Pyruvate Kinase M2 Overexpression Research Articles

Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation

The endogenous mitochondrial quality control (MQC) system serves to protect mitochondria against cellular stressors. Although mitochondrial dysfunction contributes to cardiac damage during many pathological conditions, the regulatory signals influencing MQC disruption during septic cardiomyopathy (SC) remain unclear. This study aimed to investigate the involvement of pyruvate kinase M2 (PKM2) and prohibitin 2 (PHB2) interaction followed by MQC impairment in the pathogenesis of SC. We utilized LPS-induced SC models in PKM2 transgenic (PKM2TG) mice, PHB2S91D-knockin mice, and PKM2-overexpressing HL-1 cardiomyocytes. After LPS-induced SC, cardiac PKM2 expression was significantly downregulated in wild-type mice, whereas PKM2 overexpression in vivo sustained heart function, suppressed myocardial inflammation, and attenuated cardiomyocyte death. PKM2 overexpression relieved sepsis-related mitochondrial damage via MQC normalization, evidenced by balanced mitochondrial fission/fusion, activated mitophagy, restored mitochondrial biogenesis, and inhibited mitochondrial unfolded protein response. Docking simulations, co-IP, and domain deletion mutant protein transfection experiments showed that PKM2 phosphorylates PHB2 at Ser91, preventing LPS-mediated PHB2 degradation. Additionally, the A domain of PKM2 and the PHB domain of PHB2 are required for PKM2-PHB2 binding and PHB2 phosphorylation. After LPS exposure, expression of a phosphorylation-defective PHB2S91A mutant negated the protective effects of PKM2 overexpression. Moreover, knockin mice expressing a phosphorylation-mimetic PHB2S91D mutant showed improved heart function, reduced inflammation, and preserved mitochondrial function following sepsis induction. Abundant PKM2 expression is a prerequisite to sustain PKM2-PHB2 interaction which is a key element for preservation of PHB2 phosphorylation and MQC, presenting novel interventive targets for the treatment of septic cardiomyopathy.

Apoptin Inhibits Glycolysis and Regulates Autophagy by Targeting Pyruvate Kinase M2 (PKM2) in Lung Cancer A549 Cells.

Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis and plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, can stably express apoptin in tumor cells and selectively causes cell death in tumor cells. The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and the energy metabolism of tumor cells has not been clarified. In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement in apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62, and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors' tissue. As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway. This study confirmed the necessary role of Ad-apoptin in the energy metabolism of A549 cells.

Hypoxia-Challenged Pancreatic Adenocarcinoma Cell-Derived Exosomal circR3HCC1L Drives Tumor Growth Via Upregulating PKM2 Through Sequestering miR-873-5p.

Pancreatic adenocarcinoma (PAAD) is a fatal disease with poor survival. Increasing evidence show that hypoxia-induced exosomes are associated with cancer progression. Here, we aimed to investigate the function of hsa_circ_0007678 (circR3HCC1L) and hypoxic PAAD cell-derived exosomal circR3HCC1L in PAAD progression. Through the exoRBase 2.0 database, we screened for a circular RNA circR3HCC1L related to PAAD. Changes of circR3HCC1L in PAAD samples and cells were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, migration, invasion were analyzed by colony formation, cell counting, and transwell assays. Measurements of glucose uptake and lactate production were done using corresponding kits. Several protein levels were detected by western blotting. The regulation mechanism of circR3HCC1L was verified by dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. Exosomes were separated by differential ultracentrifugation. Animal experiments were used to verify the function of hypoxia-derived exosomal circR3HCC1L. CircR3HCC1L was upregulated in PAAD samples and hypoxic PAAD cells. Knockdown of circR3HCC1L decreased hypoxia-driven PAAD cell proliferation, migration, invasion, and glycolysis. Hypoxic PAAD cell-derived exosomes had higher levels of circR3HCC1L, hypoxic PAAD cell-derived exosomal circR3HCC1L promoted normoxic cancer cell malignant transformation and glycolysis in vitro and xenograft tumor growth in mouse models in vivo. Mechanistically, circR3HCC1L regulated pyruvate kinase M2 (PKM2) expression via sponging miR-873-5p. Also, PKM2 overexpression or miR-873-5p silencing offset circR3HCC1L knockdown-mediated effects on hypoxia-challenged PAAD cell malignant transformation and glycolysis. Hypoxic PAAD cell-derived exosomal circR3HCC1L facilitated PAAD progression through the miR-873-5p/PKM2 axis, highlighting the contribution of hypoxic PAAD cell-derived exosomal circR3HCC1L in PAAD.

