Abstract B19: LKB1-AMPK axis regulates the switch of Pyruvate Kinase M isoforms to tolerate nutritional stress

Abstract

Abstract Background: Cancer cells are known to acquire a unique metabolic strategy of aerobic glycolysis (Warburg effect), where they rely more on glycolysis rather than oxidative phosphorylation, regardless of ample amount of oxygen. Aerobic glycolysis is presumed as an inefficient way to generate energy, yet believed to sustain equilibrium between catabolism and anabolism to achieve limitless proliferative competence in cancer cells. Conversely, glucose deprivation is an inevitable characteristic feature of tumors due to their rate of proliferation and poor vasculature. Thus, how cancer cells with an often encountered microenvironment within the tumor of hypoglycemia co-ordinate their growth and proliferates with an abnormal bio-energetics remains ambiguous. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase, known to get activated by sensing bioenergetic stress and remodels the core of cellular metabolism to preserve energy homeostasis. Recent studies have appreciated the critical importance of Liver kinase B1 (LKB1) – AMPK pathway in tumorigenesis, where the axis of LKB1-AMPK has been shown to provide tolerance to nutrient deprived cancer, but how AMPK contributes to cancer cell metabolism is largely unknown, except for the established role in enzymes, like PFK2 (phospho fructokinase 2); and more work is needed to define the role of AMPK in regulation of other essential glycolytic enzymes: Hexokinase (HK), Pyruvate Kinase (M2 PK) and Lactate Dehydrogenase (LDH), associated with aerobic glycolysis. Methodology: Lung cancer (H1299 & A549) and breast cancer (MCF-7 & MDA MB 231) cell lines were used in this study. qPCR and Immunoblotting analysis were performed to measure the gene expression status. Oligomeric status and the distribution of PKM isoforms was assessed using density gradient ultracentrifugation followed by immunoblotting. PKM1 and PKM2 knockdowns were generated by lentiviral transduction. Metabolites levels were assayed using enzyme coupled assays. Cell proliferation was assessed using MTB assay; and apoptosis measured by developing immunoblot for Cleaved PARP and Cleaved Caspase 3. Results: We show how activation of AMPK in cultured cancer cells through relative glucose deprivation (25, 5 & 1 mM) increased the relative activity of PKM, but not of HK and LDH. Further, we demonstrate AMPK facilitates PK activity by enhancing their subunit association, which is the consequential outcome of PKM isoform switch from a catalytically less active M2 isoform to more active M1 isoform. Other than the response in glucose deprivation, the role of AMPK in the PKM isoform switch is confirmed by experimenting with AMPK agonist, inhibitor and constitutively active mutant of AMPK in cell culture. We also show the coordinated axis between LKB1 and AMPK in mediating alternative splicing of PKM isoforms, where transfection of LKB1 cDNA was carried out in cell lines devoid of LKB1. We for the first time report the indirect involvement of AMPK in regulation of alternative splicing of PKM isoforms, independent of c-Myc. Moreover, we show either impairment of AMPK activation through pharmacological agents or knockdown of PKM1 isoform in cancer cells cultured in glucose deprived medium (5mM glucose), diminishes the rate of glucose uptake, lactate production and cellular pyruvate and ATP level, as a result of attenuated cell proliferation and enhanced onset of apoptosis. Together our findings suggest that AMPK –PKM1 axis enhances the aerobic glycolysis and favors cancer cells in culture by conserving the energy homeostasis to endure nutritional stress.” Conclusion: The study highlights the integral role of LKB1-AMPK axis in dynamically regulating the preferential expression of either PKM2 or PKM1 in response to nutritional status in lung cancer and breast cancer cell line models, drawing an inference with regard to real life tumor microenvironment. Citation Format: Gopinath Prakasam, Rameshwar N.K. Bamezai. LKB1-AMPK axis regulates the switch of Pyruvate Kinase M isoforms to tolerate nutritional stress. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B19.