M2型丙酮酸激酶非代谢功能在肿瘤治疗中的研究进展

丙酮酸激酶(pyruvate kinase,PK),作为糖酵解的关键酶之一,可以编码四个不同亚型的基因,其中M2型丙酮酸激酶(PKM2)主要表达在正常人类胚胎发育中,和组织修复、再生密切相关,随着研究的深入,PKM2在肿瘤组织中的作用受到越来越多的关注。PKM2除了代谢作用外,还可以通过PKM2抑制剂和激活剂变构调节四聚体和二聚体,二聚体状态的PKM2可以调节细胞核中的基因表达及细胞增殖。本文综述了PKM2表达调控,重点介绍了PKM2非代谢功能及在抗肿瘤治疗中的临床应用。     

M2型丙酮酸激酶的抗肿瘤作用研究进展

摘 要：糖酵解（Warburg效应）是肿瘤细胞的重要特征，其相比于线粒体氧化磷酸化来说更高效地产生能量及大量的中间产物用于生物合成，还可抑制活性氧的生成。M2型丙酮酸激酶（pyruvate kinase M2，PKM2）多以低活性的二聚体形式存在于肿瘤细胞等快速增殖细胞中，作为催化糖酵解最后一个步骤的限速酶，是肿瘤细胞Warburg效应的一个关键调节因子。PKM2在肿瘤细胞中也发挥着重要的非代谢作用。PKM2被诱导转移入细胞核后，通过磷酸化特定的核蛋白后激活诸多基因的转录，促进肿瘤的生长。基于这些研究成果，该文综述了PKM2在肿瘤生长中的作用，并对PKM2的抑制剂和激活剂的抗肿瘤疗效分别进行讨论，并预测PKM2可能成为癌症治疗的潜在靶点。     

基于生物信息学分析PKM2在肺腺癌中的表达及临床意义

目的：通过生物信息学方法分析M2型丙酮酸激酶（Pyruvate kinase M2, PKM2）在肺腺癌（lung adenocarcinoma,LUAD）中的表达及临床意义。方法：利用Oncomine和TCGA数据库荟萃分析PKM2 mRNA在LUAD组织和正常肺组织中的表达差异,验证HPA数据库中PKM2蛋白表达;分析该基因与LUAD患者生存的关系;LinkedOmics和Metascape平台做PKM2与LUAD临床病理特征及KEGG通路分析;String-DB网站构建PPI网络。结果：LUAD组织中PKM mRNA表达水平是正常肺组织的1.95倍（P<0.05）。免疫组化结果显示PKM2蛋白在LUAD组织中高表达。PKM2表达与LUAD的病理分级、Ｔ分期和Ｎ分期呈正相关（均P<0.05）,且高表达者总生存率低于低表达者（P<0.001,HR=2.56）。PKM2共表达基因主要与糖酵解、癌症中央碳代谢、HIF-1等信号通路有关。结论：PKM2在LUAD组织中高表达,且与LUAD患者不良预后相关,有望成为LUAD诊断和治疗的有效生物标志物。     

M2型丙酮酸激酶及其在肿瘤生长中的作用

肿瘤细胞的快速增殖导致它和正常细胞比较需要更多的代谢原料。为维持其自身的高度增殖,肿瘤细胞往往过表达M2型丙酮酸激酶（pyruvate kinase subtypeM2,PKM2）。在肿瘤的糖代谢通路中,二聚体形式的PKM2（抑制型）促进葡萄糖中间产物进行生物大分子合成代谢,而四聚体结构的PKM2（激活型）则促进葡萄糖的氧化磷酸化为细胞提供能量,二聚体PKM2在肿瘤细胞中占主要地位。PKM2的过表达以及PKM2二聚体、四聚体之间的转化使肿瘤细胞能在各种氧含量和营养环境下生存并增殖,与肿瘤的发生和扩散有密切的关系。     