Apatinib weakens proliferation, migration, invasion, and angiogenesis of thyroid cancer cells through downregulating pyruvate kinase M2

Thyroid cancer (TC) is the most frequent malignancy of the endocrine system. Apatinib, as an anti-angiogenic agent, has been applied in the therapy of several cancers. However, the function and mechanism of Apatinib in TC have not been clearly elucidated. After processing with Apatinib alone or combined PKM2 overexpression plasmids, cell proliferation, migration, and invasion were analyzed by EdU staining, CCK-8, wound healing, and Transwell. Meanwhile. HUVECs were incubated with the conditioned medium prepared from cell culture medium, and tube formation and VEGFR2 expression in HUVECs were examined using tube formation and immunofluorescence (IF) assays. Besides, we established a nude mouse xenograft model by lentivirus-mediated PKM2 shRNAs, and tested the growth of tumors; the pathological structure was analyzed with H&E staining. And the expressions of N-cadherin, Vimentin, E-cadherin, PKM2, VEGFA, VEGFR2, and Ki67 were determined by immunohistochemistry or Western blot. Apatinib could prominently suppress proliferation, migration, invasion, and HUVEC tube formation in SW579 and TPC-1 cells. Besides, we discovered that Apatinib had a significant inhibitory role on the expression of pyruvate kinase M2 (PKM2) in TC cells. And PKM2 overexpression also could notably reverse Apatinib-mediated inhibition of TC progression. Moreover, PKM2 shRNAs were applied to TC xenografts, resulting in significant reduction in tumor volume and suppression of angiogenesis-related protein expression. In summary, Apatinib has a regulatory role in TC progression, and Apatinib can block cancer cell angiogenesis by downregulating PKM2. This will provide a theoretical basis for therapy of TC.

Endothelial Cell-Derived Lactate Triggers Bone Mesenchymal Stem Cell Histone Lactylation to Attenuate Osteoporosis.

Blood vessels play a role in osteogenesis and osteoporosis; however, the role of vascular metabolism in these processes remains unclear. The present study finds that ovariectomized mice exhibit reduced blood vessel density in the bone and reduced expression of the endothelial glycolytic regulator pyruvate kinase M2 (PKM2). Endothelial cell (EC)-specific deletion of Pkm2 impairs osteogenesis and worsens osteoporosis in mice. This is attributed to the impaired ability of bone mesenchymal stem cells (BMSCs) to differentiate into osteoblasts. Mechanistically, EC-specific deletion of Pkm2 reduces serum lactate levels secreted by ECs, which affect histone lactylation in BMSCs. Using joint CUT&Tag and RNA sequencing analyses, collagen type I alpha 2 chain (COL1A2), cartilage oligomeric matrix protein (COMP), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and transcription factor 7 like 2 (TCF7L2) as osteogenic genes regulated by histone H3K18la lactylation are identified. PKM2 overexpression in ECs, lactate addition, and exercise restore the phenotype of endothelial PKM2-deficient mice. Furthermore, serum metabolomics indicate that patients with osteoporosis have relatively low lactate levels. Additionally, histone lactylation and related osteogenic genes of BMSCs are downregulated in patients with osteoporosis. In conclusion, glycolysis in ECs fuels BMSC differentiation into osteoblasts through histone lactylation, and exercise partially ameliorates osteoporosis by increasing serum lactate levels.

