent cells, resist apoptosis and chemotherapy. Recently, the IAP family member survivin has been identified as a transcriptional target of HIF-1α (12). Being up-regulated in many cancers, survivin is reported to be involved in the regulation of both apoptosis and cell division. Thus, HIF-1α overexpression (either constitutive or hypoxia-induced) may promote tumorigenesis by exerting double effects on key members of major gene families controlling cell death and proliferation; that is, the inhibition of cell death by up-regulating BCL-xL and survivin and promotion of cell proliferation by up-regulation of survivin. Elucidation of HIF-1α-dependent BCL-xL expression may provide a new dimension for understanding BCL-xL regulation.

Acknowledgments

We thank Dr. Xianming Mo of the Stem Cell Research Laboratory, Dr. Yidong Huang and Dr. Ying Huang in the Laboratory of Pathology, the State Key Laboratory of Biotherapy, for assistance.

Notes

*This work was supported by Natural Science Foundation of China Grants NSFC 30570692, 30871383, and 30800637, Ministry of Education PhD Program Fund 20060610053, and Postdoctoral Fund 20060401022 of China.

Footnotes

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.

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