CircPHGDH downregulation decreases papillary thyroid cancer progression through miR-122-5p/PKM2 axis.

BACKGROUND: Although papillary thyroid carcinoma (PTC) has a favorable prognosis, it could affect patient life quality and become a serious threat because of invasion and metastasis. Many investigations have suggested that circular RNAs (circRNAs) are involved in different cancer regulations. Nevertheless, circRNAs role in invasive PTC remains unclear. METHODS: In the present investigation, next-generation sequencing was applied to explore abnormal circRNA expression. The expression of circRNA phosphoglycerate dehydrogenase (circPHGDH) in PTC cell lines and tissues were examined. Then, we investigated regulatory mechanism and circPHGDH downstream targets using bioinformatics analysis and luciferase reporting analysis. Then transwell migration, Cell Counting Kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used for cells migration and proliferation analysis. In vivo metastasis and tumorigenesis assays were also employed to evaluate the circPHGDH role in PTC. RESULTS: The data showcased that circPHGDH expression increased in both PTC cell lines and tissues, which suggested that circPHGDH functions in PTC progression. circPHGDH downregulation suppressed PTC invasion and proliferation in both in vivo and in vitro experiments. Bioinformatics and luciferase reporter results confirmed that both microRNA (miR)-122-5p and pyruvate kinase M2 subtype (PKM2) were downstream targets of circPHGDH. PKM2 overexpression or miR-122-5p suppression reversed PTC cell invasion and proliferation post silencing circPHGDH by restoring aerobic glycolysis. CONCLUSION: Taken together, our research found that circPHGDH downregulation reduced PTC progression via miR-122-5p/PKM2 axis regulation mediated by aerobic glycolysis.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

Ubiquitinome Analysis Uncovers Alterations in Synaptic Proteins and Glucose Metabolism Enzymes in the Hippocampi of Adolescent Mice Following Cold Exposure.

Cold exposure exerts negative effects on hippocampal nerve development in adolescent mice, but the underlying mechanisms are not fully understood. Given that ubiquitination is essential for neurodevelopmental processes, we attempted to investigate the effects of cold exposure on the hippocampus from the perspective of ubiquitination. By conducting a ubiquitinome analysis, we found that cold exposure caused changes in the ubiquitination levels of a variety of synaptic-associated proteins. We validated changes in postsynaptic density-95 (PSD-95) ubiquitination levels by immunoprecipitation, revealing reductions in both the K48 and K63 polyubiquitination levels of PSD-95. Golgi staining further demonstrated that cold exposure decreased the dendritic-spine density in the CA1 and CA3 regions of the hippocampus. Additionally, bioinformatics analysis revealed that differentially ubiquitinated proteins were enriched in the glycolytic, hypoxia-inducible factor-1 (HIF-1), and 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathways. Protein expression analysis confirmed that cold exposure activated the mammalian target of rapamycin (mTOR)/HIF-1α pathway. We also observed suppression of pyruvate kinase M2 (PKM2) protein levels and the pyruvate kinase (PK) activity induced by cold exposure. Regarding oxidative phosphorylation, a dramatic decrease in mitochondrial respiratory-complex I activity was observed, along with reduced gene expression of the key subunits NADH: ubiquinone oxidoreductase core subunit V1 (Ndufv1) and Ndufv2. In summary, cold exposure negatively affects hippocampal neurodevelopment and causes abnormalities in energy homeostasis within the hippocampus.

Search progress of pyruvate kinase M2 (PKM2) in organ fibrosis.

Fibrosis is characterized by abnormal deposition of the extracellular matrix (ECM), leading to organ structural remodeling and loss of function. The principal cellular effector in fibrosis is activated myofibroblasts, which serve as the main source of matrix proteins. Metabolic reprogramming, transitioning from mitochondrial oxidative phosphorylation to aerobic glycolysis, is widely observed in rapidly dividing cells such as tumor cells and activated myofibroblasts and is increasingly recognized as a fundamental pathogenic basis in organ fibrosis. Targeting metabolism represents a promising strategy to mitigate fibrosis. PKM2, a key enzyme in glycolysis, plays a pivotal role in metabolic reprogramming through allosteric regulation, impacting both metabolic and non-metabolic pathways. Therefore, metabolic reprogramming induced by PKM2 activation is involved in the occurrence and development of fibrosis in various organs. A comprehensive understanding of the role of PKM2 in fibrotic diseases is crucial for seeking new anti-fibrotic therapeutic targets. In this context, we summarize PKM2's role in glycolysis, mediating the intricate mechanisms underlying fibrosis in multiple organs, and discuss the potential value of PKM2 inhibitors and allosteric activators in future clinical treatments, aiming to identify novel therapeutic targets for proliferative fibrotic diseases.

