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Mitochondrial phosphoenolpyruvate carboxykinase (PCK2), a mitochondrial isoenzyme, supports the growth of cancer cells under glucose deficiency conditions in vitro. This study investigated the role and potential mechanism of PCK2 in the occurrence and development of Hepatocellular carcinoma (HCC). The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and other databases distinguish the expression of PCK2 and verified by qRT-PCR and Western blotting. Kaplan–Meier was conducted to assess PCK2 survival in HCC. The potential biological function of PCK2 was verified by enrichment analysis and gene set enrichment analysis (GSEA). The correlation between PCK2 expression and immune invasion and checkpoint was found by utilizing Tumor Immune Estimation Resource (TIMER). Lastly, the effects of PCK2 on the proliferation and metastasis of hepatocellular carcinoma cells were evaluated by cell tests, and the expressions of Epithelial mesenchymal transformation (EMT) and apoptosis related proteins were detected. PCK2 is down-regulated in HCC, indicating a poor prognosis. PCK2 gene mutation accounted for 1.3% of HCC. Functional enrichment analysis indicated the potential of PCK2 as a metabolism-related therapeutic target. Subsequently, we identified several signaling pathways related to the biological function of PCK2. The involvement of PCK2 in immune regulation was verified and key immune checkpoints were predicted. Ultimately, after PCK2 knockdown, cell proliferation and migration were significantly increased, and N-cadherin and vimentin expression were increased. PCK2 has been implicated in immune regulation, proliferation, and metastasis of hepatocellular carcinoma, and is emerging as a novel predictive biomarker and metabolic-related clinical target.

Acquired resistance remains a bottleneck for molecular‐targeted therapy in advanced hepatocellular carcinoma (HCC). Metabolic adaptation and epigenetic remodeling are recognized as hallmarks of cancer that may contribute to acquired resistance. In various lenvatinib‐resistant models, increased glycolysis leads to lactate accumulation and lysine lactylation of IGF2BP3. This lactylation is crucial for capturing PCK2 and NRF2 mRNAs, thereby enhancing their expression. This process reprograms serine metabolism and strengthens the antioxidant defense system. Additionally, altered serine metabolism increases the availability of methylated substrates, such as S‐adenosylmethionine (SAM), for N6‐methyladenosine (m6A) methylation of PCK2 and NRF2 mRNAs. The lactylated IGF2BP3‐PCK2‐SAM‐m6A loop maintains elevated PCK2 and NRF2 levels, enhancing the antioxidant system and promoting lenvatinib resistance in HCC. Treatment with liposomes carrying siRNAs targeting IGF2BP3 or the glycolysis inhibitor 2‐DG restored lenvatinib sensitivity in vivo. These findings highlight the connection between metabolic reprogramming and epigenetic regulation and suggest that targeting metabolic pathways may offer new strategies to overcome lenvatinib resistance in HCC. This study reveals that in lenvatinib‐resistant hepatocellular carcinoma, increased glycolysis results in lactate accumulation and lysine lactylation of IGF2BP3, which increase the expression of PCK2 and NRF2. This leads to a reprogramming of serine metabolism, S‐adenosylmethionine (SAM) production, RNA m6A modification, and the antioxidant system. The IGF2BP3 lactylation‐PCK2‐SAM‐m6A loop sustains the upregulation of PCK2 and NRF2 expression and ultimately confers lenvatinib resistance.

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets (GSE28829, GSE43292, GSE100927, and GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation . Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 ( ) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in knockout (KO) mice by wire injury and identified expression in human femoral artery atheroma. Among six DEGs, only and showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with siRNA. RNA sequencing of siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of -KO mice and was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.

Despite the growing interest in Phosphoenolpyruvate carboxykinase 2 (PCK2) as a potential biomarker in cancer research, studies on its clinical relevance and biological processes in glioblastoma are still unexplored. Three main glioma cohorts (TCGA, CGGA, Rembrandt) were extracted to exploit the association between PCK2 expression and clinical relevance through Kaplan-Meier survival analysis, univariate and multivariate cox regression analysis. Immunohistochemistry was used to detect PCK2 expression in glioma samples. GSEA, Pearson correlation and ROC analysis were performed to verify the specificity of PCK2 in mesenchymal GBM. Gene set variation analysis and CIBERSORT were used to explore the correlation of tumor-infiltrating immune cells according to PCK2 expression. Double immunofluorescence was performed to testify the co-expression patterns across PCK2, CD11C and PD-L1 in GBM tissues. PCK2 is increasingly expressed in GBM tissues and could serve as an independent poor prognostic indicator for glioma patients. PCK2 is preferentially expressed in mesenchymal subtype and correlates with immune infiltrates and immunosuppression in glioblastoma. Furthermore, PCK2 exhibits a positive correlation with dendritic cell infiltration and is co-expressed with CD11C and PD-L1 in the peritumoral region of the GBM tissues. Additionally, the enrichment of dendritic cell signature is associated with poor prognosis in glioblastoma patients. Our study highlights the potential therapeutic applicability of PCK2 and PCK2 mediated dendritic cell infiltration as a mechanism for glioblastoma immunosuppression.

