Pyruvate Kinase M2 Knockdown Research Articles

Pyruvate Kinase M2 Knockdown Suppresses Migration, Invasion, and Epithelial-Mesenchymal Transition of Gastric Carcinoma via Hypoxia-Inducible Factor Alpha/B-Cell Lymphoma 6 Pathway.

Gastric carcinoma is a common malignant cancer. Pyruvate kinase M2 (PKM2) is highly expressed in cancers, including gastric carcinoma. However, its function and molecular mechanism in gastric carcinoma remains unclear. Here, we aimed to explore the function and the underlying mechanism of PKM2 on malignant phenotypes in gastric carcinoma. In this study, the mRNA levels and protein levels of PKM2 in gastric carcinoma cell lines and normal gastric mucosa epithelial cell lines were detected using quantitative real-time PCR and western blot, respectively. PKM2 was downregulated by siRNA transfection. HIF-1α or BCL-6 was upregulated by corresponding overexpression plasmid. Cell viability was detected using CCK-8 assay. Cell invasion and migration were determined using transwell assay. Higher expression of PKM2 was observed in human gastric carcinoma cell lines MKN-45 and SGC-7901 than in the normal gastric mucosa epithelial cell line GES-1. PKM2 knockdown suppressed cancer cell invasion and migration and inhibited the epithelial-mesenchymal transition (EMT) phenotype by inhibiting E-cadherin and promoting vimentin and N-cadherin expression. Also, we observed that PKM2 knockdown suppressed the hypoxia-inducible factor alpha (HIF-1α) and B-cell lymphoma 6 (BCL-6) signaling pathway. HIF-1α overexpression reversed the function of PKM2 silencing on cell invasion, migration, EMT, and BCL-6 expression. BCL-6 overexpression also reversed the function of PKM2 silencing on cell invasion, migration, and EMT but did not affect HIF-1α expression. Taken together, data from our study suggest that PKM2 knockdown impeded cell migration, invasion, and EMT of gastric carcinoma cells via the HIF-1α/BCL-6 pathway.

Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition

Background: Aerobic glycolysis and epidermal–mesenchymal transition (EMT) play key roles in the development of bladder cancer. This study aimed to investigate the function and the underlying mechanism of dihydropyrimidinase like 2 (DPYSL2) in bladder cancer progression. Methods: The expression pattern of DPYSL2 in bladder cancer and the correlation of DPYSL2 expression with clinicopathological characteristics of bladder cancer patients were analyzed using the data from different databases and tissue microarray. Gain- and loss-of-function assays were performed to explore the role of DPYSL2 in bladder cancer progression in vitro and in mice. Proteomic analysis was performed to identify the interacting partner of DPYSL2 in bladder cancer cells. Findings: The results showed that DPYSL2 expression was upregulated in bladder cancer tissue compared with adjacent normal bladder tissue and in more aggressive cancer stages compared with lower stages. DPYSL2 promoted malignant behavior of bladder cancer cells in vitro, as well as tumor growth and distant metastasis in mice. Mechanistically, DPYSL2 interacted with pyruvate kinase M2 (PKM2) and promoted the conversion of PKM2 tetramers to PKM2 dimers. Knockdown of PKM2 completely blocked DPYSL2-induced enhancement of the malignant behavior, glucose uptake, lactic acid production, and epithelial-mesenchymal transition in bladder cancer cells. Funding Statement: The present study was financially supported by the Natural Science Foundation of Guangdong Province (grant no. 2018A0303130327) and The Third Affiliated Hospital of Guangzhou Medical University (grant no. 2018Q10). Declaration of Interests: The authors declare no competing interests in this study. Ethics Approval Statement: Each patient provided written informed consent before sample collection. This study was approved by the Internal Review and Ethics Boards at the Cancer Center of Guangzhou Medical University. All animal experiments were conducted according to the Principles of Laboratory Animal Care (National Society for Medical Research). These experiments were approved by the Internal Research Ethics Board at the Third Affiliated Hospital of Guangzhou Medical University.

