PKM2 Knockdown Research Articles

Tanshinone analog inhibits castration-resistant prostate cancer cell growth by inhibiting glycolysis in an AR-dependent manner

Androgen receptor (AR) is one of the key targets for the treatment of Castration-resistant prostate cancer (CRPC). Current endocrine therapy can greatly improve CRPC patients. However, with the change of pathogenic mechanism, acquired resistance often leads to the failure of treatment. Studies have shown that tanshinone IIA (TS-IIA) and its derivatives have significant antitumor activity, and have certain AR targeting effects, but the mechanism is unknown. In this study, the TS-IIA analog TB3 was found to significantly inhibit the growth of CRPC in vitro and in vivo. Molecular docking, cellular thermal shift assay and CHX experiments confirmed that AR was the target of TB3 and promoted the degradation of AR. Furthermore, TB3 can significantly inhibit glycolysis metabolism by targeting the AR/PKM2 axis..The addition of pyruvic acid could significantly alleviate the inhibitory effect of TB3 on CRPC cells. Besides, the knockdown of AR or PKM2 also could reverse the effect of TB3 on CRPC cells. Taken together, our study suggests that TS-IIA derivative TB3 inhibits glycolysis to prevent the CRPC process by targeting the AR/PKM2 axis.

Long noncoding RNA TMPO-AS1 accelerates glycolysis by regulating the miR-1270/PKM2 axis in colorectal cancer

BackgroundLong noncoding RNA thymopoietin-antisense RNA 1 (TMPO-AS1) is recognized as a participant in cancer progression. Nevertheless, its biological function in colorectal cancer remains obscure and needs further elucidation.Methods and ResultsFirst, we discovered enriched TMPO-AS1 in the tumor tissues that were related to poor prognosis. TMPO-AS1 knockdown enhanced SW480 cell apoptosis but inhibited invasion, proliferation, migration, and glucose metabolism. Further, MiR-1270 is directly bound with TMPO-AS1. MiR-1270 mimics were confirmed to inhibit cell proliferation, invasion, and glucose metabolism in our study. Mechanistically, miR-1270 directly is bound with the 3' untranslated regions (3'UTR) of PKM2 to downregulate PKM2. MiR-1270 inhibitors reversed the TMPO-AS1 knockdown’s effect on suppressing the tumor cell proliferation, invasion, and glycolysis, while the knockdown of PKM2 further inverted the function of miR-1270 inhibitors on the TMPO-AS1 knockdown.ConclusionsThis study illustrated that TMPO-AS1 advanced the development and the glycolysis of colorectal cancer by modulating the miR-1270/PKM2 axis, which provided a new insight into the colorectal cancer therapeutic strategy.

PKM2 controls the translation of TFE3 to maintain the integrity of the Golgi apparatus for the survival of HeLa and ME-180 cervical cancer cells.

The M2 isoform of pyruvate kinase (PKM2) is abundantly expressed in various cancer cells and associated with tumorigenesis, tumour proliferation and tumour progression. However, the role of PKM2 in these oncological processes is not fully understood. In the present study, we depleted PKM2 expression using RNA interference (RNAi), which induced apoptotic cell death and was accompanied by the downregulation of GM130, giantin, and p115 in HeLa and ME-180 cervical cancer cells. The decreased expression of these proteins caused structural and functional disturbances in the Golgi apparatus, which manifested as the dispersion of the Golgi apparatus and delayed anterograde trafficking from the ER to the Golgi. The transcription factor, TFE3, which functions in the Golgi stress response, was responsible for the expression of GM130, giantin, and p115 that maintained the integrity of the organelle under normal growth conditions. In PKM2-knockdown cells, the translation of TFE3 was markedly reduced. Knockdown of TFE3 by RNAi resulted in the downregulation of GM130, giantin, and p115, dispersion of the Golgi apparatus, and apoptotic cell death, similar to those observed following PKM2 knockdown. Conversely, the exogenous expression of TFE3 in PKM2 knockdown cells partially mitigated the aforementioned effects. We also demonstrated that PKM2 bound to the 5' UTR on TFE3 mRNA and promoted translation. This study is the first to identify a new function for PKM2, which activates the basal Golgi stress response to maintain the integrity of the Golgi apparatus through the translation of TFE3 and promote cancer cell survival.