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M–dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation.

Gluconeogenesis, mediated by phosphoenolpyruvate carboxykinase (PCK2), promotes anabolic metabolism in lung cancer cells in the absence of glucose. Here we show considerable heterogeneity of the utilization of gluconeogenesis or glycolysis in human non‐small cell lung cancers (NSCLC) and NSCLC metastases and a localization of PCK2 at tumor margins. We identify hypoxia as an important modulator of both pathways in NSCLC cells. Inhibition of glycolysis has been considered as a therapeutic approach in aggressive cancers including lung cancer. Abbreviated gluconeogenesis, mediated by phosphoenolpyruvate carboxykinase (PEPCK), was recently discovered to partially circumvent the need for glycolysis in lung cancer cells. However, the interplay of glycolysis and gluconeogenesis in lung cancer is still poorly understood. Here, we analyzed the expression of GLUT1, the prime glucose transporter, and of PCK1 and PCK2, the cytoplasmic and mitochondrial isoforms of PEPCK, in 450 samples of non‐small cell lung cancer (NSCLC) and in 54 NSCLC metastases using tissue microarrays and whole tumor sections. Spatial distribution was assessed by automated image analysis. Additionally, glycolytic and gluconeogenic gene expression was inferred from The Cancer Genome Atlas (TCGA) datasets. We found that PCK2 was preferentially expressed in the lung adenocarcinoma subtype, while GLUT1 expression was higher in squamous cell carcinoma. GLUT1 and PCK2 were inversely correlated, GLUT1 showing elevated expression in larger tumors while PCK2 was highest in smaller tumors. However, a mixed phenotype showing the presence of both, glycolytic and gluconeogenic cancer cells was frequent. In lung adenocarcinoma, PCK2 expression was associated with significantly improved overall survival, while the opposite was found for GLUT1. The metabolic tumor microenvironment and the 3‐dimensional context play an important role in modulating both pathways, since PCK2 expression preferentially occurred at the tumor margin and hypoxia regulated both, glycolysis and gluconeogenesis, in NSCLC cells in vitro, albeit in opposite directions. PCK1/2 expression was enhanced in metastases compared to primary tumors, possibly related to the different environment. The results of this study show that glycolysis and gluconeogenesis are activated in NSCLC in a tumor size and oxygenation modulated manner and differentially correlate with outcome. The frequent co‐activation of gluconeogenesis and glycolysis in NSCLC should be considered in potential future therapeutic strategies targeting cancer cell metabolism.

Osteosarcoma (OS) is the most common primary bone malignancy, predominately affecting children and adolescents. Due to the introduction of chemotherapy, the 5-year survival rate of OS patients has dramatically improved to 60-70%. Unfortunately, OS patients with recurrence or metastatic disease have less than a 20% chance of long-term survival, despite aggressive therapies. In this study, we aimed to identify gene expression patterns associated with metastasis and recurrence in order to identify potential biomarkers with prognostic power. We found that high expression of polyglutamine tract-binding protein 1 ( ) and low expression of phosphoenolpyruvate carboxykinase 2 ( ) were related to a high probability of recurrence and metastasis in OS patients and also predicted shorter recurrence-free survival (RFS) and metastasis-free survival (MFS) after adjustment for other clinical variables. Prediction models based on the combination of and expression had good and robust predictive power for recurrence and metastasis. A PQBP1 and PCK2-centered protein interaction network was built, and the hypothetical regulatory path between them was identified and termed the PQBP1-SF3A2-UBA52-PCK2 axis. Gene enrichment analysis indicated that aberrations of metabolism might play an important role in recurrence and metastasis in OS patients. Accordingly, PQBP1 and PCK2 are crucial for recurrence and metastasis in OS, and these findings provide a molecular basis for the exploitation of diagnostic and therapeutic strategies for overcoming recurrence and metastasis in OS.