Pyruvate Kinase M2 Mediates Glycolysis in the Lymphatic Endothelial Cells and Promotes the Progression of Lymphatic Malformations

Metabolism plays a pivotal role in the formation of the lymphatic vasculature. Pyruvate kinase M2 (PKM2) is typically a metabolic marker of proliferating cells and maintains the growth of vascular endothelial cells. In this study, the potential status of PKM2 in lymphatic endothelial cells and the pathogenesis of lymphatic malformations (LMs) was investigated. The glycolysis index, including glucose uptake, ATP, and lactate production, stayed at a relatively high level in human dermal lymphatic endothelial cells (HDLECs) compared with human umbilical vein endothelial cells, whereas the inhibition of PKM2 by shikonin or PKM2 knockdown significantly suppressed glycolysis, migration, tubular formation, and invasion of HDLECs. Moreover, compared with lymphatic vessels in healthy skin, lymphatic vessels of LMs expressed PKM2 highly, and this expression correlated with infection of LMs. Meanwhile, the overexpression of PKM2 in HDLECs strengthened the proliferation, migration, tubular formation, and invasion of HDLECs. The findings from further experiments in a rat LM model support that targeting PKM2 by shikonin significantly impedes the progression of LMs, even in an infected LM rat model. Taken together, these results indicate that PKM2 plays a pivotal role in the activation of LECs and promotes the progression of LMs, whereas the inhibition of PKM2 can effectively suppress the pathogenesis of LM lesions in the rat model.

PKM2 Drives Hepatocellular Carcinoma Progression by Inducing Immunosuppressive Microenvironment.

Background and AimsPyruvate kinase M2 (PKM2) is an essential regulator of the Warburg effect, but its biological function promoting immune escape of hepatocellular carcinoma (HCC) is unclear.MethodsGEPIA web tool and immunohistochemistry (IHC) analysis were employed to evaluate the clinical relevance of PKM2 in HCC patients. Both in vitro CCK-8, colony formation, and transwell assays, and in vivo xenografts were performed to evaluate the malignancy of HCC cells. PKM2 and PD-L1 levels were examined by Western blot, qRT-PCR, and IHC. The role of PKM2 on in vivo immune response was also investigated.ResultsPKM2 was significantly upregulated in HCC and associated with a poor prognosis of HCC patients. Knockdown of PKM2 inhibited in vitro proliferation, migration, and invasion of HCC cells, as well as in vivo tumor growth. Strikingly, PKM2 showed a strong correlation with the expression of immune inhibitory cytokines and lymphocyte infiltration in HCC. The overexpression of PKM2 sensitized HCC to immune checkpoint blockade, which enhanced IFN-γ positive CD8 T cells in HCC mice models.ConclusionPKM2 might be a predictor and a potential therapeutic target for immune checkpoint inhibitors in HCC.

Pyruvate Kinase M2 Tetramerization Protects against Hepatic Stellate Cell Activation and Liver Fibrosis

Liver fibrosis is an increasing health problem worldwide, for which no effective antifibrosis drugs are available. Although the involvement of aerobic glycolysis in hepatic stellate cell (HSC) activation has been reported, the role of pyruvate kinase M2 (PKM2) in liver fibrogenesis still remains unknown. We examined PKM2 expression and location in liver tissues and primary hepatic cells. The invitro and invivo effects of a PKM2 antagonist (shikonin) and its allosteric agent (TEPP-46) on liver fibrosis were investigated in HSCs and liver fibrosis mouse model. Chromatin immunoprecipitation sequencing and immunoprecipitation were performed to identify the relevant molecular mechanisms. PKM2 expression was significantly up-regulated in both mouse and human fibrotic livers compared with normal livers, and mainly detected in activated, rather than quiescent, HSCs. PKM2 knockdown markedly inhibited the activation and proliferation of HSCs invitro. Interestingly, the PKM2 dimer, rather than the tetramer, induced HSC activation. PKM2 tetramerization induced by TEPP-46 effectively inhibited HSC activation, reduced aerobic glycolysis, and decreased MYC and CCND1 expression via regulating histone H3K9 acetylation in activated HSCs. TEPP-46 and shikonin dramatically attenuated liver fibrosis invivo. Our findings demonstrate a nonmetabolic role of PKM2 in liver fibrosis. PKM2 tetramerization or suppression could prevent HSC activation and protects against liver fibrosis.