PKM2 promotes lymphatic metastasis of hypopharyngeal carcinoma via regulating epithelial-mesenchymal transition: an experimental research.

BACKGROUND: Patients with hypopharyngeal carcinoma (HPC) have a poor prognosis mainly because of lymphatic metastasis. This research aimed to determine the PKM2 role in lymphatic metastasis in HPC and the underlying molecular mechanism contributing to this phenomenon. METHODS: PKM2 in HPC was studied for its expression and its likelihood of overall survival using TCGA dataset. Western blotting, qRT-PCR, and IHC were employed to confirm PKM2 expression. Methods including gain- and loss-of-function were used to examine the PKM2 role in HPC metastasis in vitro and in vivo. In vitro and in vivo studies also confirmed lymphatic metastasis's mechanism. RESULTS: Prominent PKM2 overexpression was seen in patients with lymphatic metastasis of HPC, and there was an inherent relationship between a high PKM2 level and poor prognosis. In vitro research showed that knocking down PKM2 decreased tumor cell invasion, migration, and proliferation while promoting apoptosis and inhibiting epithelial-mesenchymal transition, but overexpressing PKM2 had the reverse effect. Animal studies suggested that PKM2 may facilitate tumor development and lymphatic metastasis. CONCLUSIONS: Our findings suggest that PKM2 may be a tumor's promoter gene of lymphatic metastasis, which may promote lymphatic metastasis of HPC by regulating epithelial-mesenchymal transition. PKM2 may be a biomarker of metastatic potential, ultimately providing a basis for exploring new therapeutic targets.

Sulfone-based human liver pyruvate kinase inhibitors - Design, synthesis and in vitro bioactivity.

Non-alcoholic fatty liver disease (NAFLD) is a prevalent pathological condition characterised by the accumulation of fat in the liver. Almost one-third of the global population is affected by NAFLD, making it a significant health concern. However, despite its prevalence, there is currently no approved drug specifically designed for the treatment of NAFLD. To address this critical gap, researchers have been investigating potential targets for NAFLD drug development. One promising candidate is the liver isoform of pyruvate kinase (PKL). In recent studies, Urolithin C, an allosteric inhibitor of PKL, has emerged as a potential lead compound for therapeutic intervention. Building upon this knowledge, our team has conducted a comprehensive structure-activity relationship of Urolithin C. In this work, we have employed a scaffold-hopping approach, modifying the urolithin structure by replacing the urolithin carbonyl with a sulfone moiety. Our structure-activity relationship analysis has identified the sulfone group as particularly favourable for potent PKL inhibition. Additionally, we have found that the presence of catechol moieties on the two aromatic rings further improves the inhibitory activity. The most promising inhibitor from this new series displayed nanomolar inhibition, boasting an IC50 value of 0.07 µM. This level of potency rivals that of urolithin D and significantly surpasses the effectiveness of urolithin C by an order of magnitude. To better understand the molecular interactions underlying this inhibition, we obtained the crystal structure of one of the inhibitors complexed with PKL. This structural insight served as a valuable reference point, aiding us in the design of inhibitors.

Mechanistic Investigation of Thiazole-Based Pyruvate Kinase M2 Inhibitor Causing Tumor Regression in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a deadly breast cancer with a poor prognosis. Pyruvate kinase M2 (PKM2), a key rate-limiting enzyme in glycolysis, is abnormally highly expressed in TNBC. Overexpressed PKM2 amplifies glucose uptake, enhances lactate production, and suppresses autophagy, thereby expediting the progression of oncogenic processes. A high mortality rate demands novel chemotherapeutic regimens at once. Herein, we report the rational development of an imidazopyridine-based thiazole derivative 7d as an anticancer agent inhibiting PKM2. Nanomolar range PKM2 inhibitors with favorable drug-like properties emerged through enzyme assays. Experiments on two-dimensional (2D)/three-dimensional (3D) cell cultures, lactate release assay, surface plasmon resonance (SPR), and quantitative real-time polymerase chain reaction (qRT-PCR) validated 7d preclinically. In vivo, 7d outperformed lapatinib in tumor regression. This investigation introduces a lead-based approach characterized by its clear-cut chemistry and robust efficacy in designing an exceptionally potent inhibitor targeting PKM2, with a focus on combating TNBC.

