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.     

Nuclear translocation of PKM2 modulates astrocyte proliferation via p27 and β-catenin pathway after spinal cord injury

Aberrant functionality of the cell cycle has been implicated in the pathology of traumatic SCI. Although it has been reported that the expressions of various cell cycle related proteins were altered significantly following SCI, detailed information on the subject remains largely unclear. The embryonic pyruvate kinase M2 (PKM2) is an important metabolic kinase in aerobic glycolysis or the warburg effect, however, its functions in central nervous system (CNS) injury remains elusive. Here we demonstrate that PKM2 was not only significantly upregulated by western blot and immunohistochemistry but certain traumatic stimuli also induced translocation of PKM2 into the nucleus in astrocytes following spinal cord injury (SCI). Furthermore, the expression levels and localization of p-β-catenin, p27, cyclin D1 and PCNA were correlated with PKM2 after SCI. In vitro, we also found that PKM2 co-immunoprecipitation with p-β-catenin and p27 respectively. Knockdown of PKM2 apparently decreased the level of PCNA, cyclinD1, p27 in primary astrocyte cells. Taken together, our findings indicate that nuclear translocation of PKM2 promotes astrocytes proliferation after SCI through modulating cell cycle signaling. These discoveries firstly uncovered the role of PKM2 in spinal cord injury and provided a potential therapeutic target for CNS injury and repair.     

PKM2促进肿瘤发展的作用机制研究

为了分析丙酮酸激酶M2型（pyruvate kinase M2,PKM2）在不同肿瘤中的表达情况及其与肿瘤患者临床预后的关系,并探索PKM2对肿瘤细胞增殖和迁移的影响及其作用机制,用TCGA数据库和免疫印迹分析了33种肿瘤中PKM2的表达情况,探索了PKM2与不同肿瘤患者预后的关系。在肺癌细胞系中过表达PKM2,利用CCK8和Transwell方法分析PKM2对肺癌细胞增殖和迁移能力的影响。利用免疫印迹检测不同肿瘤细胞中过表达和敲低PKM2对热休克蛋白90α(Hsp90α)分泌的影响以及上皮-间质转化（epithelial-mesenchgmal transition,EMT）相关蛋白的变化。TCGA数据分析显示,PKM2在包括乳腺癌、肺癌等15种肿瘤中高表达,且9种肿瘤中PKM2的高表达与肿瘤的预后具有显著相关性。在肺癌细胞中过表达PKM2后,肺癌细胞的增殖和迁移能力显著增强。过表达PKM2能够显著增加乳腺癌和肺癌中Hsp90α的分泌。敲低PKM2能够抑制N-钙黏蛋白（N-cadhesion）和波形蛋白（Vimentin）的表达,促进E-钙黏蛋白（E-cadhesion）的表达。研究...     

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.     

The multifaceted regulation and functions of PKM2 in tumor progression

Tumor cells undergo metabolic rewiring from oxidative phosphorylation towards aerobic glycolysis to maintain the increased anabolic requirements for cell proliferation. It is widely accepted that specific expression of the M2 type pyruvate kinase (PKM2) in tumor cells contributes to this aerobic glycolysis phenotype. To date, researchers have uncovered myriad forms of functional regulation for PKM2, which confers a growth advantage on the tumor cells to enable them to adapt to various microenvironmental signals. Here the richness of our understanding on the modulations and functions of PKM2 in tumor progression is reviewed, and some new insights into the paradoxical expression and functional differences of PKM2 in distinct cancer types are offered.     

Translocalization of enhanced PKM2 protein into the nucleus induced by cancer upregulated gene 2 confers cancer stem cell-like phenotypes

Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Ex-pression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of β-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound heal-ing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator.     