Abstract 1916: Targeting Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma

Abstract Background: A major target for esophageal adenocarcinoma (EAC) therapies is the human epidermal growth factor receptor 2 (HER2). EAC overexpresses HER2 and lapatinib, a dual tyrosine kinase inhibitor blocking HER1 and HER2 pathways fails to improve patient survival. Molecular mechanisms of this lapatinib resistance are still unclear. We therefore explored the role of glycolytic enzyme pyruvate kinase M2 (PKM2), a key regulator of Warburg effect in lapatinib resistance mechanism of EAC cells. Methods: First we established lapatinib-resistant OE19 (LPR-OE19) cells from OE19 EAC cells and characterized it. We then investigated the comparative cell growth inhibition, apoptotic and lactate production effects of HER2 inhibitor lapatinib and PKM2 inhibitor shikonin either alone or in combinations, both in OE19 and LPR-OE19 cells with and without knockdown of PKM2 and HSP40 by siRNAs. We then explored the antitumor efficacy following lapatinib and shikonin mono and combination therapies in murine subcutaneous xenograft model of human EAC. Results: Lapatinib-resistant OE19 (LPR-OE19) cells were established from OE19 cells by intermittent exposure to increasing concentrations of lapatinib for a period of total six months. Lapatinib resistant LPR-OE19 cells showed downregulation of HSP40 both at protein and mRNA levels whereas showed upregulation of PKM2 only at protein level. LPR-OE19 cells showed significantly reduced sensitivity to lapatinib induced cell growth inhibition, apoptosis and decreased cell migration compared to parent OE19 cells. Interestingly, augmented cell growth inhibition, apoptosis and decreased cell migration were observed in LPR-OE19 cells compared to parent OE19 cells when lapatinib was combined with shikonin. More interestingly, knockdown of PKM2 in LPR-OE19 cells abolished the reduced sensitivity of lapatinib induced cell growth inhibition and also abolished augmented cell growth inhibition response when lapatinib and shikonin were combined. Contrary to that, knockdown of HSP40 in OE19 cells enhanced PKM2 expression leading to significantly reduced sensitivity to lapatinib induced growth inhibition with augmented growth inhibition when lapatinib and shikonin were combined. Moreover, LPR-OE19 cells showed enhanced lactate production compared to parent OE19. Interestingly, PKM2 and HSP40 co-localize with each other in the cell nucleus suggesting that PKM2 bound to molecular chaperone HSP40. In subcutaneous xenografts using LPR-OE19 cells, average net tumor growth after two weeks in different therapy groups was 494.72 mm3 in control, 403.11 mm3 after lapatinib (p=0.241), 339.48 mm3 after shikonin (p=0.0478), and 141.59 mm3 after shikonin plus lapatinib (p=0.0003). Conclusions: In conclusion, these data suggest that combination therapy with lapatinib and glycolytic inhibitor shikonin should be tested as a potent option for lapatinib resistant HER2-positive EAC patients and could be a novel treatment strategy for specific EAC. In addition, it is suggested that targeting HSP40-PKM2 interaction as an innovative therapeutic approach to overcome lapatinib resistance in EAC. Citation Format: Md Sazzad Hassan, Samantha Holmes, Victoria Makuru, Chloe Johnson, Urs von Holzen. Targeting Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1916.

Ablation of PKM2 ameliorated ER stress-induced apoptosis and associated inflammation response in IL-1β-treated chondrocytes via blocking Rspo2-mediated Wnt/β-catenin signaling.

