Insulin Increases Migration and Invasion in Androgen Deprived Prostate Cancer Cells.

Advanced prostate cancer (PCa) is treated with androgen deprivation therapy (ADT), as prostate tumours are dependent on androgens for survival. Unfortunately, ADT offers a temporary remission and prostate tumours begin to capitalise on additional intratumoural or adrenal sources of androgens. This has led to continued development of antagonists to the androgen receptor (AR) and inhibitors of androgen synthesis to combat PCa which result in extended time to progression and cancer specific survival in advanced PCa patients. A major side effect of ADT is the induction of features of the metabolic syndrome, including persistent hyperinsulinaemia, which is specifically associated with poor outcomes, including rapid progression and increased cancer mortality. Using patient databases (Oncomine™, Compendia Bioscience, Ann Arbor, MI) we observed that insulin receptor and IRS-2 are upregulated from primary versus metastatic primary tumours across eight clinical datasets. We hypothesized that following androgen withdrawal, high insulin levels promote an adaptive response that drives castrate resistance PCa progression. To address this we undertook transcriptional profiling of AR-responsive LNCaP cell line following insulin treatment with or without androgens. We identified a number of pathways uniquely up-regulated by insulin including genes associated with increased potential for migration and invasion. We observed that in the absence of androgens, insulin promoted up to 3.4 fold increased migration through transwell and wound healing assays in LNCaP, DU145 and 22RV1 cells; and increased Matrigel invasion of LNCaP (2.5 fold, p=0.0106), DU145 (1.9 fold, p=0.007) and 22RV1 (2.4 fold, p=0.004) cells. AR inhibitor, bicalutamide, increased cell migration of LNCaP and 22RV1 by 72% and 45%, respectively. Insulin and bicalutamide do not appear to be additive suggesting a common pathway. Importantly, insulin- induced migration was blocked by inhibitors of insulin receptor, BMS-745807-04 (48%) and CP-751871 (42%), and by the MAPK inhibitor U0126 (46%). In agreement with our microarray data, QRT-PCR revealed insulin-induced molecular changes within the cells reminiscent of an epithelial-to-mesenchymal transition (EMT); androgen deprivation alone increased expression of vimentin and the SNAIL and ZEB1 transcription factors, which was further increased with insulin. Changes to protein expression and transcription factor localisation were consistent with mRNA. Recent studies report ‘obesity factors’ promoting EMT in prostate cancer cells. Our data indicates insulin can promote this plasticity. These novel results show insulin promotes PCa cell migration and invasion, possibly by inducing EMT-like changes within PCa cells and make a strong case for investigating the clinical efficacy of using insulin-sensitizing drugs such as metformin as an adjuvant therapy for PCa treatment.