Resveratrol inhibits cancer cell metabolism by down regulating pyruvate kinase M2 via inhibition of mammalian target of rapamycin

Metabolism of cancer cells with pyruvate kinase M2 (PKM2) at its centre stage has assumed a prime significance in cancer research in recent times. Cancer cell metabolism, characterized by enhanced glucose uptake, production of lactate and anabolism is considered an ideal target for therapeutic interventions. Expression of PKM2 switches metabolism in favor of cancer cells, therefore, the present study was designed to investigate the hitherto unknown effect of resveratrol, a phytoalexin, on PKM2 expression and resultant implications on cancer metabolism. We observed that resveratrol down-regulated PKM2 expression by inhibiting mTOR signaling and suppressed cancer metabolism, adjudged by decreased glucose uptake, lactate production (aerobic glycolysis) and reduced anabolism (macromolecule synthesis) in various cancer cell lines. A contingent decrease in intracellular levels of ribose-5-phosphate (R5P), a critical intermediate of pentose phosphate pathway, accounted for a reduced anabolism. Consequently, the state of suppressed cancer metabolism resulted in decreased cellular proliferation. Interestingly, shRNA-mediated silencing of PKM2 inhibited glucose uptake and lactate production, providing evidence for the critical role of PKM2 and its mediation in the observed effects of resveratrol on cancer metabolism. Further, an over-expression of PKM2 abolished the observed effects of resveratrol, signifying the role of PKM2 downregulation as a critical function of resveratrol. The study reports a novel PKM2-mediated effect of resveratrol on cancer metabolism and provides a new dimension to its therapeutic potential.     

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.     

A Novel Pyruvate Kinase M2 Activator Compound that Suppresses Lung Cancer Cell Viability under Hypoxia

Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.     

Inhibition of PKM2 sensitizes triple-negative breast cancer cells to doxorubicin

Cancer cells alter regular metabolic pathways in order to sustain rapid proliferation. One example of metabolic remodeling in cancerous tissue is the upregulation of pyruvate kinase isoenzyme M2 (PKM2), which is involved in aerobic glycolysis. Indeed, PKM2 has previously been identified as a tumor biomarker and as a potential target for cancer therapy. Here, we examined the effects of combined treatment with doxorubicin and anti-PKM2 small interfering RNA (siRNA) on triple-negative breast cancer (TNBC). The suppression of PKM2 resulted in changes in glucose metabolism, leading to decreased synthesis of adenosine triphosphate (ATP). Reduced levels of ATP resulted in the intracellular accumulation of doxorubicin, consequently enhancing the therapeutic efficacy of this drug in several triple-negative breast cancer cell lines. Furthermore, the combined effect of PKM2 siRNA and doxorubicin was evaluated in an in vivo MDA-MB-231 orthotopic breast cancer model. The siRNA was systemically administered through a polyethylenimine (PEI)-based delivery system that has been extensively used. We demonstrate that the combination treatment showed superior anticancer efficacy as compared to doxorubicin alone. These findings suggest that targeting PKM2 can increase the efficacy of chemotherapy, potentially providing a new approach for improving the outcome of chemotherapy in patients with TNBC.     

Proviral Insertion in Murine Lymphomas 2 (PIM2) Oncogene Phosphorylates Pyruvate Kinase M2 (PKM2) and Promotes Glycolysis in Cancer Cells

Pyruvate kinase M2 (PKM2) is a key player in the Warburg effect of cancer cells. However, the mechanisms of regulating PKM2 are not fully elucidated. Here, we identified the protein-serine/threonine kinase PIM2, a known oncogene, as a novel binding partner of PKM2. The interaction between PIM2 and PKM2 was confirmed by multiple biochemical approaches in vitro and in cultured cells. Importantly, we found that PIM2 could directly phosphorylate PKM2 on the Thr-454 residue, resulting in an increase of PKM2 protein levels. Compared with wild type, PKM2 with the phosphorylation-defective mutation displayed a reduced effect on glycolysis, co-activating HIF-1α and β-catenin, and cell proliferation, while enhancing mitochondrial respiration of cancer cells. These findings demonstrate that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer, highlighting PIM2 as a potential therapeutic target.     

Nuclear PKM2 regulates the Warburg effect

Pyruvate kinase is a rate-limiting glycolytic enzyme. The PKM1 and PKM2 isoforms result from mutually exclusive alternative splicing of the PKM pre-mRNA. PKM2 rather than PKM1 regulates the Warburg effect and tumorigenesis by poorly understood mechanisms. Emerging evidence has revealed that ERK1/2 phosphorylates PKM2, but not PKM1, leading to PIN1-dependent cis–trans isomerization and conversion of PKM2 from a tetramer to a monomer. Monomeric PKM2 translocates into the nucleus, where it functions as a histone kinase and upregulates the expression of c-Myc and cyclin D1, thereby promoting the Warburg effect and cell cycle progression, respectively. Thus, nuclear PKM2 is essential for tumorigenesis and may serve as a target for treating human cancer.     