Endoplasmic reticulum (ER) stress and the related apoptosis and inflammation damage play key roles in osteoarthritis development. The aim of the present work was to investigate the exact role and potential underlying mechanism of pyruvate kinase M2 (PKM2) in rat chondrocytes exposed to interleukin-Iβ (IL-1β). We observed that IL-1β stimulation resulted in an apparent enhancement in PKM2 expression. Additionally, loss of PKM2 evidently ascended cell viability in response to IL-1β exposure. Simultaneously, elimination of PKM2 manifestly repressed IL-1β-stimulated chondrocyte apoptosis, concomitant with attenuated in the proapoptotic protein markers Bax and cleaved caspase-3, and elevated the antiapoptotic protein Bcl-2. In the meanwhile, knockdown of PKM2 ameliorated ER stress in IL-1β-treated chondrocytes, as evidenced by reduced expression of the ER stress-associated proteins GRP78, CHOP, and cleaved caspase-12. Furthermore, PKM2 silencing protected chondrocytes against IL-1β-triggered inflammatory response, as reflected by the downregulated release of proinflammatory mediators, including tumor necrosis factor-α, IL-6, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2, as well as decreased nitric oxide generation. More important, abrogating PKM2 expression caused a marked decline in Rspo2 expression, and subsequently blocked Wnt/β-catenin signaling. Mechanistically, the Wnt/β-catenin signaling activator Licl effectively impeded the beneficial effects of PKM2 ablation on IL-1β-stimulated apoptosis and inflammatory response. These findings collectively implicated that PKM2 inhibition protected against ER stress-mediated cell apoptosis and inflammatory injury in rat chondrocytes stimulated with IL-1β by inactivating Rspo2-mediated Wnt/β-catenin pathway, and may represented a novel therapeutic target for osteoarthritis.

Cutting off the fuel supply to calcium pumps in pancreatic cancer cells: role of pyruvate kinase-M2 (PKM2)

BackgroundPancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby preventing Ca2+-dependent cell death. The ATP-generating pyruvate kinase-M2 (PKM2) is oncogenic and overexpressed in PDAC. This study investigated the PKM2-derived ATP supply to the PMCA as a potential therapeutic locus.MethodsPDAC cell growth, migration and death were assessed by using sulforhodamine-B/tetrazolium-based assays, gap closure assay and poly-ADP ribose polymerase (PARP1) cleavage, respectively. Cellular ATP and metabolism were assessed using luciferase/fluorescent-based assays and the Seahorse XFe96 analyzer, respectively. Cell surface biotinylation identified membrane-associated proteins. Fura-2 imaging was used to assess cytosolic Ca2+ overload and in situ Ca2+ clearance. PKM2 knockdown was achieved using siRNA.ResultsThe PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca2+ overload. PKM2 associates with plasma membrane proteins providing a privileged ATP supply to the PMCA. PKM2 knockdown reduced PMCA activity and reduced the sensitivity of shikonin-induced cell death.ConclusionsCutting off the PKM2-derived ATP supply to the PMCA represents a novel therapeutic strategy for the treatment of PDAC.

Knockdown of Pyruvate Kinase M2 Inhibits Cell Proliferation, Metabolism, and Migration in Renal Cell Carcinoma.

Emerging evidence indicates that the activity of pyruvate kinase M2 (PKM2) isoform is crucial for the survival of tumor cells. However, the molecular mechanism underlying the function of PKM2 in renal cancer is undetermined. Here, we reveal the overexpression of PKM2 in the proximal tubule of renal tumor tissues from 70 cases of patients with renal carcinoma. The functional role of PKM2 in human renal cancer cells following small-interfering RNA-mediated PKM2 knockdown, which retarded 786-O cell growth was examined. Targeting PKM2 affected the protein kinase B (AKT)/mechanistic target of the rapamycin 1 (mTOR) pathway, and downregulated the expression of glycolytic enzymes, including lactate dehydrogenase A and glucose transporter-1, and other downstream signaling key proteins. PKM2 knockdown changed glycolytic metabolism, mitochondrial function, adenosine triphosphate (ATP) level, and intracellular metabolite formation and significantly reduced 786-O cell migration and invasion. Acridine orange and monodansylcadaverine staining, immunocytochemistry, and immunoblotting analyses revealed the induction of autophagy in renal cancer cells following PKM2 knockdown. This is the first study to indicate PKM2/AKT/mTOR as an important regulatory axis mediating the changes in the metabolism of renal cancer cells.

Epigenetic regulation of the Warburg effect by H2B monoubiquitination.