Empagliflozin mediated miR-128-3p upregulation promotes differentiation of hypoxic cancer stem-like cells in breast cancer

AimsThe hsa-miR-128-3p expression is downregulated in advanced breast cancer patients. Empagliflozin (EMPA) is an anti-diabetic drug with anticancer potential. The present study investigated the effect of EMPA on cancer cell differentiation by acting as a miR-128-3p mimicking drug in breast cancer. Main methodsOur results first demonstrate SP1 and PKM2 as the downstream effectors of hsa-miR-128-3p. Further, transfection with siPKM2, miR-128-3p mimics, and inhibitors was performed to assess their involvement in cancer stemness using flow cytometry. Further, EMPA as miR-128-3p mimicking drug was screened and explored on cancer cell differentiation. Then, we treated the 4T1-Red-FLuc allograft breast tumor with EMPA to assess its inhibitory potential toward tumor growth using IVIS® Spectrum. Immunohistochemistry was performed to evaluate cancer cell differentiation and cell proliferation. Key findingsWe found that hsa-miR-128-3p is the upstream regulator of SP1 and PKM2 in hypoxic breast cancer cells. Overexpression of miR-128-3p with mimics downregulate SP1 and PKM2, whereas miR-128-3p inhibitor shows an opposite effect. The enhanced expression of miR-128-3p and PKM2 knockdown diminishes hypoxia-induced CD44 expression and enhance CD44+/CD24+ differentiated cells. We also identified EMPA as the miR-128-3p mimicking drug that can enhance the differentiated cell population. Further, EMPA suppressed in vivo tumor growth, lung metastasis, tumor bioluminescence, and cell proliferation. Therefore, EMPA abrogates breast cancer stemness by inactivating SP1 and PKM2 via enhanced miR-128-3p expression. SignificanceEMPA could be a promising drug in combination with other chemotherapeutic drugs in advanced breast cancer.

Knockdown of pyruvate kinase M2 suppresses bladder cancer progression

Abstract Objectives Bladder cancer (BCa) is one of the most frequently diagnosed cancers of the urinary tract and has a high mortality. The M2 splice isoform of pyruvate kinase (PKM2) is a key regulator of the Warburg effect in cancer cells. This study aimed to evaluate metabolic alterations and biological behaviours after knocking down PKM2. Methods In this study, 36 pairs of BCa tissues and adjacent normal tissues were collected to analyse the expression level of PKM2 and to explore the relationship between PKM2 level and tumour and patient status. After PKM2 knockdown in T24 cells, cell survival, migration, invasion, glucose uptake, lactate production, and apoptosis were detected. The tumour-forming ability of PKM2-reducing T24 cells was examined in vivo. Results The results showed that PKM2 expression correlates with BCa stage and grade. PKM2 knockdown decreases glucose consumption and lactate production and suppresses cell proliferation, migration, and invasion while increasing reactive oxygen species levels and apoptosis in T24 BCa cells in vitro. In nude mouse models, PKM2 knockdown reduced xenograft and orthotopic tumour size. Moreover, PKM2 knockdown decreased vimentin and fibronectin expression and increased E-cadherin expression. Analysis of high-throughput sequencing data revealed that PKM2 may also be associated with biological processes and diseases. Conclusions Overall, these results indicate that PKM2 may be a therapeutic target for BCa patients.

Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

Ischemic preconditioning (IPC) is a potential intervention known to protect the heart against ischemia/reperfusion injury, but its role in the no-reflow phenomenon that follows reperfusion is unclear. Dihydrotanshinone I (DT) is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC. Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation (OGD), but the protection was prevented by a ROS scavenger. In addition, DT administration protected the heart against isoprenaline challenge. Mechanistically, PKM2 reacted to transient ROS via oxidization at Cys423/Cys424, leading to glutathionylation and nuclear translocation in dimer form. In the nucleus, PKM2 served as a co-factor to promote HIF-1α-dependent gene induction, contributing to adaptive responses. In mice subjected to permanent coronary ligation, cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection, which was rescued by overexpression of wild-type Pkm2, rather than Cys423/424-mutated Pkm2. In conclusion, PKM2 is sensitive to oxidation, and subsequent glutathionylation promotes its nuclear translocation. Although IPC has been viewed as a protective means against reperfusion injury, our study reveals its potential role in protection of the heart from no-reflow ischemia.

CircCYP24A1 facilitates esophageal squamous cell carcinoma progression through binding PKM2 to regulate NF-κB-induced CCL5 secretion

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignant tumor, while the molecular mechanisms have not been fully elucidated. Multiple circular RNAs have been reported to involve in the onset and progression of malignant tumors through various molecular mechanisms. However, the clinical significance and functional mechanism of most circRNAs involved in the progression of ESCC remains obscure.MethodsRNA-Seq was used to explore potential circRNAs in participated in 5 pairs of ESCC and their corresponding normal esophageal tissues. The up-regulated circCYP24A1 was selected. Fluorescence in situ hybridization was cunducted to verificated the expression and intracellular localization of circCYP24A1 by using the tissue microarray. The Kaplan–Meier method and Cox proportional hazards model was used to examine the potential prognostic value of circCYP24A1 on overall survival of ESCC patients. The biological function were confirmed by gain- and loss-of-function approaches in vivo. mRNA expression profile microarray was proformed to investigate the downstream signaling pathways involved in circCYP24A1. RNA pull-down assay and mass spectrometry were performed to identify the proteins associated with circCYP24A1. Rescue experiments were carried out to identified hypothetical regulatory role of circCYP24A1 on ESCC progression in vivo and in virto.ResultsIn this study, we identified circCYP24A1 in ESCC tissues by RNA sequencing, which is up-regulated in 114 cases of ESCC tissues and acts as a novel prognosis-related factor. Moreover, circCYP24A1 promoted the ability of proliferation, migration, invasion and clone formation in vitro, as well as tumor growth in vivo. Mechanistically, chemokine (C-Cmotif) ligand 5 (CCL5) is functional downstream mediator for circCYP24A1, which is screened by mRNA microarray. Moreover, circCYP24A1 physically interacts with M2 isoform of pyruvate kinase (PKM2). Rescue experiments showed that PKM2 knockdown partly reverses the promotional effects of circCYP24A1. It was revealed that circCYP24A1 increases secretion of CCL5 through the mechanism mainly by interacting with PKM2, an activator of NF-κB pathway, and thereby accelerate malignant progression of ESCC.ConclusionsUp-regulated circCYP24A1 could activate NF-κB pathway by binding PKM2, which promotes the secretion of CCL5 and accelerate malignant progression of ESCC. Our fndings recommended a novel function for circCYP24A1 as a potential effective biomarker for judging prognosis and a therapeutic target in ESCC.

Fibroblast Programmed Cell Death Ligand 1 Promotes Osteoclastogenesis in Odontogenic Keratocysts

