Pyruvate kinase type M2 is upregulated in colorectal cancer and promotes proliferation and migration of colon cancer cells

Pyruvate kinase type M2 (PKM2) has been reported to be involved in aerobic glycolysis and cell growth in various tumors. However, the expression pattern of PKM2 in colorectal cancer (CRC) and the correlation between PKM2 expression and CRC remains unclear. The aim of this study is to investigate PKM2 expression and its possible role in CRC. We found that expression of PKM2 was increased in CRC and the increased PKM2 expression was associated with later stage and lymph metastasis of the tumors. Knockdown of PKM2 suppressed the aerobic glycolysis and decreased lactate production of colon cancer RKO cells. Knockdown of PKM2 repressed proliferation and migration of the cells. Inhibition of PKM2 suppressed xenograft tumor growth of RKO cells in vivo. These results suggest that the expression of PKM2 plays a critical role in development of CRC, and it may provide a growth advantage for colon cancer cells. Thus, PKM2 might be a potential therapeutic target for CRC. ? 2012 IUBMB Life, 64(9): 775-782, 2012.     

Rocking cell metabolism: revised functions of the key glycolytic regulator PKM2 in cancer

Cancer cell metabolism is exemplified by high glucose consumption and lactate production. Pyruvate kinase (PK), which catalyzes the final step of glycolysis, has emerged as a potential regulator of this metabolic phenotype. The M2 isoform of PK (PKM2) is highly expressed in cancer cells. However, the mechanisms by which PKM2 coordinates high energy requirements with high anabolic activities to support cancer cell proliferation are still not completely understood. Current research has elucidated novel regulatory mechanisms for PKM2, contributing to its important role in cancer. This review summarizes the current understanding and explores future directions in the field, highlighting controversies regarding the activity and specificity of PKM2 in cancer. In light of this knowledge, the potential therapeutic implications and strategies are critically discussed.     

MUC1-C oncoprotein regulates glycolysis and pyruvate kinase M2 activity in cancer cells

Aerobic glycolysis in cancer cells is regulated by multiple effectors that include Akt and pyruvate kinase M2 (PKM2). Mucin 1 (MUC1) is a heterodimeric glycoprotein that is aberrantly overexpressed by human breast and other carcinomas. Here we show that transformation of rat fibroblasts by the oncogenic MUC1-C subunit is associated with Akt-mediated increases in glucose uptake and lactate production, consistent with the stimulation of glycolysis. The results also demonstrate that the MUC1-C cytoplasmic domain binds directly to PKM2 at the B- and C-domains. Interaction between the MUC1-C cytoplasmic domain Cys-3 and the PKM2 C-domain Cys-474 was found to stimulate PKM2 activity. Conversely, epidermal growth factor receptor (EGFR)-mediated phosphorylation of the MUC1-C cytoplasmic domain on Tyr-46 conferred binding to PKM2 Lys-433 and inhibited PKM2 activity. In human breast cancer cells, silencing MUC1-C was associated with decreases in glucose uptake and lactate production, confirming involvement of MUC1-C in the regulation of glycolysis. In addition, EGFR-mediated phosphorylation of MUC1-C in breast cancer cells was associated with decreases in PKM2 activity. These findings indicate that the MUC1-C subunit regulates glycolysis and that this response is conferred in part by PKM2. Thus, the overexpression of MUC1-C oncoprotein in diverse human carcinomas could be of importance to the Warburg effect of aerobic glycolysis.     

Crosstalk of mTOR/PKM2 and STAT3/c-Myc signaling pathways regulate the energy metabolism and acidic microenvironment of gastric cancer

Cancer cells consume large amounts of glucose to produce lactate, even in the presence of ample oxygen. This phenomenon is called the Warburg effect. c-Myc is an important member of the Myc gene family and is involved in the development of various tumors. It plays an important role in the regulation of tumor energy metabolism, which can regulate glycolysis to promote the Warburg effect in a tumor. Our study aimed to improve the malignant biological behavior by controlling the energy metabolism of gastric cancer through the mTOR/PKM2 and signal transduction and activator 3 (STAT3)/c-Myc signaling pathways through a series of in vitro experiments. Human gastric cancer AGS and HGC-27 cells were treated with PKM2 and c-Myc lentivirus, and the effects of the knockdown of PKM2 and/or c-Myc were analyzed on cell proliferation, cell apoptosis, the ability of cell migration, and the growth signaling pathway in vitro. The expressions of PKM2, c-Myc, LDHA, STAT3, P-STAT3, GLUT-1 gene were identified by the quantitative real-time polymerase chain reaction and Western blot analysis. Lactate and glucose levels were tested by the corresponding kit. Our findings showed that PKM2 and c-Myc were upregulated in human gastric cancer. Knockdown of c-Myc in gastric cancer cells suppressed cell proliferation capacity and glycolysis level, and the inhibitory effects on gastric cancer cells upon co-knockdown of PKM2 and c-Myc were more obvious compared with knockout of PKM2 or c-Myc alone. And there was a correlation between the mTOR/PKM2 and the STAT3/c-Myc signaling pathways. Our results suggested that c-Myc might be considered a potential therapeutic target for gastric cancer and PKM2 combined with c-Myc could better inhibit the malignant biological behaviors of gastric cancer.     