PKM2 deficiency exacerbates gram-negative sepsis-induced cardiomyopathy via disrupting cardiac calcium homeostasis

Sepsis is a life-threatening syndrome with multi-organ dysfunction in critical care medicine. With the occurrence of sepsis-induced cardiomyopathy (SIC), characterized by reduced ventricular contractility, the mortality of sepsis is boosted to 70–90%. Pyruvate kinase M2 (PKM2) functions in a variety of biological processes and diseases other than glycolysis, and has been documented as a cardioprotective factor in several heart diseases. It is currently unknown whether PKM2 influences the development of SIC. Here, we found that PKM2 was upregulated in cardiomyocytes treated with LPS both in vitro and in vivo. Pkm2 inhibition exacerbated the LPS-induced cardiac damage to neonatal rat cardiomyocytes (NRCMs). Furthermore, cardiomyocytes lacking PKM2 aggravated LPS-induced cardiomyopathy, including myocardial damage and impaired contractility, whereas PKM2 overexpression and activation mitigated SIC. Mechanism investigation revealed that PKM2 interacted with sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a), a key regulator of the excitation-contraction coupling, to maintain calcium homeostasis, and PKM2 deficiency exacerbated LPS-induced cardiac systolic dysfunction by impairing SERCA2a expression. In conclusion, these findings highlight that PKM2 plays an essential role in gram-negative sepsis-induced cardiomyopathy, which provides an attractive target for the prevention and treatment of septic cardiomyopathy.

Regeneration of glomerular metabolism and function by podocyte pyruvate kinase M2 in diabetic nephropathy.

Diabetic nephropathy (DN) arises from systemic and local changes in glucose metabolism and hemodynamics. We have reported that many glycolytic and mitochondrial enzymes, such as pyruvate kinase M2 (PKM2), were elevated in renal glomeruli of DN-protected patients with type 1 and type 2 diabetes. Here, mice with PKM2 overexpression specifically in podocytes (PPKM2Tg) were generated to uncover the renal protective function of PPKM2Tg as a potential therapeutic target that prevented elevated albumin/creatinine ratio (ACR), mesangial expansion, basement membrane thickness, and podocyte foot process effacement after 7 months of streptozotocin-induced (STZ-induced) diabetes. Furthermore, diabetes-induced impairments of glycolytic rate and mitochondrial function were normalized in diabetic PPKM2Tg glomeruli, in concordance with elevated Ppargc1a and Vegf expressions. Restored VEGF expression improved glomerular maximal mitochondrial function in diabetic PPKM2Tg and WT mice. Elevated VEGF levels were observed in the glomeruli of DN-protected patients with chronic type 1 diabetes and clinically correlated with estimated glomerular filtration (GFR) — but not glycemic control. Mechanistically, the preservations of mitochondrial function and VEGF expression were dependent on tetrameric structure and enzymatic activities of PKM2 in podocytes. These findings demonstrate that PKM2 structure and enzymatic activation in podocytes can preserve the entire glomerular mitochondrial function against toxicity of hyperglycemia via paracrine factors such as VEGF and prevent DN progression.

Combination Treatment Using Pyruvate Kinase M2 Inhibitors for the Sensitization of High Density Triple-negative Breast Cancer Cells.

Few studies have examined the correlation between pyruvate kinase M2 (PKM2) overexpression and triple-negative breast cancer (TNBC). TNBC is considered incurable with the currently available treatments, highlighting the need for alternative therapeutic targets. PKM2 expression was examined immunohistochemically in human breast tumor samples. Furthermore, we studied the effect of three PKM2 inhibitors (gliotoxin, shikonin, and compound 3K) in the MDA-MB-231 TNBC cell line. PKM2 overexpression correlates with TNBC. Interestingly, most TNBC tissues showed increased levels of PKM2 compared to those of receptor-positive breast cancer tissues. This suggests that PKM2 overexpression is an important factor in the development of TNBC. MDA-MB-231 TNBC cells are resistant to anticancer drugs, such as vincristine (VIC) compared to other cancer cells. We found that the recently developed PKM2 inhibitor gliotoxin sensitized MDA-MB-231 cells at a relatively low dose to the same extent as the known PKM2 inhibitor shikonin, suggesting that PKM2 inhibitors could be an effective treatment for TNBC. Detailed sensitization mechanisms were also analyzed. Both gliotoxin and shikonin highly increased late apoptosis in MDA-MB-231 cells, as revealed by annexin V staining. However, MDA-MB-231 cells with high cellular density inhibited the sensitizing effect of PKM2 inhibitors; therefore, we investigated ways to overcome this inhibitory effect. We found that gliotoxin+shikonin co-treatment highly increased toxicity in MDA-MB-231 cells with high density, whereas either VIC+gliotoxin or VIC+shikonin were not effective. Thus, combination therapy with various PKM2 inhibitors may be more effective than combination therapy with anticancer drugs. Gliotoxin+shikonin co-treatment did not increase S or G2 arrest in cells, suggesting that the co-treatment showed a high increase in apoptosis without S or G2 arrest. We confirmed that another recently developed PKM2 inhibitor compound 3K had similar mechanisms of sensitizing MDA-MB-231 cells, suggesting that PKM2 inhibitors have similar sensitization mechanisms in TNBC. PKM2 is a regulator of the oncogenic function of TNBC, and combination therapy with various PKM2 inhibitors may be effective for high-density TNBC. Targeting PKM2 in TNBC lays the foundation for the development of PKM2 inhibitors as promising anti-TNBC agents.