Cancer cells reprogram their energy metabolic system from the mitochondrial oxidative phosphorylation (OXPHOS) pathway to a glucose-dependent aerobic glycolysis pathway. This metabolic reprogramming phenomenon is known as the Warburg effect, a significant hallmark of cancer. However, the detailed mechanisms underlying this event or triggering this reprogramming remain largely unclear. Here, we found that histone H2B monoubiquitination (H2Bub1) negatively regulates the Warburg effect and tumorigenesis in human lung cancer cells (H1299 and A549 cell lines) likely through controlling the expression of multiple mitochondrial respiratory genes, which are essential for OXPHOS. Moreover, our work also suggested that pyruvate kinase M2 (PKM2), the rate-limiting enzyme of glycolysis, can directly interact with H2B in vivo and in vitro and negatively regulate the level of H2Bub1. The inhibition of cell proliferation and nude mice xenograft of human lung cancer cells induced by PKM2 knockdown can be partially rescued through lowering H2Bub1 levels, which indicates that the oncogenic function of PKM2 is achieved, at least partially, through the control of H2Bub1. Furthermore, PKM2 and H2Bub1 levels are negatively correlated in cancer specimens. Therefore, these findings not only provide a novel mechanism triggering the Warburg effect that is mediated through an epigenetic pathway (H2Bub1) but also reveal a novel metabolic regulator (PKM2) for the epigenetic mark H2Bub1. Thus, the PKM2-H2Bub1 axis may become a promising cancer therapeutic target.

Targeting the Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma

Background: Esophageal adenocarcinoma (EAC) overexpresses HER2. Lapatinib, a dual tyrosine kinase inhibitor blocking HER1 and HER2 pathways failed to improve patient survival. Molecular mechanisms of this lapatinib resistance are still unclear. We therefore explored the role of glycolytic enzyme pyruvate kinase M2 (PKM2), a key regulator of the Warburg effect in the lapatinib resistance mechanism of EAC cells.
 Methods: First we established a lapatinibresistant OE19 (LPR-OE19) cell line from OE19 EAC cells. We then investigated the comparative cell growth inhibition, apoptotic and lactate production effects of HER2 inhibitor lapatinib and PKM2 inhibitor shikonin either alone or in combinations, both in OE19 and LPR-OE19 cells with and without knockdown of PKM2 and HSP40 by siRNAs.
 Results: Lapatinib resistant LPR-OE19 cells showed downregulation of HSP40 both at protein and mRNA levels, but showed upregulation of PKM2 only at the protein level. LPR-OE19 cells showed significantly reduced sensitivity to lapatinib induced cell growth inhibition, apoptosis and decreased cell migration compared to parent OE19 cells. Interestingly, augmented cell growth inhibition, apoptosis and decreased cell migration were observed in LPR-OE19 cells compared to parent OE19 cells when lapatinib was combined with shikonin. Knockdown of PKM2 in LPR-OE19 cells abolished the reduced sensitivity of lapatinib induced cell growth inhibition, and also abolished augmented cell growth inhibition response when lapatinib and shikonin were combined. Contrary to that, knockdown of HSP40 in OE19 cells enhanced PKM2 expression leading to significantly reduced sensitivity to lapatinib induced growth inhibition with augmented growth inhibition when lapatinib and shikonin were combined. Moreover, LPR-OE19 cells showed enhanced lactate production compared to parent OE19. Interestingly, PKM2 and HSP40 co-localize with each other in the cell nucleus suggesting that PKM2 binds to molecular chaperone HSP40.
 Conclusions: These data suggest that targeting HSP40PKM2 interaction could be an innovative therapeutic approach to overcome lapatinib resistance in EAC.

PKM2 upregulation promotes malignancy and indicates poor prognosis for intrahepatic cholangiocarcinoma

Although pyruvate kinase M2 (PKM2) has been shown to be among the crucial enzymes that regulate aerobic glycolysis in multiple tumour cells, its role in the treatment and prognosis of intrahepatic cholangiocarcinoma (ICC) remains unclear. This study primarily aimed to determine whether the expression status of PKM2 is potentially associated with the clinical outcomes of ICC. PKM2 expression was evaluated in ICC cell lines and tissues via real-time quantitative reverse-transcription polymerase chain reaction, immunofluorescence assays, and Western blot, and its prognostic value was determined according to its impact on the overall survival of patients. We found that PKM2 is highly expressed in ICC, and this was correlated with patient survival. Moreover, we found that PKM2 knockdown could considerably inhibit ICC cell proliferation, invasion, and migration in vitro. PKM2 was overexpressed in ICC, and it may regulate proliferation, invasion, and migration and lead to poor prognosis. Thus, PKM2 might be a potential independent prognostic factor for ICC.