Local aggressive growth of odontogenic keratocysts (OKCs) can cause serious bone destruction, even resulting in pathologic fractures of the mandible. The mechanism of osteoclastogenesis in OKCs was explored by investigating the role of programmed cell death ligand 1 (PD-L1), a key immune checkpoint, in OKCs and its relationship with the M2 isoform of pyruvate kinase (PKM2), a key enzyme of glycolysis. The data from immunohistochemistry, real-time quantitative PCR, Western blot, and flow cytometry indicated that the expression level of PD-L1 was significantly increased in the stroma and fibroblasts of OKCs (OKC-Fs) when compared with oral mucosa. Double-labeling staining demonstrated that osteoclasts in OKCs spatially interacted with PD-L1-positive OKC-Fs. Exogenous expression of PD-L1 in OKC-Fs promoted osteoclastogenesis when OKC-Fs were co-cultured with osteoclast precursors (RAW264.7 cells). Because OKC-Fs exhibit energy dependency and acquire energy from PKM2-mediated glycolysis, this study generated stable PKM2 knockdown OKC-Fs using shRNAs against PKM2, and found that PD-L1 expression level was decreased by PKM2 knockdown. Furthermore, Spearman rank correlation analysis showed that there was a positive correlation between the immunostaining of PKM2 and PD-L1 in OKC samples. In addition, double-labeling immunofluorescence showed colocalizations between PKM2 and PD-L1 in the fibrous tissue walls of OKCs. In conclusion, PD-L1 in fibroblasts promotes osteoclastogenesis in OKCs, which is regulated by PKM2.

LncRNA NEAT1 promotes the proliferation and metastasis of hepatocellular carcinoma by regulating the FOXP3/PKM2 axis.

ObjectiveHepatocellular carcinoma (HCC) is a malignant tumor. The occurrence of HCC is involved in the alteration of a variety of oncogenes or tumor suppressor genes, but the specific molecular mechanism remains unknown. This research proved the effects of long non-coding RNA NEAT1 (lncRNA NEAT1) on the viability, proliferation, migration, and invasion of hepatocellular carcinoma cells and explored the mechanism behind these effects.MethodsNEAT1 in 97H and Huh7 cell lines was overexpressed or knocked down, respectively. The expression of FOXP3 and its target gene PKM2 was hinged on qRT-PCR and Western blot, respectively. RNA pulldown and RNA immunoprecipitation experiments were carried out to detect the interaction between NEAT1 and proteins. Finally, the effect of NEAT1 on the tumor volume of HCC was verified by animal experiments.ResultsA series of experiments have shown that NEAT1 knockdown can inhibit the viability, proliferation, migration, and invasion of HCC cells; NEAT1 can bind FOXP3 to promote PKM2 transcription; PKM2 knockdown can inhibit the viability, proliferation, migration, and invasion of HCC cells; and PKM2 knockdown reversed the function of NEAT1.ConclusionlncRNA NEAT1 can promote the malignant behavior of HCC cells, while silencing of NEAT1 can inhibit that behavior of HCC cells. Mechanically, NEAT1 promotes the transcriptional activation of PKM2 by binding FOXP3, and PKM2 knockout reverses the function of NEAT1.

Liver cancer heterogeneity modeled by in situ genome editing of hepatocytes.

Mechanistic study and precision treatment of primary liver cancer (PLC) are hindered by marked heterogeneity, which is challenging to recapitulate in any given liver cancer mouse model. Here, we report the generation of 25 mouse models of PLC by in situ genome editing of hepatocytes recapitulating 25 single or combinations of human cancer driver genes. These mouse tumors represent major histopathological types of human PLCs and could be divided into three human-matched molecular subtypes based on transcriptomic and proteomic profiles. Phenotypical characterization identified subtype- or genotype-specific alterations in immune microenvironment, metabolic reprogramming, cell proliferation, and expression of drug targets. Furthermore, single-cell analysis and expression tracing revealed spatial and temporal dynamics in expression of pyruvate kinase M2 (Pkm2). Tumor-specific knockdown of Pkm2 by multiplexed genome editing reversed the Warburg effect and suppressed tumorigenesis in a genotype-specific manner. Our study provides mouse PLC models with defined genetic drivers and characterized phenotypical heterogeneity suitable for mechanistic investigation and preclinical testing.

Pyruvate kinase M1 regulates butyrate metabolism in cancerous colonocytes

Colorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non-cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re-addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 suppresses glycolysis and inhibits hypoxia-inducible factor-1 alpha. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.

