Metformin regulated genes in human prostate cancer cells

The biguanide metformin has been shown to not only reduce circulating glucose levels but also suppress in vitro and in vivo growth of prostate cancer. However, the mechanisms underlying the anti-tumor effects of metformin in advanced prostate cancers are not fully understood. The goal of the present study was to define the signaling pathways regulated by metformin in androgen-receptor (AR) positive, castration-resistant prostate cancers. Our group used RNA sequencing (RNA-seq) to examine genes regulated by metformin within the C4-2 human prostate cancer cell line. Western blot analysis and quantitative RT-PCR were used to confirm alterations in gene expression and further explore regulation of protein expression by metformin. Data from the RNA-seq analysis revealed that metformin alters the expression of genes products involved in metabolic pathways, the spliceosome, RNA transport, and protein processing within the endoplasmic reticulum. Gene products involved in ErbB, insulin, mTOR, TGF-?, MAPK, and Wnt signaling pathways are also regulated by metformin. A subset of metformin-regulated gene products were genes known to be direct transcriptional targets of p53 or AR. Together, our results suggest metformin regulates multiple pathways linked to tumor growth and progression within advanced prostate cancer cells. Overall design: C4-2 cells were plated in 10 cm dishes at a density of ~ 750,000 million cells/dish in T medium (80% DMEM low glucose medium, 20% Hams' F12 medium, 5% heat inactivated FBS, 1% penicillin/streptomycin, 0.244 µg/ml d-biotin, 25 µg/ml adenine hemisulfate, 5 µg/ml insulin and 5 µg/ml apotransferrin) and allowed to attach for two days. The cells were then treated for 24 hours with either PBS vehicle or 5 mM metformin. Total RNA was extracted from treated cells using the Trizol reagent and further purified using the Qiagen RNeasy Mini kit. Each RNA sample (three replicates per treatment group) was validated for RNA integrity using an Agilent 2100 Bioanalyzer. RNA-sequencing (RNA-seq), including library preparation, was conducted at the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facility. A standardized TruSeq mRNA protocol was used for library preparation, followed by sequence runs on an Illumina HiSeq 2500 utilizing a 50-bp RNA-Seq protocol.