Steroid receptor coactivator-3 regulates glucose metabolism in bladder cancer cells through coactivation of hypoxia inducible factor 1α

Abstract

Cancer cell proliferation is a metabolically demanding process, requiring high glycolysis, which is known as "Warburg effect," to support anabolic growth. Steroid receptor coactivator-3 (SRC-3), a steroid receptor coactivator, is overexpressed and/or amplified in multiple cancer types, including non-steroid targeted cancers, such as urinary bladder cancer (UBC). However, whether SRC-3 regulates the metabolic reprogramming for cancer cell growth is unknown. Here, we reported that overexpression of SRC-3 accelerated UBC cell growth, accompanied by the increased expression of genes involved in glycolysis. Knockdown of SRC-3 reduced the UBC cell glycolytic rate under hypoxia, decreased tumor growth in nude mice, with reduction of proliferating cell nuclear antigen and lactate dehydrogenase expression levels. We further revealed that SRC-3 could interact with hypoxia inducible factor 1α (HIF1α), which is a key transcription factor required for glycolysis, and coactivate its transcriptional activity. SRC-3 was recruited to the promoters of HIF1α-target genes, such as glut1 and pgk1. The positive correlation of expression levels between SRC-3 and Glut1 proteins was demonstrated in human UBC patient samples. Inhibition of glycolysis through targeting HK2 or LDHA decelerated SRC-3 overexpression-induced cell growth. In summary, overexpression of SRC-3 promoted glycolysis in bladder cancer cells through HIF1α to facilitate tumorigenesis, which may be an intriguing drug target for bladder cancer therapy.

Keywords: Cancer; Cell Proliferation; Glycolysis; HIF1α; SRC-3; Transcription Coactivators; Tumor Metabolism; Urinary Bladder Cancer.

FIGURE 1.

Overexpressed SRC-3 promoted bladder cancer cell proliferation. A, Western blot analysis of SRC-3 protein expression in four bladder cancer cell lines and 11 pairs of human UBC samples. N, non-tumorous bladder tissue adjacent to UBC; T, tumor tissue. B, overexpression of SRC-3 in human UBC samples at the mRNA level. Compare the mRNA level of src-3 between normal (n = 14) and UBC samples (Tumor, n = 46) in the GEO profile dataset (GSE3167). Quantitative RT-PCR assay detected at the src-3 level in 9 human UBC samples. C, cell growth curve in SRC-3 overexpressed and control cells by the MTT assay over 4 days. D, Western blot analysis of SRC-3, Cyclin D1, and PCNA protein levels in SRC-3 overexpressed and control cells. E, colony formation capacity in soft agar of SRC-3 overexpressed and control cells. CTRL, BIU87 cells transfected empty vector; #1 and #2, two different clones from BIU87 cells stably transfected with the SRC-3 expression plasmid. F–K, SRC-3 deficiency in human UBC T24 (F-H) and 5637 (I–K) cells impairs cell proliferation and colony formation capacity. F and I, cell growth curve in SRC-3 knockdown (shSRC-3) and control (shCTRL) cells by MTT assay. G and J, Western blot analysis of SRC-3, Cyclin D1, and PCNA protein levels in SRC-3 knockdown and control cells. H and K, colony formation capacity in soft agar of the SRC-3 knockdown and control cells. **, p < 0.01; ***, p < 0.001.

FIGURE 2.

Ectopic expression of SRC-3 enhanced bladder cancer cell aerobic glycolysis. A, glucose consumption and lactate production in SRC-3 overexpressed and control cells under normoxia and hypoxia conditions. The mRNA and protein expression levels of glycolytic genes in the SRC-3 overexpressed and control cells were detected by quantitative RT-PCR assay (B) and Western blot assay (C). CTRL, BIU87 cells transfected with empty vector; #1 and #2, two stable transfectants of SRC-3. *, p < 0.05; **, p < 0.001.

FIGURE 3.