Pyruvate kinase M1 regulates butyrate metabolism in cancerous colonocytes

Colorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Butyrate is a short‐chain fatty acid produced by resident bacteria in the colon and serves as the primary energy source for non‐cancerous colonocytes. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate, and produces ATP. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non‐cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not by PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re‐addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 inhibits hypoxia‐inducible factor‐1 alpha (HIF1α) and suppresses glycolysis. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.

RASD2 promotes the development and metastasis of uveal melanoma via enhancing glycolysis

Uveal melanoma (UVM) is a primary intraocular tumor in adults with high mortality. Nearly half of primary UVM tumors metastasize to the liver and lung. RASD2 encodes a Ras-related GTP-binding protein and involves in psychiatric disorders. RASD2 has been shown to be expressed in multiple tissues including skin. However, the function of RASD2 in UVM is not fully studied. Here, we investigated the expression, functional role and expression regulation of RASD2 in UVM. RASD2 expression was significantly elevated in metastasis UVM, while high level of RASD2 indicated poor prognosis of patients with metastasis UVM. Silencing RASD2 dampened cell growth, migration and invasion of UVM cells. Additionally, xenograft tumor model suggested that RASD2 knockdown suppressed in vivo UVM xenograft tumor growth and lung metastasis. Bioinformatics analysis predicted that RASD2 regulated epithelial-mesenchymal transition and glycolysis in UVM, which was further confirmed both in vivo and in vitro. Moreover, RASD2 knockdown suppressed UVM cell metabolism, with decreased expression of glycolysis-related HK2, LDHA, GLUT1 and PKM2. In addition, we demonstrated that PKM2 knockdown antagonized the effect of RASD2 on cell growth, migration and invasion. In summary, our findings suggest that RASD2 may enhance the development and metastasis of UVM via enhancing glycolysis. Targeting RASD2 could be a novel therapeutic strategy for UVM treatment.

PKM2 Interacts With the Cdk1-CyclinB Complex to Facilitate Cell Cycle Progression in Gliomas.

PKM2 is a phosphotyrosine-binding glycolytic enzyme upregulated in many cancers, including glioma, and contributes to tumor growth by regulating cell cycle progression. We noted, however, that in multiple glioma cell lines, PKM2 knock-down resulted in an accumulation of cells in G2-M phase. Moreover, PKM2 knock-down decreased Cdk1 activity while introducing a constitutively active Cdk1 reversed the effects of PKM2 knock-down on cell cycle progression. The means by which PKM2 increases Cdk1 activity have not been described. Transient interaction of T14/Y15-phosphorylated Cdk1 with cyclin B allows Cdk7-mediated pT161 Cdk1 phosphorylation followed by cdc25C-mediated removal of pT14/Y15 and activation of Cdk1 in cycling cells. In the present course of investigation, PKM2 modulation did not influence Cdk7 activity, but phosphotyrosine binding forms of PKM2 co-immunoprecipitated with pY15-containing Cdk1-cyclinB and enhanced formation of active pT161 Cdk1-cyclin B complexes. Moreover, exogenous expression of phosphotyrosine binding forms of PKM2 reversed the effects of PKM2 knock-down on G2-M arrest. We here show that PKM2 binds and stabilize otherwise transient pY15-containing Cdk1-cyclinB complexes that in turn facilitate Cdk1-cyclin B activation and entry of cells into mitosis. These results, therefore, establish metabolic enzyme PKM2 as a direct interactor and activator of Cdk1-cyclin B complex and thereby directly controls mitotic progression and the growth of brain tumor cells.

Potential of PKM2 as a drug target in mouse models with type 1 diabetes mellitus.