Diagnosis and management of pyruvate kinase deficiency: international expert guidelines.

Pyruvate kinase (PK) deficiency is the most common cause of chronic congenital non-spherocytic haemolytic anaemia worldwide, with an estimated prevalence of one in 100 000 to one in 300 000 people. PK deficiency results in chronic haemolytic anaemia, with wide ranging and serious consequences affecting health, quality of life, and mortality. The goal of the International Guidelines for the Diagnosis and Management of Pyruvate Kinase Deficiency was to develop evidence-based guidelines for the clinical care of patients with PK deficiency. These clinical guidelines were developed by use of GRADE methodology and the AGREE II framework. Experts were invited after consideration of area of expertise, scholarly contributions in PK deficiency, and country of practice for global representation. The expert panel included 29 expert physicians (including adult and paediatric haematologists and other subspecialists), geneticists, laboratory specialists, nurses, a guidelines methodologist, patients with PK deficiency, and caregivers from ten countries. Five key topic areas were identified, the panel prioritised key questions, and a systematic literature search was done to generate evidence summaries that were used in the development of draft recommendations. The expert panel then met in person to finalise and vote on recommendations according to a structured consensus procedure. Agreement of greater than or equal to 67% among the expert panel was required for inclusion of a recommendation in the final guideline. The expert panel agreed on 31 total recommendations across five key topics: diagnosis and genetics, monitoring and management of chronic complications, standard management of anaemia, targeted and advanced therapies, and special populations. These new guidelines should facilitate best practices and evidence-based PK deficiency care into clinical practice.

Tetramerization of pyruvate kinase M2 attenuates graft-versus-host disease by inhibition of Th1 and Th17 differentiation.

Lethal graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic stem-cell transplantation (Allo-HSCT). Pyruvate kinase M2 (PKM2) is essential for CD4+ T-cell differentiation. Using the well-characterized mouse models of Allo-HSCT, we explored the effects of TEPP-46-induced PKM2 tetramerization on GVHD and graft-versus-leukemia (GVL) activity. TEPP-46 administration significantly improved the survival rate of GVHD. The severity of GVHD and histopathological damage of GVHD-targeted organs were obviously alleviated by PKM2 tetramerization. Additionally, tetramerized PKM2 inhibited the activation of NF-κB pathway and decreased the inflammation level of GVHD mice. PKM2 tetramerization blocked Th1 and Th17 cell differentiation and secretion of pro-inflammatory cytokine (IFN-γ, TNF-α, and IL-17). Meanwhile, differentiation of Treg cells and IL-10 secretion were promoted by tetramerized PKM2. These findings demonstrated that PKM2 enhanced the augment of Th1 and Th17 cells to accelerate the progression of GVHD, and allosteric activation of PKM2 targeted Th1 and Th17 cells attenuated GVHD. Furthermore, we also confirmed that TEPP-46 administration did not compromise GVL activity and resulted in slightly improvement of leukemia-free survive. Thus, targeting Th1 and Th17 cell response with PKM2 allosteric activator may be a promising therapeutic strategy for GVHD prevention while preserving the GVL activity in patients receiving Allo-HSCT.

PKM2 diverts glycolytic flux in dependence on mitochondrial one-carbon cycle.

Modeling tumor metabolism in vitro remains challenging. Here, we used galactose as an in vitro tool compound to mimic glycolytic limitation. In contrast to the established idea that high glycolytic flux reduces pyruvate kinase isozyme M2 (PKM2) activity to support anabolic processes, we have discovered that glycolytic limitation also affects PKM2 activity. Surprisingly, despite limited carbon availability and energetic stress, cells induce a near-complete block of PKM2 to divert carbons toward serine metabolism. Simultaneously, TCA cycle flux is sustained, and oxygen consumption is increased, supported by glutamine. Glutamine not only supports TCA cycle flux but also serine synthesis via distinct mechanisms that are directed through PKM2 inhibition. Finally, deleting mitochondrial one-carbon (1C) cycle reversed the PKM2 block, suggesting a potential formate-dependent crosstalk that coordinates mitochondrial 1C flux and cytosolic glycolysis to support cell survival and proliferation during nutrient-scarce conditions.

PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation.

Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.

Identification of PKM2 as a pyroptosis-related key gene aggravates senile osteoporosis via the NLRP3/Caspase-1/GSDMD signaling pathway.

BACKGROUNDS: Senile osteoporosis-alternatively labeled as skeletal aging-encompasses age-induced bone deterioration and loss of bone microarchitecture. Recent studies have indicated a potential association between senile osteoporosis and chronic systemic inflammation, and pyroptosis in bone marrow-derived mesenchymal stem cells is speculated to contribute to bone loss and osteoporosis. Therefore, targeting pyroptosis in stem cells may be a potential therapeutic strategy for treating osteoporosis. METHODS: Initially, we conducted bioinformatics analysis to screen the GEO databases to identify the key gene associated with pyroptosis in senile osteoporosis. Next, we analyzed the relationship between altered proteins and clinical data. In vitro experiments were then performed to explore whether the downregulation of PKM2 expression could inhibit pyroptosis. Additionally, an aging-related mouse model of osteoporosis was established to validate the efficacy of a PKM2 inhibitor in alleviating osteoporosis progression. RESULTS: We identified PKM2 as a key gene implicated in pyroptosis in senile osteoporosis patients through bioinformatics analysis. Further analyses of bone marrow and stem cells demonstrated significant PKM2 overexpression in senile osteoporosis patients. Silencing PKM2 expression inhibited pyroptosis in senile stem cells, of which the osteogenesis potential and angiogenic function were also primarily promoted. Moreover, the results in vivo demonstrated that administering PKM2 inhibitors suppressed pyroptosis in senile osteoporosis mice and mitigated senile osteoporosis progression. CONCLUSION: Our study uncovered PKM2, a key pyroptosis marker of bone marrow mesenchymal stem cells in senile osteoporosis. Shikonin, a PKM2 inhibitor, was then identified as a potential drug candidate for the treatment of osteoporosis.

MEK1/2-PKM2 Pathway Modulates the Immunometabolic Reprogramming of Proinflammatory Allograft-infiltrating Macrophages During Heart Transplant Rejection.

BACKGROUND: Emerging evidence has highlighted the role of macrophages in heart transplant rejection (HTR). However, the molecular signals modulating the immunometabolic phenotype of allograft-infiltrating macrophages (AIMs) during HTR remain unknown. METHODS: We analyzed single-cell RNA sequencing data from cardiac graft-infiltrating immunocytes to characterize the activation patterns and metabolic features of AIMs. We used flow cytometry to determine iNOS and PKM2 expression and MEK/ERK signaling activation levels in AIMs. We then generated macrophage-specific Mek1/2 knockout mice to determine the role of the MEK1/2-PKM2 pathway in the proinflammatory phenotype and glycolytic capacity of AIMs during HTR. RESULTS: Single-cell RNA sequencing analysis showed that AIMs had a significantly elevated proinflammatory and glycolytic phenotype. Flow cytometry analysis verified that iNOS and PKM2 expressions were significantly upregulated in AIMs. Moreover, MEK/ERK signaling was activated in AIMs and positively correlated with proinflammatory and glycolytic signatures. Macrophage-specific Mek1/2 deletion significantly protected chronic cardiac allograft rejection and inhibited the proinflammatory phenotype and glycolytic capacity of AIMs. Mek1/2 ablation also reduced the proinflammatory phenotype and glycolytic capacity of lipopolysaccharides + interferon-γ-stimulated macrophages. Mek1/2 ablation impaired nuclear translocation and PKM2 expression in macrophages. PKM2 overexpression partially restored the proinflammatory phenotype and glycolytic capacity of Mek1/2 -deficient macrophages. Moreover, trametinib, an Food and Drug Administration-approved MEK1/2 inhibitor, ameliorated chronic cardiac allograft rejection. CONCLUSIONS: These findings suggest that the MEK1/2-PKM2 pathway is essential for immunometabolic reprogramming of proinflammatory AIMs, implying that it may be a promising therapeutic target in clinical heart transplantation.

An impact of l-histidine on the phosphorylation and stability of pyruvate kinase at low NaCl level.