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

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. 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 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. 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. 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.

Pyruvate kinase M2 contributes to cell growth in gastric cancer via aerobic glycolysis

Pyruvate kinase M2 (PKM2) serves as a key enzyme that promotes aerobic glycolysis. This study investigated the function of PKM2 in tumor growth and maintenance in gastric cancer (GC). Histological staining was applied to detect PKM2 expression in GC tissues. PCR and western blotting were used to measure PKM2 expression in GC cells. PKM2 was knocked down to examine the biological behavior of tumors, glycometabolism, and apoptosis. PKM2 was upregulated in GC tissues (65%, 34/52) compared with that in adjacent normal tissues (27%, 10/37). Moreover, PKM2 knockdown inhibited proliferation of BGC823 GC cells, and elevated PKM2 levels were associated with poor survival of GC patients. Furthermore, knockdown of PKM2 altered the biological behavior of BGC823 cells through induction of apoptosis. In conclusion, the results of this study indicated that inhibition of PKM2 could represent a novel strategy for gastric cancer treatment.

Alpha-synuclein induces microglial migration via PKM2-dependent glycolysis.

After spinal cord injury, microglial cells are activated and converted to an M1 phenotype. Emerging evidence supports the hypothesis that glucose reprogramming accompanies microglial activation. What contributes to the activation of microglia and glucose reprogramming, however, remains unclear. In the current study, we investigated the role and underlying mechanism of a-synuclein in regulating the aerobic glycolysis in microglia. We found that a-synuclein contributed to the reprogramming of glucose metabolism in microglia by promoting glycolysis and inhibiting mitochondrial biogenesis and oxidative phosphorylation. Further studies demonstrated that pyruvate kinase M2 (PKM2), a rate-limiting enzyme in glycolysis, mediated glucose reprogramming regulated by a-synuclein. A co-immunoprecipitation assay and Western blot assay demonstrated that a-synuclein interacted with PKM2. Further studies demonstrated that knockdown of PKM2 in a-synuclein-exposed microglia markedly reduced glycolysis and lactate production. Additionally, a-synuclein exposure promoted migration abilities in glucose-cultured microglia, whereas migration ability was suppressed in PKM2 knockdown microglia. Additionally, the PKM2 activator TEPP-46 promoted migration ability in a-synuclein-treated microglia, compared to treatment with a-synuclein alone. In conclusion, we demonstrate a PKM2-dependent glycolysis of a-synuclein in microglial.

PKM2 Mediates Chronic Myeloid Leukemia Imatinib Resistance By Regulating Glycolysis Energy Metabolism

