M2 Isoform Research Articles

Interpretable attention-based deep learning ensemble for personalized ovarian cancer treatment without manual annotations.

Inhibition of pathological angiogenesis has become one of the first FDA approved targeted therapies widely tested in anti-cancer treatment, i.e. VEGF-targeting monoclonal antibody bevacizumab, in combination with chemotherapy for frontline and maintenance therapy for women with newly diagnosed ovarian cancer. Identification of the best predictive biomarkers of bevacizumab response is necessary in order to select patients most likely to benefit from this therapy. Hence, this study investigates the protein expression patterns on immunohistochemical whole slide images of three angiogenesis related proteins, including Vascular endothelial growth factor, Angiopoietin 2 and Pyruvate kinase isoform M2, and develops an interpretable and annotation-free attention based deep learning ensemble framework to predict the bevacizumab therapeutic effect on patients with epithelial ovarian cancer or peritoneal serous papillary carcinoma using tissue microarrays (TMAs). In evaluation with five-fold cross validation, the proposed ensemble model using the protein expressions of both Pyruvate kinase isoform M2 and Angiopoietin 2 achieves a notably high F-score (0.99±0.02), accuracy (0.99±0.03), precision (0.99±0.02), recall (0.99±0.02) and AUC (1.00±0). Kaplan-Meier progression free survival analysis confirms that the proposed ensemble is able to identify patients in the predictive therapeutic sensitive group with low cancer recurrence (p<0.001), and the Cox proportional hazards model analysis further confirms the above statement (p=0.012). In conclusion, the experimental results demonstrate that the proposed ensemble model using the protein expressions of both Pyruvate kinase isoform M2 and Angiopoietin 2 can assist treatment planning of bevacizumab targeted therapy for patients with ovarian cancer.

Recombinant Treponema pallidum Protein Tp47 Promoted the Phagocytosis of Macrophages by Activating NLRP3 Inflammasome Induced by PKM2-Dependent Glycolysis.

Glycolysis is a critical pathway in cellular glucose metabolism that provides energy and participates in immune responses. However, whether glycolysis is involved in NOD-like receptor family protein 3 (NLRP3) inflammasome activation and phagocytosis of macrophages in response to Treponema pallidum infection remains unclear. To investigate the role of glycolysis in activating the NLRP3 inflammasome for regulating phagocytosis in macrophages in response to Treponema pallidum protein Tp47 and its associated mechanisms. Interactions between activation of the NLRP3 inflammasome and phagocytosis and the role of glycolysis in Tp47-treated macrophages were investigated through experiments on peritoneal macrophages and human monocytic cell line-derived macrophages. Activation of phagocytosis and NLRP3 inflammasome were observed in Tp47-treated macrophages. Treatment with NLRP3 inhibitor MCC950 or si-NLRP3 attenuated Tp47-induced phagocytosis. Glycolysis and glycolytic capacity were enhanced by Tp47 stimulation in macrophages, and a change in the levels of glycolytic metabolites (phosphoenolpyruvate, citrate, and lactate) was induced by Tp47 in macrophages. Inhibition of glycolysis with 2-deoxy-D-glucose, a glycolysis inhibitor, decreased the activation of NLRP3. Expression of M2 isoform of pyruvate kinase (PKM2), an enzyme catalyzing a rate-limiting reaction in the glycolytic pathway, was upregulated in Tp47-stimulated macrophages. Inhibition of PKM2 with shikonin or si-PKM2 decreased glycolysis and NLRP3 activation. Tp47 promotes phagocytosis in macrophages by activating the NLRP3 inflammasome, which is induced by the enhancement of PKM2-dependent glycolysis.

PKM2 dictates the poised chromatin state of PFKFB3 promoter to enhance breast cancer progression.

