Work

Metformin targets Clusterin to control lipogenesis and inhibit the growth of bladder cancer cells through SREBP-1c/FASN axis

Year: 2021

Type: article

Abstract: Metformin, a widely prescribed drug for treating type II diabetes, has shown an important anti-cancer property. 1 However, new launched clinical trials did not achieve such desirable results. 2 One explanation could be that metformin’s anti-cancer mechanisms and precise therapeutic targets remain unclear. To identify potential therapeutic targets of metformin in bladder cancer, we screened differential proteins with metformin treatment in T24 cells by high-throughput protein chip. As shown in Fig. 1a , Clusterin was screened as a target of metformin in bladder cancer. The direct binding of metformin with Clusterin was further confirmed by Surface Plasmon Resonance (SPR) analysis (Fig. 1b ). Meanwhile, results from Western blot and RT-PCR showed that treatment with metformin resulted in significant Clusterin downregulation in UMUC3 and J82 cells (Supplementary Fig. S1a, b ). Clusterin is a conserved glycoprotein which plays key roles in cellular stress response and survival. 3 As evidenced by its key roles in cancer, Clusterin served as a therapeutic target for fighting tumor growth. Fig. 1 a High-throughput protein chip was used to screen differential proteins with metformin treatment in T24 cells for 48 h. Metf 0 metformin 0 mM, Metf 16 metformin 16 mM. b SPRi binding signal curve of metformin to Clusterin protein. c Western blot analysis of Clusterin protein expression in three human bladder cancer cells and one normal bladder cell ( n = 3). d RT-PCR analysis of Clusterin mRNA expression in three human bladder cancer cells and one normal bladder cell ( n = 3). e MTT was used to assess the cell proliferation after Clusterin knockdown and the effect of metformin on inhibiting proliferation in Clusterin-knockdown cells compared with control cells ( n = 3). f Lipid droplet were visualized using BODIPY 493/503 after metformin treated in UMUC3 and J82 cells ( n = 3). g UMUC3 and J82 cells were treated with metformin for 36 h. Western blot was used to examine the protein expression of SREBP-1c precursor (pSREBP-1c), mature forms of SREBP-1c (nSREBP-1c) and FASN. RT-PCR was used to examine the mRNA levels of SREBP-1c and FASN after treatment with metformin for 6 h ( n = 3). h UMUC3 and J82 cells were treated with metformin for 36 h. GC-MS analyzed the long-chain and short-chain fatty acids levels ( n = 3). i GC-MS determined the long-chain and short-chain fatty acids levels in Clusterin knockdown UMUC3 cells ( n = 3). j Lipid droplet were visualized using BODIPY 493/503 in Clusterin knockdown (shCLU) cells and control cells (shCtrl) ( n = 3). k Protein and mRNA expressions of Clusterin, SREBP-1c, and FASN were detected by Western blot, RT-PCR respectively in UMUC3 cells after siRNA silencing for 36 h. siNT = empty vector; siCLU = Clusterin silence ( n = 3). l The shCtrl and shCLU UMUC3 cells were injected subcutaneously into the left and right flanks of the same nude mice, respectively. Tumor growth was monitored weekly using the IVIS Spectrum imaging system ( n = 5). m Images before and after treatment with metformin or PBS ( n = 5, each group). Tumor growth was monitored weekly using the IVIS Spectrum imaging system ( n = 5). n Tissue microarray analyzed Clusterin expression in total 66 tissue samples (10 normal tissues and 56 cancer tissues). N normal bladder, NMIBC non-muscular invasive bladder cancer, MIBC muscle-invasive bladder cancer, T bladder tumor. o Relative expression of Clusterin mRNA in bladder tumor tissues obtained from TCGA database. p A diagram summarizes that metformin inhibits Clusterin-mediated fatty acid synthesis pathway. ER endoplasmic reticulum. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001 Full size image

Source: Signal Transduction and Targeted Therapy

DOI: 10.1038/s41392-021-00493-8