As one of the key rate limiting enzymes in glycolysis process, the characteristics of pyruvate kinase (PK) play an important role in regulating the muscle quality. Given the close relationship between kinase phosphorylation level and its stability, the present study investigated the impact of exogenous l-histidine (l-his) on PK phosphorylation and activity at 1% NaCl level in the early postmortem. An incubation system was also constructed and the results showed that the introduction of 0.06% l-his caused the dephosphorylation and increased the activity of PK at 1% NaCl. Compared with 1% NaCl treatment, three differential phosphorylation sites were produced when l-his was introduced. The PK secondary structure was shift from order to disorder, leading to a distinct degradation. This present study provided us with inspiration that meat quality could be improved by exogenous l-his at early postmortem under low NaCl conditions.

Metformin Increases the Response of Cholangiocarcinoma Cells to Gemcitabine by Suppressing Pyruvate Kinase M2 to Activate Mitochondrial Apoptosis.

BACKGROUND: Cholangiocarcinoma (CCA) is a malignant tumor with a high mortality rate. Resistance to chemotherapy remains a major challenge related to cancer treatment, and increasing the sensitivity of cancer cells to therapeutic drugs is a major focus of cancer treatment. AIMS: We purposed to explore the role of Metformin in CCA involved in chemotherapeutic sensitivity and Pyruvate kinase M2 (PKM2) through regulating mitochondrial apoptosis in the present study. METHODS: CCA cell lines of HCC9810 and RBE were treated with Metformin companied with antagonists or agonists of PKM2, cells sensitivity to Gemcitabine, cell migration and invasion along with apoptosis, which is mediated by JC-1 and LDH were assayed. RESULTS: Our results indicated that Metformin and Gemcitabine exhibit synergistic effect on inhibition of cholangiocarcinoma cell viability, cell migration and invasion as well as promotion apoptosis of cholangiocarcinoma cells. In vivo, Metformin combined with Gemcitabine has cooperation in inhibiting the growth of cholangiocarcinoma cell-derived tumors. Moreover, Metformin and Gemcitabine inhibited expression of PKM2 and PDHB in HCC9810 and RBE. CONCLUSION: Our study suggested that Metformin may increase the response of cholangiocarcinoma cells to Gemcitabine by suppressing PKM2 to activate mitochondrial apoptosis.

[RBMX overexpression inhibits proliferation, migration, invasion and glycolysis of human bladder cancer cells by downregulating PKM2].

OBJECTIVE: To investigate the role of RNA-binding motif protein X-linked (RBMX) in regulating the proliferation, migration, invasion and glycolysis in human bladder cancer cells. METHODS: A lentivirus vectors system and RNA interference technique were used to construct bladder cancer 1376 and UC-3 cell models with RBMX overexpression and knockdown, respectively, and successful cell modeling was verified using RT-qPCR and Western blotting. Proliferation and colony forming ability of the cells were evaluated using EdU assay and colony-forming assay, and cell migration and invasion abilities were determined using Transwell experiment. The expressions of glycolysis-related proteins M1 pyruvate kinase (PKM1) and M2 pyruvate kinase (PKM2) were detected using Western blotting. The effects of RBMX overexpression and knockdown on glycolysis in the bladder cancer cells were assessed using glucose and lactic acid detection kits. RESULTS: RT-qPCR and Western blotting confirmed successful construction of 1376 and UC-3 cell models with RBMX overexpression and knockdown. RBMX overexpression significantly inhibited the proliferation, clone formation, migration and invasion of bladder cancer cells, while RBMX knockdown produced the opposite effects. Western blotting results showed that RBMX overexpression increased the expression of PKM1 and decreased the expression of PKM2, while RBMX knockdown produced the opposite effects. Glucose consumption and lactate production levels were significantly lowered in the cells with RBMX overexpression (P < 0.05) but increased significantly following RBMX knockdown (P < 0.05). CONCLUSION: RBMX overexpression inhibits bladder cancer progression and lowers glycolysis level in bladder cancer cells by downregulating PKM2 expression, suggesting the potential of RBMX as a molecular target for diagnosis and treatment of bladder cancer.

LncRNA PWRN1 inhibits the progression of hepatocellular carcinoma by activating PKM2 activity.