The hypoxic milieu is a critical modulator of aerobic glycolysis, yet the regulatory mechanisms between the key glycolytic enzymes in hypoxic cancer cells are largely unchartered. In particular, the M2 isoform of pyruvate kinase (PKM2), the rate-limiting enzyme of glycolysis, is known to confer adaptive advantages under hypoxia. Herein, we report that non-canonical PKM2 mediates HIF-1α and p300 enrichment at PFKFB3 hypoxia-responsive elements (HREs), causing its upregulation. Consequently, the absence of PKM2 activates an opportunistic occupancy of HIF-2α, along with acquisition of a poised state by PFKFB3 HREs-associated chromatin. This poised nature restricts HIF-2α from inducing PFKFB3 while permitting the maintenance of its basal-level expression by harboring multiple histone modifications. In addition, the clinical relevance of the study has been investigated by demonstrating that Shikonin blocks the nuclear translocation of PKM2 to suppress PFKFB3 expression. Furthermore, TNBC patient-derived organoids and MCF7 cells-derived xenograft tumors in mice exhibited substantial growth inhibition upon shikonin treatment, highlighting the vitality of targeting PKM2. Conclusively, this work provides novel insights into the contributions of PKM2 in modulating hypoxic transcriptome and a previously unreported poised epigenetic strategy exhibited by the hypoxic breast cancer cells for ensuring the maintenance of PFKFB3 expression.

M2 isoform of pyruvate kinase rewires glucose metabolism during radiation therapy to promote an antioxidant response and glioblastoma radioresistance.

Resistance to existing therapies is a significant challenge in improving outcomes for glioblastoma (GBM) patients. Metabolic plasticity has emerged as an important contributor to therapy resistance, including radiation therapy (RT). Here, we investigated how GBM cells reprogram their glucose metabolism in response to RT to promote radiation resistance. Effects of radiation on glucose metabolism of human GBM specimens were examined in vitro and in vivo with the use of metabolic and enzymatic assays, targeted metabolomics, and FDG-PET. Radiosensitization potential of interfering with M2 isoform of pyruvate kinase (PKM2) activity was tested via gliomasphere formation assays and in vivo human GBM models. Here, we show that RT induces increased glucose utilization by GBM cells, and this is accompanied with translocation of GLUT3 transporters to the cell membrane. Irradiated GBM cells route glucose carbons through the pentose phosphate pathway (PPP) to harness the antioxidant power of the PPP and support survival after radiation. This response is regulated in part by the PKM2. Activators of PKM2 can antagonize the radiation-induced rewiring of glucose metabolism and radiosensitize GBM cells in vitro and in vivo. These findings open the possibility that interventions designed to target cancer-specific regulators of metabolic plasticity, such as PKM2, rather than specific metabolic pathways, have the potential to improve the radiotherapeutic outcomes in GBM patients.

The "sweet" path to cancer: focus on cellular glucose metabolism.

The hypoxia-inducible factor-1α (HIF-1α), a key player in the adaptive regulation of energy metabolism, and the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2), a critical regulator of glucose consumption, are the main drivers of the metabolic rewiring in cancer cells. The use of glycolysis rather than oxidative phosphorylation, even in the presence of oxygen (i.e., Warburg effect or aerobic glycolysis), is a major metabolic hallmark of cancer. Aerobic glycolysis is also important for the immune system, which is involved in both metabolic disorders development and tumorigenesis. More recently, metabolic changes resembling the Warburg effect have been described in diabetes mellitus (DM). Scientists from different disciplines are looking for ways to interfere with these cellular metabolic rearrangements and reverse the pathological processes underlying their disease of interest. As cancer is overtaking cardiovascular disease as the leading cause of excess death in DM, and biological links between DM and cancer are incompletely understood, cellular glucose metabolism may be a promising field to explore in search of connections between cardiometabolic and cancer diseases. In this mini-review, we present the state-of-the-art on the role of the Warburg effect, HIF-1α, and PKM2 in cancer, inflammation, and DM to encourage multidisciplinary research to advance fundamental understanding in biology and pathways implicated in the link between DM and cancer.