MicroRNAs介导丙酮酸激酶影响肿瘤细胞的糖代谢

肿瘤细胞在氧含量充足的情况下仍倾向于糖酵解,被称之为Warburg效应,这是肿瘤细胞的重要特征之一,现在认为它是癌基因和抑癌基因共同作用的结果.目前研究表明microRNAs能够在转录后水平参与相关基因的表达,通过多种途径调节肿瘤细胞的糖代谢方式.其中丙酮酸激酶参与糖酵解的最后一个阶段,不同亚型的表达水平差异能调控糖代谢方式.本文着重探讨microRNAs调控丙酮酸激酶的作用机制,及其对肿瘤细胞产生的影响,为临床治疗提供理论基础.     

M2型丙酮酸激酶与Warburg效应在肺癌中的研究进展

靶向肿瘤代谢途径是肺癌治疗的新兴策略之一。Warburg效应是肿瘤代谢的主要特征,表现为肿瘤细胞在有氧条件下通过糖酵解获得能量。最近研究表明,M2型丙酮酸激酶作为糖酵解途径关键酶的异常表达与肺癌的增殖、转移、诊断、治疗及预后密切相关,是肺癌治疗的潜在靶点。     

丙酮酸激酶 M2 在胃癌诊治和预后中的研究进展

胃癌是我国发病率与死亡率均位居前列的恶性肿瘤,5 年生存率低,胃癌治疗主要以手术联合放化疗为主。 由于 胃癌早期症状不明显,且目前传统的肿瘤标志物敏感度和特异度有限,早期胃癌患者诊断困难。 因此,早期胃癌的诊断途径, 胃癌治疗的靶点以及有效的预后评估方法成为热点。 近年来,肿瘤细胞能量代谢过程中的关键酶受到了更多的关注。 丙酮 酸激酶 M2(PKM2)是细胞糖酵解中的关键酶,在肿瘤组织、血清及其他体液中表达异常,不仅参与了肿瘤细胞的营养代谢,还 参与丝裂活化蛋白酶(MAPK)的转录、磷脂酰肌醇-3 激酶/ 蛋白激酶 B/ 雷帕霉素靶蛋白(PI3K/ Akt / mTOR)信号通路以及 NFκB/ miR-148a / 152 反馈环等多个过程,并通过上述反应促进了胃癌细胞的增殖、侵袭、转移以及血管肿瘤的生成,并抑制了胃 癌细胞的凋亡。 PKM2 可用于胃癌早期诊断、治疗及预后评估。 本文对近年来 PKM2 在胃癌的早期诊断、治疗与预后评估的 相关研究进行综述,为提高诊断的敏感度和特异度、研发新的治疗策略和预后评估提供一定的价值。     

PKM2对人胃癌细胞HMGB1释放的影响

目的:探讨M2型丙酮酸激酶(pyruvate kinase M2,PKM2)对胃癌细胞高迁移率族蛋白B-1(high mobility group box 1,HMGB1)释放的作用。方法:通过siRNA干扰抑制人胃癌BGC823细胞PKM2的表达,Western blot检测各组细胞PKM2蛋白的表达,以评价PKM2 siRNA的转染效率;CCK-8法检测各组细胞活力,分析干扰PKM2的表达对胃癌BGC823细胞增殖的影响;Western blot检测各组细胞HMGB1的表达以及ELISA检测各组细胞培养液HMGB1的水平,分析PKM2对胃癌BGC823细胞HMGB1的作用。结果:与空载体pU6组相比,PKM2 siRNA 组PKM2的表达显著降低(P＜0.01),表明siRNA有效地抑制胃癌BGC823细胞PKM2的表达;CCK-8结果显示,PKM2 siRNA 组细胞活力明显低于空载体pU6组(P＜0.001),表明干扰PKM2抑制胃癌BGC823细胞增殖;此外,PKM2 siRNA 组HMGB1的表达以及细胞外HMGB1的水平显著降低(P＜0.01),表明干扰PKM2抑制人胃癌BGC823细胞HMGB1的释放。结论:PKM2促进人胃癌BGC823细胞HMGB1的释放。     