Hepatocellular carcinoma (HCC) is one of the most prevalent and leading causes of cancer-related mortality worldwide. Long non-coding RNAs (lncRNAs) have been demonstrated to play vital roles in cancer development and progression. The lncRNA PWRN1 (PWRN1), acts as a tumor suppressor factor, which is low expressed in some cancers. However, the molecular mechanisms underlying the effects of PWRN1, especially the regulatory relationship with RNA binding protein in HCC remain largely unknown. In the present study, we demonstrated that PWRN1 was significantly down-regulated in HCC and correlated with better prognosis; furthermore, gain-of-function experiments showed that PWRN1 inhibited the proliferation of HCC cells. We further found that PWRN1 up-regulated pyruvate kinase activity and thus hinders the proliferation of HCC in vitro and in vivo. Mechanistically, pyruvate kinase M2 (PKM2) was bound to it and maintained the high activity state of PKM2, thereby hindering PKM2 from entering the nucleus in the form of low-activity dimers, reducing the expression of c-Myc downstream gene LDHA, leading to a decrease in lactate levels, and inhibiting the growth of tumor cells. In addition, PWRN1 was found to inhibit aerobic glycolysis. Finally, TEPP-46, a pyruvate kinase activator, appeared to inhibit HCC proliferation by maintaining tetramer stability and increasing pyruvate kinase activity. Taken together, our results provide new insights into the biology hindering HCC proliferation and indicate that PWRN1 in combination with PKM2 activators might represent a novel therapeutic target for HCC.

The HIF-1α/PKM2 Feedback Loop in Relation to EGFR Mutational Status in Lung Adenocarcinoma.

OBJECTIVE: Gene mutations in tumor cells can lead to several unique metabolic phenotypes, which are crucial for the proliferation of cancer cells. EGFR mutation (EGFR-mt) is the main oncogenic driving mutation in lung adenocarcinoma (LUAD). HIF-1 α and PKM2 are two key metabolic regulatory proteins that can form a feedback loop and promote cancer growth by promoting glycolysis. Here, the linkage between EGFR mutational status and HIF-1α/PKM2 feedback loop in LUAD were evaluated. METHODS: Retrospective study were performed on LUAD patients (n = 89) undergoing first-time therapeutic surgical resection. EGFR mutation was analyzed by real-time PCR. Immunohistochemistry was used to measure the expressions of HIF-1α and PKM2. RESULTS: We found that the protein expressions of HIF-1α and PKM2 were significantly higher in LUAD than normal lung tissues. In adenocarcinomas, the two protein expressions were both correlated with worse pTNM stage. Moreover, the correlation between the proteins of HIF-1α/PKM2 feedback loop and the EGFR mutational status were also analyzed. We found that EGFR-mt tumors showed higher HIF-1α and PKM2 proteins compared to tumors with EGFR wild-type. Meanwhile, HIF-1α expression was significantly correlated with higher pTNM stage, and PKM2 showed a similar trend, only in EGFR-mutated tumors. The expression of HIF-1α was positively correlated with PKM2 in LUAD, furthermore, this correlation was mainly in patients with EGFR-mt. CONCLUSION: Different expression and clinical features of HIF-1α/PKM2 feedback loop was existed between LUAD and normal lung tissues, especially in EGFR mutational tumors, supporting the relationship between EGFR mutation and the key related proteins of aerobic glycolysis (HIF-1α and PKM2) in lung adenocarcinomas.

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.

Characterization of the pyruvate kinase gene family in soybean and identification of a putative salt responsive gene GmPK21.

BACKGROUND: As a key regulatory enzyme in the glycolysis pathway, pyruvate kinase (PK) plays crucial roles in multiple physiological processes during plant growth and is also involved in the abiotic stress response. However, little information is known about PKs in soybean. RESULTS: In this study, we identified 27 PK family genes against the genome of soybean cultivar Zhonghuang13. They were classified into 2 subfamilies including PKc and PKp. 22 segmental duplicated gene pairs and 1 tandem duplicated gene pair were identified and all of them experienced a strong purifying selective pressure during evolution. Furthermore, the abiotic stresses (especially salt stress) and hormone responsive cis-elements were present in the promoters of GmPK genes, suggesting their potential roles in abiotic stress tolerance. By performing the qRT-PCR, 6 GmPK genes that continuously respond to both NaCl and ABA were identified. Subsequently, GmPK21, which represented the most significant change under NaCl treatment was chosen for further study. Its encoded protein GmPK21 was localized in the cytoplasm and plasma membrane. The transgenic Arabidopsis overexpressing GmPK21 exhibited weakened salinity tolerance. CONCLUSIONS: This study provides genomic information of soybean PK genes and a molecular basis for mining salt tolerance function of PKs in the future.