PRKAR2B-HIF-1α loop promotes aerobic glycolysis and tumour growth in prostate cancer

Abstract

Objectives: Reprogramming of cellular metabolism is profoundly implicated in tumorigenesis and can be exploited to cancer treatment. Cancer cells are known for their propensity to use glucose-dependent glycolytic pathway instead of mitochondrial oxidative phosphorylation for energy generation even in the presence of oxygen, a phenomenon known as Warburg effect. The type II beta regulatory subunit of protein kinase A (PKA), PRKAR2B, is highly expressed in castration-resistant prostate cancer (CRPC) and contributes to tumour growth and metastasis. However, whether PRKAR2B regulates glucose metabolism in prostate cancer remains largely unknown.

Materials and methods: Loss-of-function and gain-of-function studies were used to investigate the regulatory role of PRKAR2B in aerobic glycolysis. Real-time qPCR, Western blotting, luciferase reporter assay and chromatin immunoprecipitation were employed to determine the underlying mechanisms.

Results: PRKAR2B was sufficient to enhance the Warburg effect as demonstrated by glucose consumption, lactate production and extracellular acidification rate. Mechanistically, loss-of-function and gain-of-function studies showed that PRKAR2B was critically involved in the tumour growth of prostate cancer. PRKAR2B was able to increase the expression level of hypoxia-inducible factor 1α (HIF-1α), which is a key mediator of the Warburg effect. Moreover, we uncovered that HIF-1α is a key transcription factor responsible for inducing PRKAR2B expression in prostate cancer. Importantly, inhibition of glycolysis by the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) or replacement of glucose in the culture medium with galactose (which has a much lower rate than glucose entry into glycolysis) largely compromised PRKAR2B-mediated tumour-promoting effect. Similar phenomenon was noticed by genetic silencing of HIF-1α.

Conclusions: Our study identified that PRKAR2B-HIF-1α loop enhances the Warburg effect to enable growth advantage in prostate cancer.

Keywords: HIF1A; PRKAR2; RII-BETA; aerobic glycolysis; glycolytic ability.

FIGURE 1

PRKAR2B knockdown inhibits prostate cancer cell glycolysis. A, Western blotting analysis of the knockdown efficiency of PRKAR2B in DU145 and PC3 cells. B‐E, The effect of PRKAR2B knockdown on the glucose utilization (B), lactate production (C), extracellular acidification rate (D), oxygen consumption rate (E), and expression of glycolytic components (F) in DU145 and PC3 cells. *P < .05; **P < .01

FIGURE 2

PRKAR2B overexpression promotes the Warburg effect in prostate cancer in vitro. A, Western blotting analysis of the overexpression efficiency of PRKAR2B in LNCaP cells. B‐E, The effect of PRKAR2B overexpression on the glucose utilization (B), lactate production (C), extracellular acidification rate (D), oxygen consumption rate (E), and expression of glycolytic components (F) in LNCaP cells. SLC2A1, solute carrier family 2 member 1; PFKP, phosphofructokinase platelet; PKM, pyruvate kinase muscle isozyme; LDHA, lactate dehydrogenase A. *P < .05; **P < .01

FIGURE 3

Androgen‐independent LNCaP cells exhibits enhanced glycolysis. A, The basal glucose utilization in LNCaP and LNCaP‐AI cells. B, The basal lactate production in LNCaP and LNCaP‐AI cells. C, Comparison of ECAR and OCR status in LNCaP and LNCaP‐AI cells. D, Real‐time qPCR analysis of glucose transporter and key glycolytic genes in LNCaP and LNCaP‐AI cells. E, Western blotting analysis of the effect of PRKAR2B knockdown efficiency in LNCaP‐AI cells. (F‐I) The effect of PRKAR2B knockdown on the glucose utilization (F), lactate production (G), ECAR/OCR (H), and expression of glycolytic components (I) in LNCaP‐AI cells. (J) Correlation analysis of the link between PRKAR2B expression and the expression level of glucose transporter and key glycolytic genes in prostate cancer tissues (n = 492); data were obtained from the TCGA cohort. *P < .05; **P < .01

FIGURE 4

PRKAR2B regulates HIF1α expression in prostate cancer. (A‐B) Correlation analysis of the link between PRKAR2B and HIF1A expression in prostate cancer tissues; data were obtained from the TCGA cohort (A) and Ren Ji cohort (B). (C) Western blotting analysis of the effect of PRKAR2B knockdown on the HIF1A protein level in DU145 cells. (D) Western blotting analysis of the effect of PRKAR2B overexpression on the HIF1A protein level in LNCaP cells. (E) Western blotting analysis of HIF1A protein level in LNCaP and LNCaP‐AI cells. (F) Western blotting analysis of the effect of PRKAR2B knockdown on the HIF1A protein level in LNCaP‐AI cells. (G) Effects of PRKAR2B knockdown or overexpression on the HIF‐1α activity. (H) Western blotting analysis of HIF1A protein level in DU145 and PC3 cells upon H89 (50 μmol/L) treatment for 24 h. (I) Determining the effect of H89 (50 μmol/L) treatment on the HIF‐1α activity in DU145 and PC3 cells. *P < .05; **P < .01

FIGURE 5

Transcriptional regulation of PRKAR2B by HIF‐1α in prostate cancer. A, Real‐time qPCR analysis of PRKAR2B expression in DU145, PC3, and LNCaP cells under hypoxia (1% O₂) and normoxia (20% O₂) condition. B, Real‐time qPCR analysis of PRKAR2B expression in DU145, PC3, and LNCaP cells after 100 μmol/L CoCl₂ treatment for 24 h. C, Effect of HIF‐1α knockdown on the PRKAR2B expression in DU145 and PC3 cells. D, Luciferase activity of PRKAR2B gene promoter reporters in DU145 cells transfected with HIF‐1α or empty vector. Red sites represents the putative HIF‐1α‐binding sites; Mut, mutant; WT, wild‐type. E, PRKAR2B ChIP‐PCR for control input and H3K4me3 ChIP. F, The PRKAR2B gene promoter activity in DU145 and PC3 cells under hypoxia (1% O₂) and normoxia (20% O₂) condition. G, The PRKAR2B gene promoter activity in LNCaP and LNCaP‐AI cells. H, PRKAR2B ChIP‐PCR for control input and HIF‐1α ChIP. *P < .05; **P < .01; ***P < .001

FIGURE 6

The growth‐promoting role of PRKAR2B in prostate cancer is glycolysis‐dependent. A, In vivo growth assay of that sh‐Ctrl and sh‐PRKAR2B DU145 cells. B, In vivo growth assay of that ov‐vector and PRKAR2B‐overexressing LNCaP cells. C, Plate clone formation assay showed the effect of PRKAR2B on LNCaP cell proliferation in the presence or absence of 5 mmol/L 2‐DG. D, Plate clone formation assay showed the effect of PRKAR2B on LNCaP cell proliferation in the culture medium containing 25 mmol/L glucose or 25 mmol/L galactose. E, Measurement of the effect of PRKAR2B overexpression on LNCaP cell proliferation in the presence or absence of HIF‐1α knockdown. F, Real‐time qPCR analysis of PRKAR2B expression in DU145 and PC3 cells after 2‐DG treatment. G, Real‐time qPCR analysis of PRKAR2B expression in DU145 and PC3 cells after galactose treatment. *P < .05; **P < .01