PKM2 Is Overexpressed in Glioma Tissues, and Its Inhibition Highly Increases Late Apoptosis in U87MG Cells With Low-density Specificity.

Pyruvate kinase M2 (PKM2) functions as an important rate-limiting enzyme in aerobic glycolysis and is involved in tumor initiation and progression. However, there are few studies on the correlation between PKM2 expression and its role in glioma. PKM2 expression was immunohistochemically examined in human brain tumor samples. Furthermore, we studied the effects of two PKM2 inhibitors (shikonin and compound 3K) on the U87MG glioma cell line. PKM2 was overexpressed in most glioma tissues when compared to controls. Interestingly, glioma-adjacent tissues from showed slight PKM2 overexpression. This suggests that PKM2 overexpression maybe an important trigger factor for glioma tumorigenesis. We found that the PKM2 inhibitor shikonin was effective against U87MG cells at a relatively low dose and was largely dependent on low cellular density compared to the effects of the anticancer drug vincristine. Shikonin highly increased late-apoptosis of U87MG cells. We also demonstrated that autophagy was involved in the increase in late-apoptosis levels caused by shikonin. Although vincristine treatment led to a high level of G2-phase arrest in U87MG cells, shikonin did not increase G2 arrest. Co-treatment with two PKM2 inhibitors, shikonin and compound 3K, increased the inhibitory effects. Combination therapy with PKM2 inhibitors together might be more effective than combination therapy with anticancer drugs. Our findings encourage the application of PKM2-targeting in gliomas, and lay the foundation for the development of PKM2 inhibitors as promising antitumor agents for glioma.

Targeting PKM2 promotes chemosensitivity of breast cancer cells in vitro and in vivo.

Pyruvate kinase M2 (PKM2) was overexpressed in many cancers, and high PKM2 expression was related with poor prognosis and chemoresistance. We investigated the expression of PKM2 in breast cancer and analyzed the relation of PKM2 expression with chemotherapy resistance to the neoadjuvant chemotherapy (NAC). We also investigated whether PKM2 could reverse chemoresistance in breast cancer cells in vitro and in vivo. Immunohistochemistry (IHC) was performed in 130 surgical resected breast cancer tissues. 78 core needle biopsies were collected from breast cancer patients before neoadjuvant chemotherapy. The relation of PKM2 expression and multi-drug resistance to NAC was compared. The effect of PKM2 silencing or overexpression on Doxorubicin (DOX) sensitivity in the MCF-7 cells in vitro and in vivo was compared. PKM2 was intensively expressed in breast cancer tissues compared to adjacent normal tissues. In addition, high expression of PKM2 was associated with poor prognosis in breast cancer patients. The NAC patients with high PKM2 expression had short survival. PKM2 was an independent prognostic predictor for surgical resected breast cancer and NAC patients. High PKM2 expression was correlated with neoadjuvant treatment resistance. High PKM2 expression significantly distinguished chemoresistant patients from chemosensitive patients. In vitro and in vivo knockdown of PKM2 expression decreases the resistance to DOX in breast cancer cells in vitro and tumors in vivo. PKM2 expression was associated with chemoresistance of breast cancers, and could be used to predict the chemosensitivity. Furthermore, targeting PKM2 could reverse chemoresistance, which provides an effective treatment methods for patients with breast cancer.