Background: Pyruvate kinase M2 (PKM2) plays an important role in malignant tumor metabolism, and provides tumor cells with enough energy and a lot of metabolic intermediate by mediating Warburg effect. Recently, studies indicate that PKM2 protein can mediate tumor chemoresistance through reprogramming cellular metabolism pathway and participate in chemotherapy resistance mechanisms of many tumor cells. Currently, the problem of imatinib resistance is still a difficult point in the treatment of chronic myeloid leukemia. This study is aimed at investigating the function of PKM2 protein in the metabolism of chronic myeloid leukemia cells and the relationship between PKM2 and drug resistance of chronic myeloid leukemia cells.Methods: We selected the CML primary cells (the cells from new diagnosed patients and refractory patients respectively) and cell lines (KBM5 cells were served as drug-sensitive cells and KBM5 cell with T315I mutation (KBM5-STI) were served as drug-resistant cell) as research objects. We used a low dose PKM2 inhibitor and/or IM treat with CML cells, and detected cell proliferation by CCK-8, apoptosis by flow cytometry. The cell glycolysis level is detected by a real-time cell energy detector, and the results represent the level of glycolysis by the acidification rate. Western blot and RT-qPCR analyzed the protein and mRNA expression level of PKM2 in TKI-sensitive and -resistant primary cells and cell lines. After transfecting control or PKM2 siRNA into KBM5 and KBM5-STI cells for 72hrs, we extracted total RNA and proteins, and determined the mRNA and protein level of PKM2, and the protein level of mTOR、P13K/AKT.Results:PKM2 inhibitor can inhibit KBM5 and KBM5-STI cells growth with IC50 5uM, 6.8uM (P=0.001), respectively, and induce cell apoptosis.Low dose PKM2 inhibitor combined with IM greatly induced KBM5 and KBM5-STI cell growth inhibition and cell death compare to using single drug.PKM2 was higher expressed in the TKI-resistant CML primary cells and cell line KBM5-STI. In the meanwhile, we found that mTOR, HIF-1a were increased in TKI-resistant CML primary cells and cell line too.PKM2 knockdown decreased the cell proliferation and growth, and increased the apoptosis of TKI-resistant cell line after treated with IM. In the mentime, PKM2 knockdown showed significantly lower glycolytic capacity than normal cell line with lower oxygen intake and lacitic acid production, and downregulating with mTOR and P13K/AKT protein expression.Conclusion: Pyruvate kinase M2 (PKM2) acts as an important rate-limiting enzyme in the aerobic glycolytic pathway, and mediates abnormal metabolic pathways which promote tumor cell proliferation, invasion and drug resistance. Compared to the TKI-sensitive primary cell and cell line, PKM2 was increased in the TKI-resistant primary cell and cell line and related to glycolytic level. PKM2 inhibitor can inhibit CML cells growth, induce cell apoptosis, and combined with IM at a low dose can exhibited a synergistic anti-leukemia effect on TKI-resistant cells. Low dose PKM2 inhibitor combined with IM can enhance targeted killing to the CML cells, suggesting that PKM2 protein plays an important role in the formation of drug resistance. After interfering PKM2 protein, it showed a significant downregulated level of glycolysis in the cell line, and the decreased level of P13K/AKT/mTOR signaling pathway. This study demonstrates that PKM2 may be involved in the regulation of energy metabolism in leukemia cells and induce drug resistance. DisclosuresNo relevant conflicts of interest to declare.

Nuclear PKM2 promotes the progression of oral squamous cell carcinoma by inducing EMT and post-translationally repressing TGIF2.

Pyruvate kinase M2 (PKM2), a glycolytic enzyme, acts as a metabolic function leading to an energy production critical for cancer progression, known as Warburg effect. In this study we showed a pivotal role of PKM2 acting as a non-metabolic function to promote cancer cell progression in human oral squamous cell carcinoma (OSCC) through the induction of epithelial-mesenchymal transition (EMT), which is crucial for the potential in cancer cell invasion, and post-translational TGIF2 degradation. PKM2 immunoreaction was strong in the cytoplasm of invasive cancer cells, and distinct in the nucleus of spindle-shaped cancer cells with EMT characteristics. TGIF2 nuclear immunoreaction was seen in dysplastic epithelial cells but was repressed in cancer cells. In vitro analyses, cytoplasmic expression of PKM2 was translocated into the nucleus in human OSCC derived HSC-4 and SAS cells when EMT was stimulated. In addition, nuclear expression of TGIF2 was distinctively repressed in EMT induced HSC-4 and SAS cells. We recognized a mismatch in TGIF2 protein and mRNA expression in EMT induced HSC-4 and SAS cells and found that TGIF2 protein was post-translationally degraded through a ubiquitin proteasome system by an MG132 proteasome inhibition assay. Finally, promotion of HSC-4 and SAS cell progression by PKM2 was recognized in PKM2 knockdown assays. Thus, we clarified a new mechanism of non-metabolic function of PKM2 to promote the progression of OSCC through PKM2 nuclear translocation, subsequently induced EMT, and post-translationally repressed TGIF2 expression by a ubiquitin proteasome system.