Missense Mutations in Pyruvate Kinase M2 Promote Cancer Metabolism,Oxidative Endurance,Anchorage Independence,and Tumor Growth in a Dominant Negative Manner

The present study was designed to examine the functional relevance of two heterozygous mutations (H391Y and K422R), observed earlier by us in the Bloom syndrome condition. Cells stably expressing exogenous wild-type or mutant PKM2 (K422R or H391Y) or co-expressing both wild type and mutant (PKM2-K422R or PKM2-H391Y) were assessed for cancer metabolism and tumorigenic potential. Interestingly, cells co-expressing PKM2 and mutant (K422R or H391Y) showed significantly aggressive cancer metabolism as compared with cells expressing either wild-type or mutant PKM2 independently. A similar trend was observed for oxidative endurance, tumorigenic potential, cellular proliferation, and tumor growth. These observations signify the dominant negative nature of mutations. Remarkably, PKM2-H391Y co-expressed cells showed a maximal effect on all the studied parameters. Such a dominant negative impaired function of PKM2 in tumor development is not known; this study demonstrates for the first time the possible predisposition of Bloom syndrome patients with impaired PKM2 activity to cancer and the importance of studying genetic variations in PKM2 in the future to understand their relevance in cancer in general.     

IGF1 regulates PKM2 function through Akt phosphorylation

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.     

Synthesis and antitumor activity of novel 2, 3-didithiocarbamate substituted naphthoquinones as inhibitors of pyruvate kinase M2 isoform

The M2 isoform of pyruvate kinase (PKM2) is a potential antitumor therapeutic target. In this study, we designed and synthesised a series of 2, 3-didithiocarbamate substituted naphthoquinones as PKM2 inhibitors based on the lead compound 3k that we previously reported. Among them, compound 3f (IC₅₀?=?1.05?±?0.17 µM) and 3h (IC₅₀?=?0.96?±?0.18 µM) exhibited potent inhibition of PKM2, and their inhibitory activities are superior to compound 3k (IC₅₀?=?2.95?±?0.53 µM) and the known PKM2 inhibitor shikonin (IC₅₀?=?8.82?±?2.62 µM). In addition, we evaluated in vitro antiproliferative effects of target compounds using MTS assay. Most target compounds exhibited dose-dependent cytotoxicity with IC₅₀ values in nanomolar concentrations against HCT116, MCF7, Hela, H1299 and B16 cells. These small molecule PKM2 inhibitors not only provide candidate compounds for cancer therapy, but also offer a tool to probe the biological effects of PKM2 inhibition on cancer cells.     

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.     

Inhibition of PKM2 Enhances Sensitivity of Olaparib to Ovarian Cancer Cells and Induces DNA Damage

Poly (ADP-ribose) polymerase inhibitors (PARPi) have showed clinical benefit as maintenance therapy in advanced ovarian cancer by impairing the homologous recombination (HR) pathway. Pyruvate kinase M2 (PKM2), the significant cancer metabolic biomarker, integrates with DNA damage to directly promote HR. We aimed to investigate the role and molecular mechanism of PKM2 downregulation on sensitization of ovarian cancer cells to PARPi. Inhibitory effects in vitro were assessed by cell viability, clone formation, transwell assay, and flow cytometry. Downregulation of PKM2 by siRNA or small molecular inhibitor shikonin (Sk) enhanced anti-tumour activity of olaparib (Ola) in ovarian cancer cells. Silencing PKM2 or Sk synergized with Ola and reduced cell growth, colony formation and migration, and induced apoptosis. Western blot and immunofluorescence demonstrated that inhibition of PKM2 amplified Ola-induced γH2AX and phospho-ATM (p-ATM) activation and interfered with BRCA1 accumulation in the nucleus. A xenograft animal model demonstrated in vivo antitumor combination effect of Sk and Ola. Furthermore, Western blot and immunofluorenscent analyses of tissue samples revealed that treatment of Sk increased DNA damage, reduced expression of BRCA1 and PKM2. Therefore, this study identified that PKM2 downregulation is a novel therapeutic strategy to enhance Ola effectiveness in treating ovarian cancer.