Development of isoselenazolium chlorides as selective pyruvate kinase isoform M2 inhibitors

Alterations in cancer metabolic pathways open up an opportunity for targeted and effective elimination of tumor cells. Pyruvate kinase M2 (PKM2) is predominantly expressed in proliferating cells and plays an essential role in directing glucose metabolism in cancer. Here, we report the design of novel class of selective PKM2 inhibitors as anti-cancer agents and their mechanism of action. Compound 5c being the most active with IC50 = 0.35 ± 0.07 μM, also downregulates PKM2 mRNA expression, modulates mitochondrial functionality, induces oxidative burst and is cytotoxic for various cancer types. Isoselenazolium chlorides have an unusual mechanism of PKM2 inhibition, inducing a functionally deficient tetrameric assembly, while exhibiting a competitive inhibitor character. The discovery of robust PKM2 inhibitors not only offers candidates for anticancer therapy but is also crucial for studying the role of PKM2 in cancer.

Scutellarein treats neuroblastoma by regulating the expression of multiple targets.

The aim of this study is to investigate the effect of scutellarein on the proliferation of neuroblastoma cells and the underlying mechanism.Six cell lines were used with drug intervention.Cell Counting Kit-8 was used to select the best, namely,SH-SY5Y,and thenits IC50 value was determined. To further investigate the mechanism of scutellarin affecting SH-SY5Y proliferation, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of 11 factors.Scutellarin administration with 300 μM significantly reduced the number of SH-SY5Y, especially on the 3rd day of exposure to scutellarin. TheIC50 value of scutellarin in SH-SY5Y cells was determined to be117.8 μM. But the practical results showed that 300 μM was the optimal concentration of scutellarin. qRT-PCR further detectedupregulated maternally expressed gene 3(MEG3), oncogene c-Fos(c-FOS),and c-jun and downregulated M2 isoform of pyruvate kinase (PKM2), non-SMC Condensin I Complex Subunit H(NCAPH), epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1, and TGF-α, suggesting that scutellarin with 300 μM volume inhibited the survival of SH-SY5Y by regulating the expression of these 8 factors.Scutellarin could be a novel drug for the treatment of neuroblastoma, and its underlying mechanism may be related to the upregulated levels of MEG3, c-FOS,and c-jun and downregulatedthe expressionof PKM2, NCAPH, EGFR, TGF-β1,and TGF-α.

Design, Synthesis and Biological Evaluation of Quinoline-8-Sulfonamides as Inhibitors of the Tumor Cell-Specific M2 Isoform of Pyruvate Kinase: Preliminary Study.

Cancer cells need to carefully regulate their metabolism to keep them growing and dividing under the influence of different nutrients and oxygen levels. Muscle isoform 2 of pyruvate kinase (PKM2) is a key glycolytic enzyme involved in the generation of ATP and is critical for cancer metabolism. PKM2 is expressed in many human tumors and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various modulators regulate PKM2, shifting it between highly active and less active states. In the presented work, a series of 8-quinolinesulfonamide derivatives of PKM2 modulators were designed using molecular docking and molecular dynamics techniques. New compounds were synthesized using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Compound 9a was identified in in silico studies as a potent modulator of muscle isoform 2 of pyruvate kinase. The results obtained from in vitro experiments confirmed the ability of compound 9a to reduce the intracellular pyruvate level in A549 lung cancer cells with simultaneous impact on cancer cell viability and cell-cycle phase distribution. Moreover, compound 9a exhibited more cytotoxicity on cancer cells than normal cells, pointing to high selectivity in the mode of action. These findings indicate that the introduction of another quinolinyl fragment to the modulator molecule may have a significant impact on pyruvate levels in cancer cells and provides further directions for future research to find novel analogs suitable for clinical applications in cancer treatment.