BackgroundThis study aimed to determine the effect of PKM2 knockout in STZ induced type 1 diabetes mellitus (T1D) mouse models and to explore the possible mechanism.MethodPKM2fl/fl C57BL/6 mouse was backcrossed with Ins‐1cre C57BL/6 mouse to generate β‐cell‐specific PKM2 knockout mouse after tamoxifen administration. The expression level of PKM2 in pancreas tissues was detected by quantitative reverse‐transcription polymerase chain reaction and western blot analysis. The blood glucose levels in STZ induced T1D mouse models were measured to validate the establishment of T1D models. The pathological changes of T1D mouse were examined by hematoxylin and eosin. The oxidative stress (OS) and inflammatory response in T1D mouse were determined by measuring the expression levels of malondialdehyde, superoxide dismutase, and 8‐OHdG in pancreatic tissues and the serum levels of interleukin‐6 and tumor necrosis factor‐α. The ability to catabolize glucose was assessed through intraperitoneal glucose tolerance test and insulin tolerance test.Resultsβ‐cell‐specific PKM2 knockout was successfully achieved in PKM2fl/flcre+ mouse. T1D mouse with PKM2 knockdown had decreased blood glucose level and suppressed cell apoptosis. PKM2 knockout in T1D mouse attenuated β cell injury. OS and inflammatory response in T1D mouse with PKM2 knockout were also suppressed compared with T1D mouse without PKM2 knockout.ConclusionPKM2 knockout in T1D mouse can attenuate OS and inflammatory response as well as decrease blood glucose level, suggesting the potential of PKM2 as a drug target for T1D treatment.

Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to mechanical ventilation-induced pulmonary fibrosis.

Background: Mechanical ventilation (MV) can induce pulmonary fibrosis. This study aims to investigate whether MV-induced pulmonary fibrosis is associated with aerobic glycolysis and seeks to uncover the underlying mechanisms mediated by integrin β3-pyruvate kinase M2 (PKM2) pathway.Methods: PKM2 knockdown or inhibition, integrin β3 knockout or inhibition and wild-type mice were exposed to MV (20 mL/kg) for 2 h.Results: Mice exposed to MV exhibited increased expression of collagen deposition, and upregulation of α-smooth muscle actin and collagen I in lung tissues. Single cells analysis showed that MV-induced pulmonary fibrosis was associated with increased gene expression of integrin and glycolysis in pulmonary fibroblasts, as well as upregulation of glycolytic products tested by metabolomics. Meanwhile, increased protein level of integrin β3 and PKM2 was confirmed by western blot and immunohistochemistry. Double immunofluorescence staining and flow cytometric analysis showed increased number of fibronectin+/integrin β3+ and fibronectin+/PKM2+ fibroblasts in lung tissues. Furthermore, MV-induced aerobic glycolysis and pulmonary fibrosis were ameliorated after treatment with PKM2 knockdown-AAV and inhibition, or in integrin β3 knockout and inhibition mice.Conclusions: Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to MV-induced pulmonary fibrosis. The inhibition of aerobic glycolysis targeting integrin β3-PKM2 pathway may be a promising treatment for MV-induced pulmonary fibrosis.

The Effect of si-PKM2 on Proliferation and Apoptosis of Acute Leukemic Cells and Its Molecular Mechanism

