Characterising cell intrinsic and whole body metabolic effects of metformin in prostate cancer

Background

Metformin inhibits PCa growth; however, its mechanism of action has been controversial. Cancer cells display altered metabolism which fuels their proliferation. The resulting metabolites could play a role in oncogenic signalling or serve as potential biomarkers for advanced PCa. We therefore aimed to: 1) identify the metabolic and signalling pathways involved in the anti-tumour effect of metformin in LNCaP cells and plasma samples from PCa patients treated with metformin; 2) predict prognostic outcome by identifying metabolic biomarkers in plasma samples.

Methods

Cell viability was determined using cell counting kit 8 (CCK8) assay on androgen- dependent  LNCaP cells. Western blot was used to examine signalling pathways regulated by metformin. Untargeted metabolomics profiling of plasma samples taken from metformin treated PCa patients was performed using liquid chromatography mass spectrometry (LC-MS) to examine whole body metabolic effects. The metabolic pathways affected by metformin treatment were identified using MetaboAnalyst 3.0. One-way or two-way ANOVA was performed for the statistical analyses of in vitro data, while a paired t -test was used for statistical analysis of plasma metabolites levels.

Results

Either metformin or the anti-androgen enzalutamide alone inhibited LNCaP cell growth in a dose and time dependent manner, while a combination of both resulted in the most significant inhibition in cell growth. Metformin stimulated AMP activated protein kinase (AMPK), and downregulated insulin-like growth factor 2 (IGF2) expression, resulting in decreased androgen receptor (AR) signalling and Akt activation. The combination of metformin and enzalutamide produced the most significant activation of AMPK and reduction in IGF2 expression, AR signalling and Akt activation. Metformin treatment in PCa patients was associated with significant upregulation of leucine (p = 0.0436), isoleucine (p = 0.0282), hydroxybutyrylcarnitine (p = 0.0121) and downregulation of citrulline (p = 0.0164). The two most significantly affected pathways by metformin in vivo that were identified were aminoacyl-tRNA synthesis biosynthesis and arginine biosynthesis.

Conclusion

We discovered a novel mechanism for the PCa growth inhibitory effects of metformin via the reduction of IGF2 expression and consequently the reduction in PI3K/Akt/mTORC1 signalling and AR signalling. Metformin treatment in PCa patients was associated with significant up or down regulation of various plasma metabolites which could be markers of metformin inhibition of tumour growth or response to metformin.

Affiliations

Wang Nan Fang¹, Dr Kevin Wang¹, Dr Kate Guan¹, A/Professor Jeff Holst^1,2 

1. Translational Cancer Metabolism Laboratory, School of Medical Sciences

2. Prince of Wales Clinical School, UNSW Sydney, Sydney, NSW, Australia