The responsively decreased PKM2 facilitates the survival of pancreatic cancer cells in hypoglucose

Cancer cells predominantly produce energy at a high rate of glycolysis even in aerobic environment. It is termed as Warburg effect and is necessary for the tumorigenesis. Studies showed pyruvate kinase M2 (PKM2), a key regulator of the Warburg effect, is overexpressed and involved in numerous cancers. However, the expression and function of PKM2 in pancreatic cancer (PC) remain undefined. Our results showed that PKM2 is overexpressed in the PC tissue compared to the peritumoral tissue. Unexpected, the downregulation of PKM2 did not affect the proliferation, invasion, and chemoresistance of PC cells. Since pancreatic cancer is a hypovascular tumor with comparably insufficient energy supply, we further investigate the relationship between PKM2 and hypoglucose. Interestingly, we further discovered that decreased expression of PKM2 was detected in PC samples with lower microvessel density as well as in PC cells treated with hypoglucose condition (0.5 mM). Furthermore, the downregulation of PKM2 facilitated, while the upregulation of PKM2 inhibited, PC cells survival during hypoglucose. We further revealed that the repressed PKM2 induced autophagy, high NADPH/NADP ratio, and biomacromolecule production, but reduced ROS accumulation. Moreover, AMPKα1 knockdown repressed the autophagy and survival of PC cells during hypoglucose, which were promoted by PKM2 knockdown. Collectively, our study indicates that decreased PKM2 diverts glucose metabolism to biomacromolecule accumulation and antioxidants generation during glucose deprivation. This metabolism alteration elevates AMPKα1-dependent autophagy, which facilitates PC cell survival during glucose deprivation. Therefore, functions of PKM2 are complicated and cannot be defined as oversimplified promoter or inhibitor in PC.

Pyruvate Kinase M2 Modulates the Glycolysis of Chondrocyte and Extracellular Matrix in Osteoarthritis.

Pyruvate kinase M2 (PKM2) has been wildly verified to modulate glycolysis in tumor cells. However, the role of PKM2 on the glycolysis of osteoarthritis (OA) chondrocytes is still unclear. In present study, we investigate the function of PKM2 on OA chondrocyte glycolysis and the collagen matrix generation in vitro. Results showed that PKM2 was upregulated in OA chondrocytes compared with healthy control chondrocytes. In OA chondrocytes, ATP expression was lower compared with healthy control chondrocytes. Loss-of-function experiment showed that PKM2 knockdown mediated by lentivirus transfection could significantly suppress the glucose consumption and lactate secretion levels and decrease glucose transporter-1 (Glut-1), lactate dehydrogenase A (LDHA), and hypoxia inducible factor 1-alpha (HIF-1α), indicating the inhibition of PKM2 knockdown on glycolysis. Moreover, Cell Counting Kit-8 (CCK-8), flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay showed that PKM2 knockdown inhibited OA chondrocyte proliferation and promoted the apoptosis. Western blot and immunocytochemical staining showed that PKM2 knockdown downregulated the expression levels of COL2A1 and SOX-9. In summary, our results conclude that PKM2 modulates the glycolysis and extracellular matrix generation, providing the vital role of PKM2 on OA pathogenesis and a novel therapeutic target for OA.

PKM2 Is Required to Activate Myeloid Dendritic Cells from Patients with Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is an autoimmune disease in which bone marrow failure is mediated by activated myeloid dendritic cells (mDCs) and T lymphocytes. Recent research has identified a strong immunomodulatory effect of pyruvate kinase M2 (PKM2) on dendritic cells in immune-mediated diseases. In this study, we aimed to explore the role of PKM2 in the activation of mDCs in SAA. We observed conspicuously higher levels of PKM2 in mDCs from SAA patients compared to normal controls at both the gene and protein levels. Concurrently, we unexpectedly discovered that after the mDC-specific downregulation of PKM2, mDCs from patients with SAA exhibited weakened phagocytic activity and significantly decreased and shortened dendrites relative to their counterparts from normal controls. The expression levels of the costimulatory molecules CD86 and CD80 were also reduced on mDCs. Our results also suggested that PKM2 knockdown in mDCs reduced the abilities of these cells to promote the activation of CD8+ T cells (CTLs), leading to the decreased secretion of cytotoxic factors by the latter cell type. These findings demonstrate that mDC activation requires an elevated intrinsic PKM2 level and that PKM2 improves the immune status of patients with SAA by enhancing the functions of mDCs and, consequently, CTLs.