USP25-PKM2-glycolysis axis contributes to ischemia reperfusion-induced acute kidney injury by promoting M1-like macrophage polarization and proinflammatory response

M1-like macrophages have been reported to play critical roles in acute kidney injury (AKI). Here, we elucidated the role of ubiquitin-specific protease 25 (USP25) in M1-like macrophages polarization and AKI. High USP25 expression was correlated with a decline in renal function in patients with acute kidney tubular injury and in mice with AKI. In contrast, USP25 knockout reduced M1-like macrophage infiltration, suppressed M1-like polarization, and improved AKI in mice, indicating that USP25 was necessary for M1-like polarization and proinflammatory response. Immunoprecipitation assay and liquid chromatography-tandem mass spectrometry showed that the M2 isoform of pyruvate kinase, muscle (PKM2) was a target substrate of USP25. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated the USP25 regulated aerobic glycolysis and lactate production during M1-like polarization via PKM2. Further analysis showed that the USP25-PKM2-aerobic glycolysis axis positively regulated M1-like polarization and exacerbated AKI in mice, providing potential therapeutic targets for AKI treatment.

Neurotoxicity of ischemic astrocytes involves STAT3-mediated metabolic switching and depends on glycogen usage.

Astrocytic responses are critical for the maintenance of neuronal networks in health and disease. In stroke, reactive astrocytes undergo functional changes potentially contributing to secondary neurodegeneration, but the mechanisms of astrocyte-mediated neurotoxicity remain elusive. Here, we investigated metabolic reprogramming in astrocytes following ischemia-reperfusion in vitro, explored their role in synaptic degeneration, and verified the key findings in a mouse model of stroke. Using indirect cocultures of primary mouse astrocytes and neurons, we demonstrate that transcription factor STAT3 controls metabolic switching in ischemic astrocytes promoting lactate-directed glycolysis and hindering mitochondrial function. Upregulation of astrocytic STAT3 signaling associated with nuclear translocation of pyruvate kinase isoform M2 and hypoxia response element activation. Reprogrammed thereby, the ischemic astrocytes induced mitochondrial respiration failure in neurons and triggered glutamatergic synapse loss, which was prevented by inhibiting astrocytic STAT3 signaling with Stattic. The rescuing effect of Stattic relied on the ability of astrocytes to utilize glycogen bodies as an alternative metabolic source supporting mitochondrial function. After focal cerebral ischemia in mice, astrocytic STAT3 activation was associated with secondary synaptic degeneration in the perilesional cortex. Inflammatory preconditioning with LPS increased astrocytic glycogen content, reduced synaptic degeneration, and promoted neuroprotection post stroke. Our data indicate the central role of STAT3 signaling and glycogen usage in reactive astrogliosis and suggest novel targets for restorative stroke therapy.

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.

Structural and mechanistic insights into cancer promoting activities of pyruvate kinase muscle isoform-2.

Pyruvate Kinase catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate with the production of ATP in the final reaction of glycolysis. The M2 isoform of the PK gene is significantly upregulated in cancer tissues and is a critical regulator of Warburg effect. The tetrameric form of PKM2 has higher kinase activity than the dimer, which supports the growth of tumor by nuclear translocation. The study aims to determine how different conditions such as an alkaline pH (prevalent in cancer cells), and a hyperthermic environment (shown to kill tumor cells) can aid or alleviate cancer progression, by acting via PKM2 which is a central protein in cancer. An alkaline pH of 8.0 was shown to alter the oligomerization state of the protein, promoting the formation of the nuclear-translocating and cancer-promoting dimeric form. The kinase activity was also hampered supporting the higher oncoprotein function. On the other hand, a hyperthermic environment of 42°C was shown to induce changes in the overall structure and stability of PKM2 at secondary and tertiary level, ascertained via Circular Dichroism and Thermal Fluorimetry. As the protein was unable to retain its structure at the slightly higher temperature, the oncoprotein activity of the protein was also obliterated, which may hint towards a possible pathway through which the therapeutic hyperthermia works on cancer cells. Deeper analysis of these mechanisms was performed by testing these conditions with cancer patient-derived mutants of PKM2. The mutants were also crystallized and diffracted at <3Å resolution, which will be solved to gain in-depth structural insights on this protein. This study will unravel novel mechanisms and structural details of PKM2 which will aid in better understanding of the role of PKM2 in the manifestation of disease.

S-palmitoylation dependent self assembly of human aquaporin-4 orthogonal arrays of particles in biomimetic membranes.