Aerobic glycolysis supports hepatitis B virus protein synthesis through interaction between viral surface antigen and pyruvate kinase isoform M2

As an intracellular pathogen, the reproduction of the hepatitis B virus (HBV) depends on the occupancy of host metabolism machinery. Here we test a hypothesis if HBV may govern intracellular biosynthesis to achieve a productive reproduction. To test this hypothesis, we set up an affinity purification screen for host factors that interact with large viral surface antigens (LHBS). This identified pyruvate kinase isoform M2 (PKM2), a key regulator of glucose metabolism, as a binding partner of viral surface antigens. We showed that the expression of viral LHBS affected oligomerization of PKM2 in hepatocytes, thereby increasing glucose consumption and lactate production, a phenomenon known as aerobic glycolysis. Reduction of PKM2 activity was also validated in several different models, including HBV-infected HepG2-NTCP-C4 cells, adenovirus mediated HBV gene transduction and transfection with a plasmid containing complete HBV genome on HuH-7 cells. We found the recovery of PKM2 activity in hepatocytes by chemical activators, TEPP-46 or DASA-58, reduced expressions of viral surface and core antigens. In addition, reduction of glycolysis by culturing in low-glucose condition or treatment with 2-deoxyglucose also decreased expressions of viral surface antigen, without affecting general host proteins. Finally, TEPP-46 largely suppressed proliferation of LHBS-positive cells on 3-dimensional agarose plates, but showed no effect on the traditional 2-dimensional cell culture. Taken together, these results indicate that HBV-induced metabolic switch may support its own translation in hepatocytes. In addition, aerobic glycolysis is likely essential for LHBS-mediated oncogenesis. Accordingly, restriction of glucose metabolism may be considered as a novel strategy to restrain viral protein synthesis and subsequent oncogenesis during chronic HBV infection.     

Specific Pyruvate Kinase M2 Inhibitor,Compound 3K,Induces Autophagic Cell Death through Disruption of the Glycolysis Pathway in Ovarian Cancer Cells

Ovarian cancer is a common cause of death among gynecological cancers. Although ovarian cancer initially responds to chemotherapy, frequent recurrence in patients remains a therapeutic challenge. Pyruvate kinase M2 (PKM2) plays a pivotal role in regulating cancer cell survival. However, its therapeutic role remains unclear. Here, we investigated the anticancer effects of compound 3K, a specific PKM2 inhibitor, on the regulation of autophagic and apoptotic pathways in SK-OV-3 (PKM2-overexpressing human ovarian adenocarcinoma cell line). The anticancer effect of compound 3K was examined using MTT and colony formation assays in SK-OV-3 cells. PKM2 expression was positively correlated with the severity of the tumor, and expression of pro-apoptotic proteins increased in SK-OV-3 cells following compound 3K treatment. Compound 3K induced AMPK activation, which was accompanied by mTOR inhibition. Additionally, this compound inhibited glycolysis, resulting in reduced proliferation of SK-OV-3 cells. Compound 3K treatment suppressed tumor progression in an in vivo xenograft model. Our findings suggest that the inhibition of PKM2 by compound 3K affected the Warburg effect and induced autophagic cell death. Therefore, use of specific PKM2 inhibitors to block the glycolytic pathway and target cancer cell metabolism represents a promising therapeutic approach for treating PKM2-overexpressing ovarian cancer.     

Beta‐elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta‐elemene (β‐elemene), an approved drug for complementary cancer therapy, exerted distinct anti‐metastatic activity dependent on PKM2. The results indicated that β‐elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. β‐Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that β‐elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of β‐elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose‐1,6‐bisphosphate (FBP) and L‐cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β‐elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti‐metastatic agent from natural compounds.     