307-OR: Activation of Mitochondrial Activities by Pyruvate Kinase M2 (PKM2) in Podocytes Improved Glomerular Metabolism and Diabetic Nephropathy

Diabetic nephropathy (DN) is the result of systemic and local changes in metabolism and hemodynamics. We have found that the elevation of glycolytic enzymes, such as pyruvate kinase M2 (PKM2) in the renal glomeruli is correlated with preservation of renal function in human type 1 and type 2 diabetes. A small-molecule PKM2 activator, TEPP46, prevented or reversed glomerular pathology in diabetic mice of chronic duration. Mice with PKM2 overexpression specifically in podocytes (PPKM2Tg) were generated, which after 7 months of diabetes induced by streptozotocin, exhibited significantly lower albumin-creatinine ratio (ACR), kidney weight-body weight ratio, and glomeruli pathology such as reduced glomeruli size, mesangial expansion, and basement membrane thickness compared to diabetic WT mice. Furthermore, the glomeruli of diabetic PPKM2Tg mice exhibited lower expressions of fibrotic (FN, TGFβ1 and Col4a) and oxidative stress (p47phox, NOX2 and NOX4) genes compared to WT diabetic mice. Interestingly, the decreased expression of endothelial-trophic genes and mitochondrial-related genes were improved in the whole glomeruli of diabetic PPKM2Tg mice. Oxygen consumption rate (OCR) of glomeruli as measured by Seahorse showed significant improvement in diabetic PPKM2Tg mice versus WT mice (p < 0.05). In vitro studies showed that the activation of PKM2 in mouse podocyte cell line by TEPP-46 decreased high glucose-induced ROS production, improved mitochondrial membrane potential (MMP) by increased the level of mitochondrial OCR and enhanced glycolytic rate. Furthermore, PKM-knockdown stable podocyte cell line, mimicking diabetic conditions, showed dramatical decline of OCR and glycolytic rate. These results showed that PKM2 specific overexpression in podocytes can significantly reduce diabetes-induced mitochondrial abnormalities and oxidative stress in the whole glomeruli, and delay the progression of diabetic nephropathy. Disclosure J. Fu: None. T. Shinjo: None. R. St-Louis: None. Q. Li: None. K. Park: None. H. Yokomizo: None. Y. Zaitsu: Employee; Self; Sunstar Group. M. Yu: None. I. Wu: None. H. Shah: None. G.L. King: Research Support; Self; Janssen Pharmaceuticals, Inc.

OSU-A9 induced-reactive oxygen species cause cytotoxicity in duodenal and gastric cancer cells by decreasing phosphorylated nuclear pyruvate kinase M2 protein levels

Pyruvate kinase M2 (PKM2) is a key enzyme responsible for the final step of glycolysis. It is still unclear whether PKM2 is involved in reactive oxygen species (ROS)-mediated cytotoxicity in gastrointestinal cancer, and what mechanisms are involved. One duodenal (AZ521) and two gastric (NUGC and SCM-1) cancer cell lines were treated with an indole-3-carbinol derivative OSU-A9, which caused cytotoxicity in acute myeloid leukemia through ROS generation. OSU-A9 caused a dose- and time-dependent cytotoxicity and induced apoptosis in duodenal and gastric cancer cells through ROS generation. Pretreatment with ROS scavengers rescued cancer cells from apoptosis and concomitant poly (ADP-ribose) polymerase cleavage, implying a key role of ROS in OSU-A9-induced cell death. Moreover, OSU-A9-induced ROS generation decreased protein levels of pTyr105-PKM2, and this effect was rescued by pretreatment with ROS scavengers. Interestingly, pTyr105-PKM2 protein levels decreased in the cell nucleus rather than in the cytoplasm. PKM2 overexpression partially rescued the survival of duodenal and gastric cancer cells treated with OSU-A9. Furthermore, the anticancer activity of OSU-A9 extended in vivo, as OSU-A9 administered by oral gavage suppressed the growth of AZ521 xenograft tumors in nude mice without obvious toxicity. In conclusion, OSU-A9 inhibited duodenal and gastric cancer cell proliferation through ROS generation and caused a subsequent decrease in nuclear pTyr105-PKM2 protein. These findings provide evidence for the non-canonical activity of PKM2 in cancer cell survival. Furthermore, they highlight the potential role of PKM2 as a future therapeutic target for duodenal and gastric cancer.