Aquaporin-4 (AQP4) is a water channel protein located in the plasma membrane of astrocytes in the central nervous system (CNS) and helps to regulate water-ion homeostasis. Isoforms of AQP4 further order themselves into aggregates known as orthogonal arrays of particles (OAPs). AQP4 exists in two main isoforms, M1 and M23, with M23 being the isoform that favors stabilization by aggregation into OAPs. It has been hypothesized that the aggregation size is regulated by the amount of M1 isoform present in the membrane. In turn, the palmitoylation state of two cysteine residues on the N-terminal tail of the M1 isoform greatly affects its mobility and ability to inhibit OAP formation. In this study, M1 was chemically cleaved and palmitoylated in vitro, and inserted in biomimetic lipid bilayers to study kinetics and aggregation behavior using single-protein fluorescence tracking. This study showed that when M1 was palmitoylated, it displays fast diffusion through the membrane with short lived protein-protein interactions and no evidence of aggregation. The depalmitoylated form of M1 showed evidence of extensive aggregation and long-lived protein-protein interactions. This study is the first of its kind to investigate human AQP4 in vitro and to extract the kinetic rate constants for protein-protein interactions as well as estimates of their equilibrium constants.

Expression of PKM2 in wound keratinocytes is coupled to angiogenesis during skin repair in vivo and in HaCaT keratinocytes in vitro

An injured skin is rapidly restored in a manner of wound healing. We have previously shown that intact insulin signaling and glucose uptake are fundamental to proper wound closure. Consequently, under exacerbated inflammation, compromised insulin action and glucose uptake lead to impaired healing. However, in spite of the increased attention to cell metabolism during tissue regeneration, metabolic mediators that govern cellular and physiological processes throughout skin repair remained largely elusive. Through assessment of mRNA using real-time PCR and protein blot analysis, we report that healing of cutaneous wounds comprise a boosted expression of genes involved in glycolysis, oxidative phosphorylation, pentose phosphate shunt, and glutamine anaplerosis. We further focused on the functional role of pyruvate kinase M (PKM) isoenzymes that catalyze the final and rate-limiting step of glycolysis. Whereas the expression of the metabolic constitutively active Pkm1 isozyme remained almost unchanged, Pkm2 is augmented during the inflammatory phase of healing. The immunohistochemistry and RNA in situ hybridization analysis showed a confined Pkm2 expression to keratinocytes of the hyperproliferative epithelium and, to a lesser extent, infiltrating neutrophils and monocytes as well as later on in macrophages. Notably, the expression of Pkm2 in keratinocytes facing the wound bed side colocalized with VEGF expression. The in vitro knockdown of PKM2 in HaCaT keratinocytes using small interfering (si) RNA confirmed an acute role for PKM2 in facilitating the complete induction of VEGF mRNA and protein expression in keratinocytes; this function is mainly HIF-1α independent.Key messages• Wound healing involves activation of glycolysis, oxidative phosphorylation, pentos-phosphate shunt, and replenishment of tri-carboxylic acid (TCA) cycle through glutamine anaplerosis.• The pyruvate kinase M2 (PKM2) isoform is upregulated during the inflammatory phase of cutaneous healing, mainly in keratinocytes of hyperproliferative epithelia.• In vivo, the expression of VEGF in wound keratinocytes is colocalized with PKM2.• PKM2 is required for full induction of VEGF in HaCaT keratinocytes in vitro.

Screening Kinase-Dependent Phosphorylation of Key Metabolic Reprogramming Regulators.

