Clinical significance, functional characterization and therapeutic implication of PFKFB3 and PDK4 in the regulation of cancer stem cell-like properties in ovarian cancer

Abstract

Ovarian cancer is a leading cause of mortality among gynecological malignancies worldwide. Late presentation with frequent metastasis develops and drug resistance development contribute to its poor prognosis. Cancer stem-like cells (CSCs) is a small subpopulation of tumor cells with stem-like properties and is responsible for tumor growth, metastasis, chemoresistance and recurrence. Thus, targeting CSCs is considered as an effective way to treat cancer. Increased aerobic glycolysis is commonly found in cancers. The inducible 6-phosphofructo- 2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is the main provider of fructose-2,6-bisphosphate which control the first committed step in glycolysis. Pyruvate dehydrogenase kinase 4 (PDK4) is a gate-keeping enzyme which shift oxidation in the mitochondria to glycolysis. PFKFB3 and PDK4 in ovarian cancer are still not explored. Accumulating evidence show that CSCs in many cancers bear an altered metabolic phenotype. However, such metabolic switch in ovarian CSCs remains unclear. This study attempted to investigate the functional roles, in particularly CSC properties and metabolic switch, clinical significance, and the underlying mechanisms of PFKFB3 and PDK4 in ovarian CSCs. Unlike other tumors, ovarian cancer prefers to metastasize through ascites. We found that tumor cells derived from ascites showed enhanced sphere-forming and metastasis capacities, commitment with ALDH and CD44 subset enrichment, and induced PFKFB3 and PDK4 expression when compared to primary tumors, suggesting ascites are enriched with cells bear CSC characteristics and altered cell metabolism. Clinically, up-regulation of both PFKFB3 and PDK4 were found to be correlated with advanced tumor stages, metastasis and poor prognosis. We then isolated CSC subpopulation using ovarian cancer cells/cell lines derived from ascites by two methods: sphere-forming culture and ALDH+CD44+ fluorescence-activated cell sorting. Similar to mammospheres, we noticed increased CSC-related genes expression in ALDH+CD44+ subpopulation. Moreover, ALDH+CD44+ subpopulation showed enhanced tumor initiation, selfrenewal capacity, migration/invasion abilities, clonogenicity and resistance to chemotherapy in vitro and in vivo compared with ALDH-CD44- subpopulation. Notably, mammospheres or ALDH+CD44+ subpopulation demonstrated upregulated PFKFB3 and PDK4 expression, increased lactate production/extracellular acidification, and decreased oxygen consumption rate. All these findings suggested ovarian CSCs derived from ascites preferentially rely on glycolysis for energy demand. Knockdown or overexpression of PFKFB3 and PDK4, and treatment of their inhibitors (PFK158, 3PO, and DCA) on isolated CSC subpopulation followed by subsequent in vitro and in vivo functional assays showed that PFKFB3 and PDK4 play critical roles in shifting metabolism from oxidative phosphorylation to glycolysis and regulating cancer and stemness properties including tumor initiation, migration/invasion abilities, clonogenicity, and resistance to chemotherapy. CSC-related genes expression was also altered. Next, by RT2 ProfilerTM PCR array and phospho-kinase array, we found PFKFB3 and PDK4 driven ovarian CSCs via STAT3 and NF-κΒ signaling pathways, which was then substantiated by DMAPT (NF-κΒ inhibitor) treatment. Furthermore, miR-16-5p down-regulated PDK4 expression and inhibited stemness in ovarian CSCs. To conclude, PFKFB3 and PDK4 exert essential roles in CSC properties and metabolic switch on mammospheres and ALDH+CD44+ ovarian CSCs through STAT3 and NF-κΒ signaling pathways. PFKFB3 and PDK4 can be therapeutic molecular targets and glycolytic inhibitors (PFK158, 3PO, and DCA) hold potential therapeutic effects for ovarian cancer.