231-OR: ADA Presidents' Select Abstract: Pyruvate Kinase M2 Overexpression in Podocytes Ameliorates Nephropathy in Diabetic Mice

Elevated expression of pyruvate kinase isoform M2 (PKM2) in the renal glomeruli of people with diabetes is associated with protection from nephropathy even with hyperglycemia. Furthermore, systemic use of PKM2 activators (TEPP46) can prevent or reverse glomerular pathology in diabetic mice. To determine specifically the role of PKM2 in the development or progression of diabetic glomerulopathy, we generated transgenic mice overexpressing PKM2 specific targeted to the podocytes (PPKM2Tg mice) driven by the podocin promoter. Analysis of the glomeruli from these mice showed 1.5-fold increases in mRNA and protein levels without changes in PKM1, compared to wild type (WT) littermates (p<0.05). Pyruvate kinase (PK) activity in the glomeruli of the PPKM2Tg mice was increased by 40% compared to those in WT mice (p<0.05). Diabetes was induced in eight-week-old WT and PPKM2Tg mice by streptozotocin (STZ) and maintained with sustained glucose levels >400mg/dL for 7 months. Diabetic PPKM2Tg mice showed significantly lower urinary albumin-creatinine ratio (ACR) and kidney weight-body weight ratios (p<0.05) compared to diabetic WT mice even with similar hyperglycemia. PK activity was lowered in the glomeruli of diabetic WT mice by 30%, whereas levels in diabetic PPKM2Tg mice were comparable to nondiabetic WT mice. For renal pathology, diabetic PPKM2Tg mice exhibited smaller glomeruli compared to diabetic WT mice. Similarly, expressions of inflammatory genes including TNFα and MCP-1, and fibrotic genes including fibronectin and TGF-β1 were downregulated in the glomeruli of diabetic PPKM2Tg mice compared to diabetic WT mice. In contrast, mitochondrial genes such as PGC-1α, Opa1, and cytochrome b in the glomeruli of diabetic PPKM2Tg mice were significantly upregulated as compared to diabetic WT mice. These findings strongly suggest that upregulation or activation of PKM2 and glycolysis may improve mitochondrial function in podocytes and prevent the progression of diabetic nephropathy. Disclosure T. Shinjo: None. R. St-Louis: None. J. Fu: None. K. Park: None. Q. Li: None. H. Yokomizo: None. M. Yu: None. H. Wang: None. A. Kannt: Employee; Self; Sanofi. T. Sadowski: Employee; Self; Sanofi-Aventis Deutschland GmbH. G.L. King: Research Support; Self; Sanofi. Funding National Institutes of Health; Sanofi

Antitumor activity of SR splicing-factor 5 knockdown by downregulating pyruvate kinase M2 in non-small cell lung cancer cells.

SR splicing-factors (SRSFs) play a vital role in carcinogenesis. SRSF5 was demonstrated to be upregulated in lung cancer and identified as a novel prognostic indicator for small-cell lung cancer. However, the role of SRSF5 in the pathogenesis of non-small cell lung cancer (NSCLC) and the molecular mechanism involved are still undefined. The expression of SRSF5 in NSCLC cells was detected by quantitative real-time polymerase chain reaction and Western blot analysis. The proliferation of cells was evaluated by cell counting kit-8 and BrdU assays. Apoptosis was assessed by flow cytometry and Western blot analysis of apoptosis-associated proteins including B-cell lymphoma 2 (Bcl-2), Bax, and cytochrome C (Cyt C). Glycolysis was detected by determining glucose consumption, lactate production, and pyruvate kinase M2 (PKM2) expression. We found that SRSF5 messenger RNA and protein levels were elevated in NSCLC cells. SRSF5 knockdown inhibited the proliferation and Ki67 expression in NSCLC cells. SRSF5 silencing increased the apoptotic rate, upregulated Bax and Cyt C, and decreased Bcl-2 level in NSCLC cells. Moreover, Knockdown of SRSF5 repressed glycolysis in NSCLC cells via reducing PKM2 expression. Enhanced glycolysis by PKM2 overexpression attenuated the effects of SRSF5 silencing on NSCLC cell proliferation and apoptosis. Overall, knockdown of SRSF5 inhibited proliferative ability and induced apoptosis by suppressing PKM2 expression in NSCLC cells.