Knockdown of SRC-3 decreased glycolysis in bladder cancer cell under hypoxia. Glucose consumption, lactate production, medium pH value, and ATP production in SRC-3 knockdown and control cells under normoxia and hypoxia were detected in both T24 (A) and 5637 (B) cells. The mRNA and protein expression levels of glycolytic genes in SRC-3 knockdown and shCTRL T24 cells were detected by quantitative RT-PCR assay (B) and Western blot assay (C). shSRC-3 and shCTRL, SRC-3-targeting shRNA and control shRNA stably transduced cells. *, p < 0.05.; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Knockdown of SRC-3 abrogated tumorigenicity of T24 bladder cancer cell in nude mice. A, in vivo tumor growth curve of SRC-3 knockdown and control xenografts over 7 weeks. Tumor size was measured by caliper and tumor volume was calculated by the formula: volume = (L × W²)/2. B, the average tumor weight of the SRC-3 knockdown (shSRC-3, n = 8) and control (shCTRL, n = 6) xenografts at the end point. C, Western blotting assay of SRC-3, PCNA, and LDHA protein expression levels in SRC-3 knockdown and control xenografts. Data, mean ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

SRC-3 coactivated HIF1α in vitro. A, ectopic SRC-3 protein interacted with HIF1α in T24 cells. T24 cells were transiently transfected with FLAG-tagged SRC-3. Co-immunoprecipitation (IP) assay was carried out in cells treated under either normoxia or hypoxia conditions. The precipitant by the IgG antibody was used as a negative control. B, schematic representation of five fragments of SRC-3 representing different domains. C, five fragments of SRC-3 were generated in E. coli BL21(DE3) as GST fusion proteins. The purified GST fused proteins were incubated with HEK293T lysate, which contained the ectopic expression of HA-HIF1α. Western blot (WB) was performed to detect the pulled down HIF1α by HA antibody. D–H, luciferase activity assay: D, in the HRE-Luc-transfected 293T cells co-transfected with SRC-3 expression plasmid, with or without DFO treatment, ***, p < 0.001, compared empty vector transfection group; ###, p < 0.001 compared with the SRC-3 transfection group; E, in HRE-Luc 293T cells with co-transfection of SRC-3 and HIF1α expression plasmids with the indicated amount of plasmids, ***, p < 0.001; F, in the PGK1-reporter 293T cells co-transfected with the SRC-3 expression plasmid, with or without DFO treatment, ***, p < 0.001, compared with the empty vector transfection group; ###, p < 0.001, compared with the SRC-3 transfection group; G, in the PGK1-reporter 293T cells with co-transfection of SRC-3 and HIF1α expression plasmids. PGK1-Luc, wild type PGK1 reporter; ΔPGK1-Luc, PGK1 reporter without HRE; followed by transfection with or without HIF1α. H, in HRE-Luc T24 cells transduced with control (−) or SRC-3 shRNA (+), with or without HIF1α overexpression, ***, p < 0.001, compared with the empty vector transfection group; ###, p < 0.001, compared with the group with SRC-3 knockdown and HIF1α overexpression. I, presence of HIF1α and SRC-3 on the endogenous glut1 promoter and pgk1 promoter from the pgk1-luc reporter detected by the ChIP assay. Parental T24 cells (left panel) and T24 wells transfected with the pgk1-luc reporter plasmid (right) were incubated under normoxia (N) or hypoxia (H), respectively. The band intensity was shown under each blot, which was quantified by ImageJ software. The precipitant by IgG antibody was used as a negative control. RLU, relative light units.

FIGURE 6.

Knockdown of glycolytic genes or HIF1α reduced SRC-3 overexpression accelerated bladder cancer cell proliferation. A, efficiency of RNA interference targeting HK-2 (siHK-2) and LDHA (siLDHA) in SRC-3 overexpressed (#1) and control cells determined by Western blot analysis. B–E, cell proliferation in SRC-3 overexpressed and control cells with the treatment of siHK-2 (B), siLDHA (C), 2-DG (12.5 mg/ml) (D), and sodium oxamate (80 mm) (E). F, efficiency of RNA interference-targeting HIF1α (siHIF1α) in SRC-3 overexpressed (#1) and control cells determined by Western blot analysis. G, cell proliferation in SRC-3 overexpressed (#1) and control (CTRL) cells with treatment of siHIF1α were detected by MTT assay. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

Correlation between expression levels of SRC, HIF1α target genes, and proliferative marker gene, pcna, in clinical human UBC samples. A, two representative UBC specimens for immunohistochemistry staining of SRC-3 and Glut1 in adjacent sections. Case 1, SRC-3 negative and Glut1 negative; case 2, positive nuclear staining of SRC-3 and positive membrane staining of Glut1. Bar, 100 μm. B, correlation between expression levels of SRC-3 and Glut1 in a cohort of human UBC specimens (n = 20, Spearman rank correlation coefficient). C and D, linear regression of src-3, HIF1α target genes (pgk1 and vegfa), and proliferative marker gene (pcna), using UBC samples from the GEO profiles database (GSE3167, n = 46).