Protein Phosphatase 2A Activation Dependent Down Regulation of PGC1-Alpha and FOXO1 Phosphorylation with OSU-2S in Human Lymphoma Cells

Metabolic reprogramming has been recognized to provide survival advantage in cancer cells. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is involved in regulation of multiple cellular pathways including metabolic events. OSU-2S, is a novel PP2A activator that exhibited potent anti-cancerous properties against human and canine lymphoma cell lines and primary human and canine lymphoma patient samples. It has been shown to activate PP2A in Ramos human lymphoma cell line leading to cytotoxicity that is prevented by inhibition of PP2A with okadaic acid (OA). Peroxisome proliferative activated receptor-gamma co-activator 1 (PPARGC1, also known as PGC1a), is a transcriptional co-activator that serves as a positive regulator of mitochondrial biogenesis and respiration, gluconeogenesis as well as many other metabolic processes such as lipid and energy metabolism. FOXO1 is a transcription factor that directly binds to the promoters of PGC1a and gluconeogenic genes involved in activation of gluconeogenesis. Activated PP2A has been shown to directly interact with FOXO1 and dephosphorylate it, leading to its delayed nuclear translocation. Given the role of PP2A in dephosphorylation of pFOX01, a regulator of PGC1a gene transcription, we hypothesized that PP2A activator OSU-2S, will down regulate PGC-1a expression through PP2A dependent FOXO1 regulation. Consistent with this hypothesis OSU-2S treatment inhibited PGC1a mRNA and protein expression in Jeko, OCI-ly18 and OCI-ly19 and raji lymphoma cell lines 24 hours post treatment. OSU-2S mediated downregulation of PGC1a and mitochondria biogenesis genes (NRF1, ERR alpha and TFAM) are dependent on PP2A activation as concentrations of OA that inhibited PP2A activation abrogated OSU-2S which induced up regulation of PGC1a and mitochondria biogenesis genes . To determine if the OSU-2S mediated inhibition of PGC1a expression is associated with its PP2A dependent modulation of phosphoFOXO1(pFOXO1), we tested the effect of OSU-2S on pFOXO1. Treatment of lymphoma cells with OSU-2S induced 60-70% decrease in pFOXO1 compared to vehicle control P =0.0001)], that is correlated with the decrease in PGC1a protein expression. Importantly OA mediated inhibition of PP2A, prevented OSU-2S-induced FOXO1 dephosphorylation. These studies suggest a role of OSU-2S induced modulation of metabolic regulator PGC1a via PP2A dependent dephosphorylation of FOXO1. Importantly, OSU-2S-induced PGC1a reduction resulted in decreased mitochondrial biogenesis as evidenced by ~43 % decrease in mitochondrial mass and ATP generation that led to reduced energy production as determined by Nonyl Acridine Orange dye staining followed by flow cytometry analysis. Interestingly, OSU-2S decreased expression of genes involved in mitochondrial biogenesis including NRF1a, ERR1a and TFAM by 75, 65 and 60% respectively P<0.0001. Ongoing mechanistic studies are aimed to define the molecular basis of OSU-2S induced transcriptional regulation of PGC1a and other genes involved in mitochondrial biogenesis in human lymphoma cell lines and primary cells.

(This work was supported by NIH-R01 CA197844-02. SMK is a recipient of Egyptian Cultural and Educational Bureau (ECEB) Award). SG is a recipient of Pelotonia Graduate Fellowship)

Keywords: PGC1- alpha, OSU-2S, PP2A, FOXO1, metabolism, lymphoma