Curcumin decreases Warburg effect in cancer cells by down-regulating pyruvate kinase M2 via mTOR-HIF1\u03b1 inhibition

Warburg effect is an emerging hallmark of cancer cells with pyruvate kinase M2 (PKM2) as its key regulator. Curcumin is an extensively-studied anti-cancer compound, however, its role in affecting cancer metabolism remains poorly understood. Herein, we show that curcumin inhibits glucose uptake and lactate production (Warburg effect) in a variety of cancer cell lines by down-regulating PKM2 expression, via inhibition of mTOR-HIF1α axis. Stable PKM2 silencing revealed that PKM2 is required for Warburg effect and proliferation of cancer cells. PKM2 over-expression abrogated the effects of curcumin, demonstrating that inhibition of Warburg effect by curcumin is PKM2-mediated. High PKM2 expression correlated strongly with poor overall survival in cancer, suggesting the requirement of PKM2 in cancer progression. The study unravels novel PKM2-mediated inhibitory effect of curcumin on metabolic capacities of cancer cells. To the best of our knowledge, this is the first study linking curcumin with PKM2-driven cancer glycolysis, thus, providing new perspectives into the mechanism of its anticancer activity.

A serine protease extracted from Trichosanthes kirilowii inhibits epithelial-mesenchymal transition via antagonizing PKM2-mediated STAT3/Snail1 pathway in human colorectal adenocarcinoma cells

Trichosanthes kirilowii extract possessed various medicinal functions. Recently, it was reported that a serine protease TKP isolated from T. kirilowii inhibits human colorectal adenocarcinoma cell proliferation through inducing apoptosis. In the present study, we studied the influence of TKP on epithelial-mesenchymal transition (EMT) of human colorectal adenocarcinoma cells. The results showed that TKP suppressed EMT of both DLD1 and HCT116 cells, which was indicated by the alteration of morphology and EMT markers including E-cadherin, N-cadherin and vimentin. Consistently, cell adhesion ability was significantly declined after TKP treatment. Mechanistically, TKP treatment inhibited signal transducer and activator of transcription 3 (STAT3)/Snail1 signaling, pyruvate kinase M2 (PKM2) expression and its nuclear translocation. However, PKM2 overexpression could partially reverse the anti-EMT effect of TKP. Taken together, these results indicated that TKP inhibited EMT program of human colorectal adenocarcinoma cells via antagonizing PKM2-mediated STAT3/Snail1 pathway.

Upregulation of pyruvate kinase M2 expression by fatty acid synthase contributes to gemcitabine resistance in pancreatic cancer.

Pancreatic cancer has one of the highest mortality rates of all cancer types. Fatty acid synthase (FASN) is a multifunctional protein homodimer that can convert acetyl coenzyme A (CoA) and malonyl-CoA into palmitate, thus regulating lipogenesis. FASN overexpression has also been shown to cause resistance to gemcitabine, a chemotherapy treatment for pancreatic cancer; however, the mechanism by which this happens is unclear. Analysis of gene expression of FASN and pyruvate kinase M2 (PKM2) in pancreatic cancer was performed using Oncomine microarray gene expression datasets, which demonstrated that FASN and PKM2 were upregulated in pancreatic cancer compared with normal tissue. Specifically, it was demonstrated that FASN enabled the upregulation of PKM2 expression at the mRNA and protein levels, increasing the glucose consumption rate in pancreatic cancer cells. The present study also revealed that decreased levels of FASN reduced resistance to gemcitabine treatment, which was induced by PKM2 overexpression in pancreatic ductal adenocarcinoma cells. Therefore, FASN may represent a novel therapeutic target in pancreatic cancer.

HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

BackgroundHeat shock protein 90 (HSP90) functions as a well-known onco-protein to regulate protein conformation, stability and degradation. Pyruvate kinase M2 (PKM2), a critical regulator of the metabolism, growth and metastasis of cancer cells, has been confirmed to be overexpressed in various human cancer including hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying the oncogenic functions of HSP90 and PKM2 overexpression in HCC remain unknown.MethodsThe expression of HSP90 and PKM2 in HCC specimens and cells were detected by immunoblotting and immunostaining. The interaction between HSP90 and PKM2 was confirmed by tandem affinity purification, co-immunoprecipitation and Glutathione S transferase (GST)-pulldown assay.ResultsIn this study, we found that HSP90 could bind to PKM2 and subsequently increased PKM2 abundance in HCC cells. Immunohistochemistry (IHC) staining showed that HSP90 level was positively correlated with PKM2 level in HCC tissues. Mechanistically, HSP90 was found to increase the phosphorylation of PKM2 at Thr-328. Protein kinase glycogen synthase kinase-3β (GSK-3β) formed a protein complex with HSP90 and PKM2, and directly mediated Thr-328 phosphorylation of PKM2 induced by HSP90. Thr-328 phosphorylation was critical for maintaining PKM2 stability and its biological functions in regulating glycolysis, mitochondria respiration, proliferation and apoptosis. Functionally, we found that HSP90 promoted the glycolysis and proliferation and inhibited apoptosis of HCC cells in a PKM2 dependent manner. In vivo experiments disclosed that PKM2 was required for the promoting effects of HSP90 on the growth of HCC cells in mice. Furthermore, we demonstrated that positive expression of HSP90 and PKM2 was correlated with poor clinicopathological features including high alpha fetoprotein (AFP) level, large tumor size, portal vein tumor thrombus (PVTT) and advanced tumor-node-metastasis (TNM) stage. Furthermore, we demonstrated that positive expression of HSP90 and PKM2, and a combination of these proteins could strongly predict the poor prognosis of HCC patients.ConclusionsWe suggest that HSP90 potentiates the glycolysis and proliferation, reduces the apoptosis and thus enhances the growth of HCC cells through PKM2.

Clinicopathological and prognostic significance of PKM2 protein expression in cirrhotic hepatocellular carcinoma and non-cirrhotic hepatocellular carcinoma

Pyruvate kinase M2 (PKM2), a key protein in glucose and lipid metabolism, has been reported to be related to carcinogenesis in various malignancies. However, its roles in hepatocellular carcinoma with cirrhotic liver (CL) and hepatocellular carcinoma with non-cirrhoticliver (NCL) haves not been investigated. In our study western bloting, qRT-PCR and immunohistochemistry were performed to evaluate the clinical significance of PKM2 protein expression in CL and NCL. The results revealed that PKM2 protein expression was significantly higher in HCC tissues than in their adjacent non-tumour tissues. The high expression rates of PKM2 were more frequently noted in CL (45. 6%) than in NCL (31. 9%) tissues. High PKM2 expression in CL and NCL tissues was significantly associated with vascular invasion (P = 0.002 and P = 0.004, respectively) and intrahepatic metastasis (P < 0.001 and P = 0.019, respectively). Importantly, Kaplan–Meier survival analysis showed that the disease-specific survival (DSS) and recurrence-free survival (RFS) were lower in CL with high PKM2 expression than in NCL with high PKM2 expression (P = 0.003 and P = 0.003, respectively). Overall, high PKM2 expression was more frequently found in CL than in NCL, and PKM2 overexpression was associated with poor survival rates in patients with CL and NCL.

Pyruvate kinase M2 deregulation enhances the metastatic potential of tongue squamous cell carcinoma.

Pyruvate kinase M2 (PKM2) has been verified to correlate with the prognosis of many types of cancer. However, its role in the development and metastasis of tongue squamous cell carcinoma (TSCC) remains unclear. The immunohistochemistry (IHC) results confirmed that PKM2 is overexpressed in patients with TSCC. PKM2 up-regulation was related to lymph node metastasis and associated with reduced overall survival. According to the microarray analysis and Western blots, PKM2 expression was up-regulated in TSCC cells with enhanced metastatic potential. PKM2 knockdown inhibited cell migration and invasion, reduced SOD2 (manganese superoxide dismutase) activity and the intracellular H2O2 level, and inhibited tumour growth and lung metastasis in vivo. PKM2 overexpression promoted cell migration and invasion, and increased SOD2 activity and the intracellular H2O2 level. Moreover, miR-138 directly targeted PKM2 and inhibited PKM2 expression. Thus, PKM2 deregulation plays an important role in TSCC and may serve as a biomarker of metastatic potential or as a therapeutic target in patients with TSCC. PKM2, a miR-138 target gene, enhances the metastatic potential of TSCC through the SOD2-H2O2 pathway.