卵巢癌组织中miR-125B-5p通过调控己糖激酶-2降低肿瘤能量代谢和抑制肿瘤细胞增殖

背景与目的 细胞内能量代谢异常是肿瘤的十大特征之一,微小RNA(microRNA,miRNA)可能通过调节有氧糖酵解相关酶的表达来介导有氧糖酵解,从而调控肿瘤细胞代谢和增殖.本研究对卵巢癌患者肿瘤组织中miR-125B-5p和己糖激酶-2(hexokinase-2,HK2)的表达水平与肿瘤能量代谢和增殖的关系进行了初步...     

HIF-1α/ALYREF/PKM2信号轴在膀胱癌糖酵解和肿瘤发生中的作用

背景与目的 丙酮酸激酶肌肉同工酶M2(pyruvate kinase muscle isozyme M2,PKM2)是糖酵解的限速酶,参与肿瘤的代谢和生长。PKM2在肿瘤中的调控网络复杂,在膀胱癌中尚未得到充分研究。PKM2 mRNA的5-甲基胞嘧啶(5-methylcytidine,m5C)修饰可能参与了膀胱癌的发病过程,机理有待深入研究。本文旨在探讨PKM2在膀胱癌中的生物学功能及其调控机制。方法 用Western blotting、qRT-PCR和免疫组织化学方法检测PKM2和Aly/REF输出因子(Aly/REF export factor,ALYREF)的表达水平。通过一系列体内实验和体外试验,研究膀胱癌细胞的生物学过程。通过RNA免疫沉淀、RNA测序和双荧光素酶报告分析,探讨PKM2在膀胱癌中的调控机制。结果 在膀胱癌中,我们首次证明了ALYREF稳定了PKM2的mRNA,并与其3’-非翻译区的m5C位点结合。ALYREF过表达通过PKM2介导的糖酵解促进膀胱癌细胞增殖。此外,PKM2和ALYREF高表达与膀胱癌患者生存不佳相关。最后,我们发现缺氧诱导因子1α(hypoxi...     

Pyruvate kinase M2 is a target of the tumor-suppressive microRNA-326 and regulates the survival of glioma cells

Emerging studies have identified microRNAs (miRNAs) as possible therapeutic tools for the treatment of glioma, the most aggressive brain tumor. Their important targets in this tumor are not well understood. We recently found that the Notch pathway is a target of miRNA-326. Ectopic expression of miRNA-326 in glioma and glioma stem cells induced their apoptosis and reduced their metabolic activity. Computational target gene prediction revealed pyruvate kinase type M2 (PKM2) as another target of miRNA-326. PKM2 has recently been shown to play a key role in cancer cell metabolism. To investigate whether it might be a functionally important target of miR-326, we used RNA interference to knockdown PKM2 expression in glioma cells. Transfection of the established glioma and glioma stem cells with PKM2 siRNA reduced their growth, cellular invasion, metabolic activity, ATP and glutathione levels, and activated AMP-activated protein kinase. The cytotoxic effects exhibited by PKM2 knockdown in glioma and glioma stem cells were not observed in transformed human astrocytes. Western blot analysis of human glioblastoma specimens showed high levels of PKM2 protein, but none was observed in normal brain samples. Strikingly, cells with high levels of PKM2 expressed lower levels of miR-326, suggestive of endogenous regulation of PKM2 by miR-326. Our data suggest PKM2 inhibition as a therapy for glioblastoma, with the potential for minimal toxicity to the brain.     