Preventing pyruvate kinase muscle expression in Chinese hamster ovary cells curbs lactogenic behavior by altering glycolysis,gating pyruvate generation,and increasing pyruvate flux into the TCA cycle

Deletion of the pyruvate kinase muscle (PKM) gene, which is involved in conversion of phosphoenolpyruvate to pyruvate, has been shown to curb lactogenic behavior in Chinese hamster ovary (CHO) cells. This study describes the generation of pyruvate kinase muscle isoforms 1 and 2 knockout (PKM-KO) and pyruvate kinase muscle isoform-1 knockout (PKM1-KO) CHO host cells to understand metabolic shifts that reduce lactate secretion in these cells. Glucose and amino acids uptake levels in wild-type (WT), PKM-KO, and PKM1-KO stable cell lines, expressing two different antibodies, were analyzed in 14-day fed-batch production assays using different vessels. PKM-KO and PKM1-KO cells consumed more glucose per cell, altered amino acids metabolism, had higher flux of pyruvate into the tricarboxylic acid (TCA) cycle, and as previously shown reduced lactate secretion levels compared with the WT cells. Additionally, both PKM-KO and PKM1-KO cells had higher specific productivity and lower cell growth rates compared with the WT cells. Our findings suggest that rewiring the flux of pyruvate to the TCA cycle by deletion of PKM or PKM1 reduced cell growth and increased specific productivity in CHO cells. Overall, PKM1-KO cells had similar product quality and comparable or better titers relative to the WT cells, hence, targeted deletion of this isoform for curbing lactogenic behavior in CHO cells is suggested.     

miR-122 Targets Pyruvate Kinase M2 and Affects Metabolism of Hepatocellular Carcinoma

In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg’s effect), with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122) is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC). Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK) gene showed the strongest anti-correlation coefficient (Pearson r = −0.6938, p = <0.0001). In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2) is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways.     

1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.     

Pyruvate kinase M2 facilitates colon cancer cell migration via the modulation of STAT3 signalling

Understanding the mechanisms of colorectal cancer (CRC) metastatic progression is essential to reducing its morbidity and mortality. Pyruvate kinase (PK) catalyses the final step of glycolysis and has been identified as a critical regulator of glucose consumption. However, the mechanisms and roles of PKM1 and PKM2 in the regulation of CRC cell migration and cell adhesion remain elusive. Here, we report that PKM2 rather than PKM1 drives CRC cell migration and cell adhesion, whereas PKM attenuation reverses these phenomena. Furthermore, the overexpression of PKM2 significantly increases the expression of N-cadherin, MMP-2, MMP-9, STAT3, Snail-2, pFAK and active β1-integrin, while E-cadherin expression is suppressed. More importantly, the results indicated that PKM2 overexpression facilitates STAT3 nuclear translocation, and it is required for PKM2 function in the regulation of migration and adhesion associated signalling. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities but possesses protein kinase activity, is critical for CRC cell migration and cell adhesion. Overall, this study suggests that PKM2 overexpression promotes CRC cell migration and cell adhesion by regulating STAT3-associated signalling and that PKM2 may serve as a therapeutic target for CRC metastasis.     

PKM2 depletion induces the compensation of glutaminolysis through β-catenin/c-Myc pathway in tumor cells

The metabolic activity in cancer cells primarily rely on aerobic glycolysis. Besides glycolysis, some tumor cells also exhibit excessive addition to glutamine, which constitutes an advantage for tumor growth. M2-type pyruvate kinase (PKM2) plays a pivotal role in sustaining aerobic glycolysis, pentose phosphate pathway and serine synthesis pathway. However, the participation of PKM2 in glutaminolysis is little to be known. Here we demonstrated that PKM2 depletion could provoke glutamine metabolism by enhancing the β-catenin signaling pathway and consequently promoting its downstream c-Myc-mediated glutamine metabolism in colon cancer cells. Treatment with 2-deoxy-d-glucose (2-DG), a glycolytic inhibitor, got consistent results with the above. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities, plays a critical role in regulating β-catenin. Moreover, we found that overexpression of PKM2 negatively regulated β-catenin through miR-200a. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon glucose metabolism impaired.     

Wnt1-inducible signaling protein 1 regulates laryngeal squamous cell carcinoma glycolysis and chemoresistance via the YAP1/TEAD1/GLUT1 pathway

Wnt1-inducible signaling protein 1 (WISP1) is a matricellular protein and downstream target of Wnt/β-catenin signaling. This study sought to determine the role of WISP1 in glucose metabolism and chemoresistance in laryngeal squamous cell carcinoma. WISP1 expression was silenced or upregulated in Hep-2 cells by the transfection of WISP1 siRNA or AdWISP1 vector. Ectopic WISP1 expression regulated glucose uptake and lactate production in Hep-2 cells. Subsequently, the expression of glucose transporter 1 (GLUT1) was significantly modulated by WISP1. Furthermore, WISP1 increased cell survival rates, diminished cell death rates, and suppressed ataxia-telangiectasia-mutated (ATM)-mediated DNA damage response pathway in cancer cells treated with cisplatin through GLUT1. WISP1 also promoted cancer cell tumorigenicity and growth in mice implanted with Hep-2 cells. Additionally, WISP1 activated the YAP1/TEAD1 pathway that consequently contributed to the regulation of GLUT1 expression. In summary, WISP1 regulated glucose metabolism and cisplatin resistance in laryngeal cancer by regulating GLUT1 expression. WISP1 may be used as a potential therapeutic target for laryngeal cancer.