To investigate the effect of glycolytic enzyme pyruvate kinase type 2 (PKM2) on the proliferation and apoptosis of human leukemia HL-60 cells. si-PKM2 plasmid was transfected into HL-60 cells (set as si-PKM2 group), and blank vector transfected cells were set as control group (si-Ctl group). The expression levels of PKM2 mRNA and protein in si-Ctl group and si-PKM2 group were detected by RT-qPCR and Western blot. CCK-8 cell detection kit was used to detect the proliferation ability of the cells in the two groups. Flow cytometry was used to detect the changes of cell cycle and apoptosis. Western blot and RT-qPCR were used to detect the changes of p-Akt and p-mTOR protein levels in PI3K/Akt/mTOR signaling pathway and the changes of glycolysis-related mRNA levels of the cells in the two groups. The changes in glucose consumption and lactic acid production of the cells were assayed. Over expressed PKM2, HL-60 cells were treated with PI3K inhibitor LY294002 or galactose, the changes in cell proliferation ability, cell cycle and apoptosis, as well as changes in glucose consumption and lactic acid production were detected. Interfered by si-PKM2, mRNA and protein levels of PKM2 in si-PKM2 group significantly decreased, and proliferation ability of the cells was also reduced (P<0.05). After PKM2 knockdown, the cells were significantly blocked at G1 phase, and cell apoptosis was obviously induced (P<0.05). p-Akt and p-mTOR levels were lower in si-PKM2 group than those in si-Ctl group. The glucose consumption and lactic acid production significantly decreased in si-PKM2 cells. Overexpressed PKM2, HL-60 cells were treated with PI3K inhibitor LY294002. The glucose consumption and lactate acid production induced by overexpressed PKM2 were reduced. Overexpressed PKM2, HL-60 cells showed no significant changes in cell proliferation, cycle and apoptosis when cultured with galactose. PKM2 knockdown can inhibit the proliferation and induce apoptosis of HL-60 cells, and its molecular mechanism may be related to the PKM2-mediated PI3K/Akt/mTOR-glycolysis, which suggesting that PKM2 may serve as a molecular target for the prevention and treatment of leukemia.

PKM2 Regulates HSP90-Mediated Stability of the IGF-1R Precursor Protein and Promotes Cancer Cell Survival during Hypoxia.

Simple SummaryGenerally, IGF-1R is overexpressed in most solid tumors, and its expression is significantly associated with poor prognosis in cancer patients. However, IGF-1R gene amplification events are extremely rare in tumors. It is, therefore, necessary to define the mechanism underlying IGR-1R overexpression to elucidate potential therapeutic targets. Our study, specifically, aimed to define the potential mechanisms associated with PKM2 function in regulating IGF-1R protein expression. PKM2 was found to be a non-metabolic protein that regulates HSP90 binding to and stabilizing the precursor IGF-1R protein, thereby promoting the basal level of mature IGF-1R protein. Consequently, PKM2 knockdown inhibits the activation of AKT, a downstream effector of IGF-1R signaling, and increases apoptosis during hypoxia. Our findings reveal a novel mechanism for regulating IGF-1R protein expression, thus suggesting PKM2 as a potential therapeutic target in cancers associated with aberrant IGF signaling.Insulin-like growth factor-1 receptor (IGF-1R), an important factor in promoting cancer cell growth and survival, is commonly upregulated in cancer cells. However, amplification of the IGF1R gene is extremely rare in tumors. Here, we have provided insights into the mechanisms underlying the regulation of IGF-1R protein expression. We found that PKM2 serves as a non-metabolic protein that binds to and increases IGF-1R protein expression by promoting the interaction between IGF-1R and heat-shock protein 90 (HSP90). PKM2 depletion decreases HSP90 binding to IGF-1R precursor, thereby reducing IGF-1R precursor stability and the basal level of mature IGF-1R. Consequently, PKM2 knockdown inhibits the activation of AKT, the key downstream effector of IGF-1R signaling, and increases apoptotic cancer cell death during hypoxia. Notably, we clinically verified the PKM2-regulated expression of IGF-1R through immunohistochemical staining in a tissue microarray of 112 lung cancer patients, demonstrating a significant positive correlation (r = 0.5208, p < 0.0001) between PKM2 and IGF-1R expression. Together, the results of a previous report demonstrated that AKT mediates PKM2 phosphorylation at serine-202; these results suggest that IGF-1R signaling and PKM2 mutually regulate each other to facilitate cell growth and survival, particularly under hypoxic conditions, in solid tumors with dysregulated IGF-1R expression.