MiR‐133b inhibits growth of human gastric cancer cells by silencing pyruvate kinase muscle‐splicer polypyrimidine tract‐binding protein 1

The metabolism in tumor cells shifts from oxidative phosphorylation to glycolysis even in an aerobic environment. This phenomenon is known as the Warburg effect. This effect is regulated mainly by polypyrimidine tract‐binding protein 1 (PTBP1), which is a splicer of the mRNA for the rate‐limiting enzymes of glycolysis, pyruvate kinase muscle 1 and 2 (PKM1 and PKM2). In the present study, we demonstrated that miR‐133b reduced PTBP1 expression at translational level and that the expression levels of miR‐133b were significantly downregulated in gastric cancer clinical samples and human cell lines, whereas the protein expression level of PTBP1 was upregulated in 80% of the 20 clinical samples of gastric cancer examined. Ectopic expression of miR‐133b and knockdown of PTBP1 in gastric cancer cells inhibited cell proliferation through the induction of autophagy by the switching of PKM isoform expression from PKM2‐dominant to PKM1‐dominant. The growth inhibition was partially canceled by an autophagy inhibitor 3‐MA or a reactive oxygen species scavenger N‐acetylcysteine. These findings indicated that miR‐133b acted as a tumor‐suppressor through negative regulation of the Warburg effect in gastric cancer cells.     

Enhanced expression of the M2 isoform of pyruvate kinase is involved in gastric cancer development by regulating cancer‐specific metabolism

Recent studies have indicated that increased expression of the M2 isoform of pyruvate kinase (PKM2) is involved in glycolysis and tumor development. However, little is known about the role of PKM2 in gastric cancer (GC). Therefore, we examined the expression and function of PKM2 in human GC. We evaluated PKM1 and PKM2 expression by quantitative RT‐PCR in gastric tissues from 10 patients who underwent gastric endoscopic submucosal dissection, 80 patients who underwent gastrectomy, and seven healthy volunteers, and analyzed the correlation with clinicopathological variables. To assess the function of PKM2, we generated PKM2‐knockdown GC cells, and investigated the phenotypic changes. Furthermore, we examined the induction of PKM2 expression by cytotoxin‐associated gene A (CagA), a pathogenic factor of Helicobacter pylori, using CagA‐inducible GC cells. We found that PKM2 was predominantly expressed not only in GC lesions but also in the normal gastric regions of GC patients and in the gastric mucosa of healthy volunteers. The PKM2 expression was significantly higher in carcinoma compared to non‐cancerous tissue and was associated with venous invasion. Knockdown of PKM2 in GC cells caused significant decreases in cellular proliferation, migration, anchorage‐independent growth, and sphere formation in vitro, and in tumor growth and liver metastasis in vivo. The serine concentration‐dependent cell proliferation was also inhibited by PKM2 silencing. Furthermore, we found that PKM2 expression was upregulated by CagA by way of the Erk pathway. These results suggested that enhanced PKM2 expression plays a pivotal role in the carcinogenesis and development of GC in part by regulating cancer‐specific metabolism.     

PKM2 uses control of HuR localization to regulate p27 and cell cycle progression in human glioblastoma cells

The M2 isoform of pyruvate kinase (PK) is upregulated in most cancers including glioblastoma. Although PKM2 has been reported to use dual kinase activities to regulate cell growth, it also interacts with phosphotyrosine (pY)‐containing peptides independently of its kinase activity. The potential for PKM2 to use the binding of pY‐containing proteins to control tumor growth has not been fully examined. We here describe a novel mechanism by which PKM2 interacts in the nucleus with the RNA binding protein HuR to regulate HuR sub‐cellular localization, p27 levels, cell cycle progression and glioma cell growth. Suppression of PKM2 in U87, T98G and LN319 glioma cells resulted in increased p27 levels, defects in entry into mitosis, increased centrosome number, and decreased cell growth. These effects could be reversed by shRNA targeting p27. The increased levels of p27 in PKM2 knock‐down cells were caused by a loss of the nuclear interaction between PKM2 and HuR, and a subsequent cytoplasmic re‐distribution of HuR, which in turn led to increased cap‐independent p27 mRNA translation. Consistent with these results, the alterations in p27 mRNA translation, cell cycle progression and cell growth caused by PKM2 suppression could be reversed in vitro and in vivo by suppression of HuR or p27 levels, or by introduction of forms of PKM2 that could bind pY, regardless of their kinase activity. These results define a novel mechanism by which PKM2 regulates glioma cell growth, and also define a novel set of potential therapeutic targets along the PKM2–HuR–p27 pathway.     

Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth

We have previously demonstrated that enhanced aerobic glycolysis and/or autophagy in the tumor stroma supports epithelial cancer cell growth and aggressive behavior, via the secretion of high-energy metabolites. These nutrients include lactate and ketones, as well as chemical building blocks, such as amino acids (glutamine) and nucleotides. Lactate and ketones serve as fuel for cancer cell oxidative metabolism, and building blocks sustain the anabolic needs of rapidly proliferating cancer cells. We have termed these novel concepts the “Reverse Warburg Effect,” and the “Autophagic Tumor Stroma Model of Cancer Metabolism.” We have also identified a loss of stromal caveolin-1 (Cav-1) as a marker of stromal glycolysis and autophagy. The aim of the current study was to provide genetic evidence that enhanced glycolysis in stromal cells favors tumorigenesis. To this end, normal human fibroblasts were genetically-engineered to express the two isoforms of pyruvate kinase M (PKM1 and PKM2), a key enzyme in the glycolytic pathway. In a xenograft model, fibroblasts expressing PKM1 or PKM2 greatly promoted the growth of co-injected MDA-MB-231 breast cancer cells, without an increase in tumor angiogenesis. Interestingly, PKM1 and PKM2 promoted tumorigenesis by different mechanism(s). Expression of PKM1 enhanced the glycolytic power of stromal cells, with increased output of lactate. Analysis of tumor xenografts demonstrated that PKM1 fibroblasts greatly induced tumor inflammation, as judged by CD45 staining. In contrast, PKM2 did not lead to lactate accumulation, but triggered a “pseudo-starvation” response in stromal cells, with induction of an NFκB-dependent autophagic program, and increased output of the ketone body 3- hydroxy-buryrate. Strikingly, in situ evaluation of Complex IV activity in the tumor xenografts demonstrated that stromal PKM2 expression drives mitochondrial respiration specifically in tumor cells. Finally, immuno-histochemistry analysis of human breast cancer samples lacking stromal Cav-1 revealed PKM1 and PKM2 expression in the tumor stroma. Thus, our data indicate that a subset of human breast cancer patients with a loss of stromal Cav-1 show profound metabolic changes in the tumor microenvironment. As such, this subgroup of patients may benefit therapeutically from potent inhibitors targeting glycolysis, autophagy and/or mitochondrial activity (such as metformin).     

No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis

The Warburg effect describes the circumstance that tumor cells preferentially use glycolysis rather than oxidative phosphorylation for energy production. It has been reported that this metabolic reconfiguration originates from a switch in the expression of alternative splice forms (PKM1 and PKM2) of the glycolytic enzyme pyruvate kinase (PK), which is also important for malignant transformation.However, analytical evidence for this assumption was still lacking. Using mass spectrometry, we performed an absolute quantification of PKM1 and PKM2 splice isoforms in 25 human malignant cancers, 6 benign oncocytomas, tissue matched controls, and several cell lines. PKM2 was the prominent isoform in all analyzed cancer samples and cell lines. However, this PKM2 dominance was not a result of a change in isoform expression, since PKM2 was also the predominant PKM isoform in matched control tissues. In unaffected kidney, lung, liver, and thyroid, PKM2 accounted for a minimum of 93% of total PKM, for 80% - 96% of PKM in colon,and 55% - 61% of PKM in bladder. Similar results were obtained for a panel of tumor and non-transformed cell lines, where PKM2 was the predominant form.Thus, our results reveal that an exchange in PKM1 to PKM2 isoform expression during cancer formation is not occurring, nor do these results support conclusions that PKM2 is specific for proliferating, and PKM1 for non-proliferating tissue.