Nucleus accumbens-associated protein 1 (NAC1), a cancer-related transcriptional regulator, is overexpressed in several malignancies, including ovarian cancer. However, its role in ovarian carcinogenesis remains unclear. We aimed to investigate whether NAC1 contributes to metabolic adaptation in endometriosis-related ovarian neoplasms (ERONs) and elucidate its regulatory mechanisms. The clinical relationship between NAC1 and its potential downstream target, phosphoenolpyruvate carboxykinase isoform 2 (PCK2), was examined using immunohistochemical analysis of ovarian cancer specimens. A cell viability assay was performed to clarify the impact of PCK2 on ovarian cancer cell viability. Reporter and chromatin immunoprecipitation (ChIP) assays were conducted to evaluate transcriptional regulation by NAC1. Metabolomic profiling was performed to assess the functional impact of the NAC1–PCK2 axis. A positive correlation between NAC1 and PCK2 expression was observed, and co-expression was associated with poor long-term survival. Knockdown of PCK2 led to a significant reduction in cell viability, indicating that PCK2 is required for maintaining cell survival. Reporter and ChIP assays confirmed that NAC1 directly binds to the PCK2 promoter via the CATG motif. The metabolomic analysis demonstrated that NAC1 promotes truncated gluconeogenesis and de novo serine synthesis through PCK2 upregulation. These findings suggest that NAC1 contributes to ovarian cancer progression by promoting metabolic adaptation, highlighting the NAC1–PCK2 axis as a potential therapeutic target for ERONs.

Purpose Metabolic rewiring in malignant transformation is often accompanied by altered expression of metabolic isozymes. Phosphoenolpyruvate carboxykinase-2 (PCK2) catalyzes the rate-limiting step of gluconeogenesis and is the dominant isoform in many cancers including triple-negative breast cancer (TNBC). Our goal was to identify small molecule inhibitors of PCK2 enzyme activity. Methods We assessed the impact of PCK2 down regulation with shRNA on TNBC cell growth in vitro and used AtomNet® deep convolutional neural network software to identify potential small molecule inhibitors of PCK2-based structure. We iteratively tested candidate compounds in an in vitro PCK-2 enzyme assay. The impact of the top hit on metabolic flux and cell viability was also assessed. Results PCK2 downregulation decreased growth of BT-549 and MDA-MB-231 cells and reduced metabolic flux through pyruvate carboxylase. The first AtomNet® in silico structural screen of 7 million compounds yielded 86 structures that were tested in PCK2 enzyme assay in vitro. The top hit ( IC 50  = 2.4 µM) was used to refine a second round of in silico screen that yielded 82 candidates to be tested in vitro, which resulted in 45 molecules with inhibition > 20%. In the second in vitro screen we also included 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, previously suggested to be PCK2 inhibitor based on structure, which emerged as the top hit. The specificity of this compound was tested in PCK1 and PCK2 enzymatic assays and showed IC 50 of 500 nM and 3.5–27 nM for PCK1 and PCK2, respectively. Conclusion 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is a high affinity PCK2 enzyme inhibitor that also has significant growth inhibitory activity in breast cell lines in vitro and represents a potential therapeutic lead compound.

Tumors have significant abnormalities in various biological properties. In renal cell carcinoma (RCC), metabolic abnormalities are characteristic biological dysfunction that cannot be ignored. Despite this, many aspects of this dysfunction have not been fully explained. The purpose of this study was to reveal a new mechanism of metabolic and energy-related biological abnormalities in RCC. Molecular screening and bioinformatics analysis were performed in RCC based on data from The Cancer Genome Atlas (TCGA) database. Regulated pathways were investigated by qRT-PCR, immunoblot analysis and immunohistochemistry. A series of functional analyses was performed in cell lines and xenograft models. By screening the biological abnormality core dataset-mitochondria-related dataset and the metabolic abnormality core dataset-energy metabolism-related dataset in public RCC databases, PCK2 was found to be differentially expressed in RCC compared with normal tissue. Further analysis by the TCGA database showed that PCK2 was significantly downregulated in RCC and predicted a poor prognosis. Through additional studies, it was found that a low expression of PCK2 in RCC was caused by methylation of its promoter region. Restoration of PCK2 expression in RCC cells repressed tumor progression and increased their sensitivity to sunitinib. Finally, mechanistic investigations indicated that PCK2 mediated the above processes by promoting endoplasmic reticulum stress. Collectively, our results identify a specific mechanism by which PCK2 suppresses the progression of renal cell carcinoma (RCC) and increases sensitivity to sunitinib by promoting endoplasmic reticulum stress. This finding provides a new biomarker for RCC as well as novel targets and strategies for the treatment of RCC.