PKM2 Knockdown Induces Autophagic Cell Death via AKT/mTOR Pathway in Human Prostate Cancer Cells

2019 Cellular Physiology and Biochemistry  
Background/Aims: Pyruvate kinase M2 (PKM2) is essential for aerobic glycolysis. Although high PKM2 expression is observed in various cancer tissues, its functional role in cancer metabolism is unclear. Here, we investigated the role of PKM2 in regulating autophagy and its associated pathways in prostate cancer cells. Methods: Immunohistochemistry was performed to compare the expression level of PKM2 in prostate cancer patients and normal human, whereas expression of PKM2 in several cell lines
more » ... everal cell lines was also examined by using western blot. PKM2 expression was silenced using various small interfering RNAs (siRNAs). Cell viability was examined using IncuCyte ZOOM™ live cell imaging system. Western blotting and immunofluorescence were performed to investigate the PKM2 knockdown on other cellular signaling molecules. Acridine orange and Monodansylcadaverine staining was performed to check effect of PKM2 knockdown on autophagy induction. High performance thin layer chromatography was carried out to quantify the level of different cellular metabolites (pyruvate and lactate). Colony formation assay was performed to determine the ability of a cells to form large colonies. Results: PKM2 was highly expressed in prostate cancer patients as compared to normal human. PKM2 siRNA-transfected prostate cancer cells showed significantly reduced viability. Acridine orange, Monodansylcadaverine staining and western blotting analysis showed that PKM2 downregulation markedly increased autophagic cell death. Results of western blotting analysis showed that PKM2 knockdown affected protein kinase B/mechanistic target of rapamycin 1 pathway, which consequently downregulated the expression of glycolytic enzymes lactate dehydrogenase A and glucose transporter 1. Knockdown of PKM2 also reduced the colony formation ability of human prostate cancer cell DU145. Conclusion: To the best of our knowledge, this is the first study to show that PKM2 inhibition alters prostate cancer cell metabolism and induces autophagy, thus providing new perspectives for developing PKM2-targeting anticancer therapies for treating prostate cancer. Introduction Prostate cancer is characterized by an abnormal growth of cells in the prostate gland and metastasis of these cancerous cells to other parts of the body, such as the lymph nodes and bone marrow. Prostate cancer generally develops in men aged over 50 years [1] . Prostate cancer is the sixth leading cause of cancer-related deaths worldwide, and >200, 000 new prostate cancer cases are predicted in the United States in 2013 [2]. According to Siegel et al. 2018, 164, 690 new case has been registered in USA and 29, 430 males has been died due to prostate cancer [3] . Tumor cells need sufficient amount of energy and biosynthetic precursors for survival and proliferation [4] . Tumor cells generally use large amounts of glucose and secrete large amounts of lactate in the presence of oxygen, a phenomenon referred to as aerobic glycolysis or "Warburg effect" [5, 6] . Glycolytic intermediates produced through the Warburg effect promote the biosynthesis of ATP and cellular macromolecular building blocks, including nucleotides, amino acids, proteins, and lipids [7, 8] . Pyruvate kinase M2 (PKM2), a ratelimiting terminal glycolytic enzyme, is a major player in aerobic glycolysis in cancer cells and catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate and releases energy, thus providing a selective growth advantage to cancer cells [9] [10] [11] [12] [13] . Pyruvate kinase has four isoforms, namely, PKM1, PKM2 (encoded by PKM), PKL, and PKR (encoded by PKLR). PKM2 is produced by the alternative splicing of PKM pre-mRNA by heterogeneous nuclear ribonucleoproteins A1 and A2 and polypyrimidine tract-binding protein splicing factor (which is upregulated by oncogenic transcription factor c-MYC) that promotes the inclusion of exon 10 and exclusion of exon 9 [14] [15] [16] . PKM1 is highly expressed in normal tissues [16], whereas PKM2 is predominantly expressed in various cancers such as colon cancer [9, 17], hepatocellular carcinoma [18], lung cancer [18], breast cancer [19], renal cell carcinoma [20-22], and gastrointestinal and cervical cancers [23], thus making it a potential hallmark of cancer metabolism. Besides its well-established role in aerobic glycolysis, PKM2 performs various nonmetabolic functions in cancer cells, including gene expression and cell cycle progression. Extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated nuclear translocation of PKM2 activates β-catenin, which in turn upregulates the expression of c-MYC and induces the expression of glycolytic enzymes, including glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA). This upregulation also induces PKM2 expression by splicing the PKM pre-mRNA into PKM2 mRNA [24] [25] [26] [27] . Nuclear PKM2 act as a transcriptional coactivator of hypoxia-inducible factor 1-alpha (HIF1α) to reprogram cancer cell metabolism [28] . Moreover, PKM2 promotes the phosphorylation and activation of STAT3 and ERK1/2 to enhance their transcriptional activity and to promote cell proliferation [29, 30] . Upregulation of these glycolytic genes increases glucose consumption and lactate production rates, thus promoting tumorigenesis [31, 32] . In the cellular environment, PKM2 is associated with protein kinase B (Akt)/mechanistic or mammalian target of rapamycin (mTOR) signaling pathway [33] , which regulates numerous cellular processes, including cell growth and survival, cell cycle progression, protein synthesis, and angiogenesis [34] . Activation of the Akt/mTOR pathway impairs autophagy in prostate cancer cells [34] . Autophagy is a well-regulated homeostatic mechanism characterized by the collection of the cytoplasmic machinery into autophagosomes, followed by its lysosomal proteolytic digestion and recycling to maintain cellular growth [35] . Under metabolic stress, autophagy promotes cancer cell survival; however, unrestricted autophagy can lead to uncontrolled cellular consumption and ultimately cell death [36] . Autophagy is
doi:10.33594/000000107 fatcat:y6e5opxpojetfdo647spdhoaje