Aerobic glycolysis has been commonly linked to cell proliferation, especially in cancer cells where it serves to generate sufficient energy and biosynthesis of new cell constituents needed for cell growth and division. The M2 isoform of pyruvate kinase (PKM2) catalyzes the last reaction of the glycolytic process. PKM2 promotes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, generating ATP and releasing pyruvate. This rate-limiting reaction relies therefore on the enzymatic activity of PKM2. The switching between the high- and low-activity states of PKM2 is subjected to a combination of allosteric mechanisms and fine-tuned regulation by oncogenes and tumor suppressor genes. These regulatory mechanisms involve primarily post-translational modifications of PKM2. Recent findings suggest that phosphorylation contributes to the regulation of PKM2 activity.Here, we describe an in vitro kinase assay we used to assess PKM2 phosphorylation by c-Jun N-terminal kinase (JNK), a master regulator of apoptosis, cell proliferation, and differentiation. While the use of phospho-specific antibodies gives information in terms of measuring the effects of a given kinase on its substrate, specific antibodies for newly identified phospho-groups are not readily available. The in vitro kinase assay allows the immediate measuring of phosphorylation of any substrate of interest. Although there are several options that do not use radioactive materials, we continue to rely on this biochemical method for robust quantitation of results. More interestingly, this protocol can be easily adapted to measure the activity of other kinases by using their specific substrates.

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.

Shikonin reverses pyruvate kinase isoform M2-mediated propranolol resistance in infantile hemangioma through reactive oxygen species-induced autophagic dysfunction.

Infantile hemangioma (IH) is the most common benign tumor in infancy. Propranolol, a nonselective β-adrenergic receptor blocker, is now the first-line therapy for IH. Recently, low sensitivity to propranolol therapy has become one major reason for the failure of IH treatment. However, the exact underlying mechanisms are yet to be fully elucidated. Here, we reported that pyruvate kinase isoform M2 (PKM2), an essential glycolytic enzyme, played a critical role in regulating the progression of IH and the therapeutic resistance of propranolol treatment. Shikonin reversed the propranolol resistance in hemangioma-derived endothelial cells and in hemangioma animal models. Moreover, shikonin combined with propranolol could induce excessive reactive oxygen species (ROS) accumulation and lead to autophagic dysfunction, which is essential for the enhanced therapeutic sensitivity of propranolol treatment. Taken together, our results indicated that PKM2 has a significant role in hemangiomas progression and therapeutic resistance; it could be a safe and effective therapeutic strategy for those hemangiomas with poor propranolol sensitivity combined with shikonin.

Protein kinase C epsilon promotes de novo lipogenesis and tumor growth in prostate cancer cells by regulating the phosphorylation and nuclear translocation of pyruvate kinase isoform M2

Protein kinase C epsilon (PKCε) belongs to a family of serine/threonine kinases that control cell proliferation, differentiation and survival. Aberrant PKCε activation and overexpression is a frequent feature of numerous cancers. However, its role in regulation of lipid metabolism in cancer cells remains elusive. Here we report a novel function of PKCε in regulating of prostate cancer cell proliferation by modulation of PKM2-mediated de novo lipogenesis. We show that PKCε promotes de novo lipogenesis and tumor cell proliferation via upregulation of lipogenic enzymes and lipid contents in prostate cancer cells. Mechanistically, PKCε interacts with NABD (1–388) domain of C-terminal deletion on pyruvate kinase isoform M2 (PKM2) and enhances the Tyr105 phosphorylation of PKM2, leading to its nuclear localization. Moreover, forced expression of mutant Tyr105 (Y105F) or PKM2 inhibition suppressed de novo lipogenesis and cell proliferation induced by overexpression of PKCε in prostate cancer cells. In a murine tumor model, inhibitor of PKM2 antagonizes lipogenic enzymes expression and prostate cancer growth induced by overexpression of PKCε in vivo. These data indicate that PKCε is a critical regulator of de novo lipogenesis, which may represent a potential therapeutic target for the treatment of prostate cancer.

131 (PB121) - The PKM2 activator and molecular glue TP-1454 modulates tumor-immune responses by destabilizing T-regulatory cells

Background: Pyruvate kinase is an important enzyme that catalyzes the last step of glycolysis. The M2 isoform (PKM2) is important for balancing respiration and biosynthesis, which can be achieved by switching between the highly active tetrameric form and the less active dimeric form through allosteric metabolite binding. In addition to its role in metabolic regulation, the dimeric form of PKM2 can translocate to the nucleus, altering transcription to enhance cancer cells’ ability to grow and evade immune detection.