FROM ROS TO PGC-1 · Roadmap to Reactive Oxygen Species (ROS) homeostasis: analysis of ROS mediated signal transduction through PGC-1alpha coactivation and mitochondrial biogenesis

Final Activity Report Summary - FROM ROS TO PGC-1 (Roadmap to Reactive Oxygen Species (ROS) homeostasis: analysis of ROS mediated signal transduction through PGC-1alpha coactivation ...)

Reactive Oxygen Species (ROS) were traditionally considered to be unavoidable by-products of aerobic respiration. It has been gradually appreciated that certain ROS derivatives, such as hydrogen-peroxide (H2O2), can act as a second messenger under selective physiological conditions. However, H2O2 mediated signalling pathways and mechanisms are poorly understood. Recently, we identified PGC-1a (PPAR? coactivator 1a) as a key regulator of anti-oxidative defence program and cellular ROS homeostasis. PGC-1a knock-out cells and mice have reduced expression of ROS detoxifying enzymes and are more sensitive to oxidative stress and MPTP induced neurodegeneration, respectively. PGC-1a is induced upon H2O2 stimulation and its induction is necessary for the anti-oxidative defence program (St-Pierre, J., Drori, S., et al. Cell 2006). Induction of anti-oxidative defence program is a critical response to cellular ROS elevation. Therefore, the inner connection between cellular H2O2 levels and PGC-1a activation is important for further understanding H2O2 mediated signalling. Toward this aim, a real-time cellular system was developed. a. PGC-1a homologous recombinant promoter fused to GFP, as an indicator of PGC-1a promoter activity. b. PGC-1a homologous recombinant promoter fused to GFP-PGC-1a, as an indicator of PGC-1a protein level. Both fibroblast (10T1/2) and cancer (RKO) cells were transfected with PGC-1a homologous recombinant promoter fused to either PGC-1a-GFP (indication of protein levels) or GFP alone (transcript levels). Positive transfected cells were subjected to clonal dilutions to identify several different expressing clones. Several cancer cell lines (RKO, Hela, U2OS) were transfected with PGC-1a homologous recombinant promoter fused to either PGC-1a-GFP (indication of protein levels) or GFP alone (transcript levels). Positive transfected cells were subjected to clonal dilutions to identify several different expressing clones. These selected clones were tested for best responsive clones. Surprisingly, Stable expression of the fluorescent-PGC-1alpha driven by 2kb homologous promoter resulted in dramatic downregulation of both endogenous PGC-1alpha and ectopic PGC-1alpha expression. This effect was not observed in other non-cancer cell line such as CHO cells. Repression of PGC-1alpha was associated with downregulation of PGC-1alpha target genes (e.g. mitochondria cytC, ATP5B, COXII and ROS detoxifying genes GPXI, SODII, catalase). More importantly, PGC-1alpha inhibition in cancer cells resulted in higher glycolytic activity and dramatic elevation of lactic acid secretion. Cellular metabolic shift that is associated with more glycolytic activity in cancer cells is known as the Warburg effect and is usually associated with more aggressive cancer development. Indeed, there are several reports that suggested a correlation between low PGC-1alpha expression levels and cancer development in breast and colon cancer. Thus, PGC-1alpha presents an altered pattern of regulation in cancer cells. This novel regulatory very likely related to a broader metabolic shift in these cells. Importantly, our data suggests that PGC-1alpha might take a positive role in cancer cells pro-apoptotic program. On the other hand, in non-malignant cell PGC-1alpha induction protects against cell death. This data propose PGC-1alpha as a selective target for inducing cancer cell death without affecting non-malignant cell. Taken together, further understanding of the altered regulatory mechanisms underlying PGC-1alpha expression in cancer cells has taken a promising twist. These novel data might be another step in the long way to providing anti-cancer treatment that would not be toxic to non-malignant cells.

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