SENP1 Interacts with HIF1α to Regulate Glycolysis of Prostatic Carcinoma Cells

Abstract

Background: Hypoxic microenvironment inside the tumor forces tumor cells to up-regulate the glycolytic pathway to maintain a sufficient energy supply for tumor growth. Activation of HIF1α under hypoxia condition is able to regulate the expression of glycolysis-related genes, and results in the proliferation and metastasis of cancer cells. However, the mechanism underlying HIF1α activation and glycolysis induction by hypoxia remains unclear. The present study is aimed to test if SENP1 regulates the glycolysis of prostate cancer cells (CaP) by improving stability of HIF1α protein. Methods: We employed qPCR and western blotting assay to analyze expression of HIF1α and SENP1. Glucose uptake assay, lactate production assay, LDH release assay and ATP production assay were utilized to evaluate cell glycolysis. The interaction between SENP1 and HIF1α was verified by co-immunoprecipitation assay. Results: We found that hypoxia condition improves glucose uptake and lactate production to sustain sufficient ATP for cellular activity in prostatic carcinoma cells. The expression of SENP1 mRNA was significantly increased in human prostatic carcinoma cell lines after exposure to hypoxia, accompanied by the up-regulation of HIF1α. Furthermore, forced expression of SENP1 was shown to regulate the glycolysis in prostatic carcinoma cells by stabilizing HIF1α. The up-regulation of SENP1 promotes tumor cell proliferation and tumorgenesis by interacting with HIF1α which was deSUMOylated and sequentially leading to a "Warburg effect". Conclusion: SENP1 interacts with HIF1α to regulate glycolysis and proliferation of prostatic carcinoma cells under hypoxia condition, which provides new insights into prostatic carcinoma therapy.

Keywords: HIF1α; SENP1; glycolysis; prostatic carcinoma; tumorgenesis..

Figure 1

Hypoxia conditions promote prostatic carcinoma cell glycolysis. (A) Morphology changes of human prostatic epithelial cell and prostatic carcinoma cell lines subjected to the treatments of different conditions. (B-E) Extreme conditions can improve glucose uptake (B), lactate production (C), LDH release (D) and ATP production (E) of prostatic carcinoma cells. * P<0.05 vs. RWPE-1 cells, ** P<0.01 vs. RWPE-1 cells or normoxia group cells.

Figure 2

Hypoxia induces up-regulation of HIF1α and SENP1 in human prostatic carcinoma. (A) HIF1α mRNA expression was increased in human prostatic carcinoma cells lines after treatment with hypoxia, anoxia and nutritional deficiency conditions. Especially, after treatment with hypoxia and anoxia, the expression of HIF1α mRNA was increased significantly in all the human prostatic carcinoma cells lines. (B) SENP1 mRNA expression was elevated in all the four extreme conditions of human prostatic carcinoma cells lines. Exposure to hypoxia and anoxia significantly increased the expression of SENP1 mRNA in all the human prostatic carcinoma cells lines. (C) The protein abundance of HIF1α and SENP1 in human prostatic carcinoma cells lines and human prostatic epithelial cell line RWPE-1 cells. * P<0.05 vs. normoxia group cells or RWPE-1 cells, ** P<0.01 vs. normoxia group cells or RWPE-1 cells.

Figure 3

SENP1 regulates cell glycolysis through stabilizing HIF1α. The SENP1 and HIF1α expression at mRNA (A, B) and protein (C) levels were measured by qRT-PCR and Western Blot assay in PC3 cells transfected with SENP1, NC, mock1, si-SENP1, si-NC and mock2 plasmids. (D) The interactions between SENP1 and HIF1α were analyzed by Co-IP. ** P<0.01 vs. NC PC3 cells, ^## P<0.01 vs. si-NC PC3 cells.

Figure 4

SENP1 promotes prostatic carcinoma growth in vivo. (A) Tumor growth curve was measured from day 4 to day 22. (B) The pictures of tumor tissues of each group xenograft model mice were taken out at day 22. (C) Hematoxylin and eosin (H&E) staining of histological sections of tumor tissues in each group (magnification 100×). ** P<0.01 vs. NC, ^## P<0.01 vs. si-NC.

Figure 5

SENP1 deSUMOylates HIF1α to regulate Warburg effect. HIF1α protein was deSUMOylated by SENP1 and subsequently combined with MCT4, which finally promotes a "Warburg effect". (A) HIF1α, SENP1 and SUMO1 proteins expressions were determined by Western Blot technique in PC3 cells treated with hypoxia or hypoxia+si-SENP1. (B) The interaction between HIF1α protein and SUMO1 protein was examined by Co-IP assay in PC3 cells after treatment with hypoxia or hypoxia+si-SENP1. (C) SENP1 activated HIF1α signaling pathway by regulating HIF1α in PC3 cells after treatment with hypoxia, SENP1, and hypoxia+SENP1 respectively. (D) The interaction between HIF1α protein and MCT4 protein was detected by Co-IP assay in PC3 cells after treatment with hypoxia. ** P<0.01 vs. Control, ^## P<0.01 vs. Hypoxia.