Lactate secreted by PKM2 upregulation promotes Galectin-9-mediated immunosuppression via inhibiting NF-\u03baB pathway in HNSCC

Pyruvate kinase M2 as a key rate-limiting enzyme in glycolysis, it plays a critical role in metabolic reprogramming and carcinogenesis. However, whether PKM2 can promote immunosuppressive microenvironment formation remains unknown in head and neck squamous cell carcinoma (HNSCC). PKM2 expression was detected using immunohistochemical staining. The biological functions of PKM2 were investigated in vitro and in vivo. Lactate production and the expression of Galectin-9, a critical immunosuppression molecule, were detected after PKM2 knockdown and overexpression in HNSCC cells. The mechanism of lactate regulating Galectin-9 expression through NF-κB signaling was explored in vitro. Overexpression of PKM2 correlates with poor prognosis in HNSCC patients. Silencing PKM2 markedly inhibits proliferation and metastasis capacity in vivo and in vitro, and vice versa. The glycolysis and glycolytic capacity are significantly decreased after PKM2 silencing. Lactate secretion induced by PKM2 significantly promotes migration and invasion capacity. Furthermore, a positive correlation between PKM2 and Galectin-9 expression is observed in HNSCC tissues. The induction of Galectin-9 expression by PKM2 can be affected by a lactate transporter inhibitor. Mechanically, lactate impeded the suppressive transcriptional complex formation of NF-κB and histone deacetylase 3 (HDAC3), which released the transcription of Galectin-9 mediated by NF-κB signaling. Our findings demonstrate that lactate produced by PKM2 upregulation promotes tumor progression and Galectin-9-mediated immunosuppression via NF-κB signaling inhibition in HNSCC, which bridges metabolism and immunosuppression. The novel PKM2-lactate-Galectin-9 axis might be a potential therapeutic target in HNSCC.

52350 PKM2 mediates anti-tumor immunity and T cell dysfunction

ABSTRACT IMPACT: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and benefit to patients; our work will identify novel mediators of this process for both therapeutic potential and underlying mechanism, allowing for both potential immediate clinical utility and identification of future targets based on new mechanistic insights. OBJECTIVES/GOALS: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and clinical benefit to the majority of patients. We aim to interrogate a novel mediator of dysfunction identified from transcriptome analyses, pyruvate kinase muscle isozyme isoform 2 (PKM2), for therapeutic utility and underlying mechanism. METHODS/STUDY POPULATION: Transcriptome analyses of CD8+ lymphocytes from tumor-bearing lungs from both murine KrasG12D p53-/- and human non-small cell lung cancer (NSCLC) patients were performed, and differentially expressed genes identified. Flow cytometric analyses for PKM isoform expression and effects of target knockdown on accumulation of dysfunctional characteristics, including checkpoint and transcription factor expression, proliferation, and cytokine production, were performed using an in vitro co-culture of murine antigen-specific T (OT-I) cells and antigen-expressing NSCLC (HKP1-ova) cells. In vivo examination of the same was performed using adoptive transfer of OT-I cells into immunocompetent recipient mice with engraftment of HKP1-ova cells, and subsequent evaluation of mouse survival and T cell phenotypes. RESULTS/ANTICIPATED RESULTS: Transcriptome analyses demonstrated that PKM expression was upregulated in dysfunctional T cells from both murine and human samples. This was confirmed both in vitro with co-culture and in vivo with adoptive transfer approaches, with both activated and dysfunctional OT-I cells expressing higher levels of isoform 2 of PKM than naive OT-I cells. Expression of PKM2 mimicked the kinetics of the transcription factor Tox, a known driver of dysfunction, and knockdown of PKM2 resulted in reduced granzyme B expression, and increased proportions of progenitors with fewer terminally differentiated dysfunctional cells. Knockdown of PKM2 in adoptively-transferred OT-I cells led to enhanced tumor control; results are being extended to other tumor models, and T cells metabolically profiled with PKM2 manipulation. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work identified a novel mediator of dysfunction whose targeting has the potential to enhance anti-tumor immunity. Mechanistically, targeting PKM2 led to altered T cell differentiation to a dysfunctional state, linking metabolic phenotypes to these traits and underlining the importance and therapeutic potential of T cell metabolic pathways.