Elevated expression of RUNX3 co-expressing with EZH2 in esophageal cancer patients from India.

BACKGROUND: Runt related transcription factor3 (RUNX3) is considered as a tumor suppressor gene (TSG) that functions through the TGF-ß dependent apoptosis. Promoter methylation of the CpG islands of RUNX3 and overexpression of enhancer of zeste homolog 2 (EZH2) has been suggested to downregulate RUNX3 in cancer. METHODS: Here, we studied the expression of RUNX3 and EZH2 in 58 esophageal tumors along with paired adjacent normal tissue. mRNA levels, protein expressions and cellular localizations of EZH2 and RUNX3 were analyzed using real-time PCR and immunohistochemistry, respectively. DNA methylation was further assessed by the methylation specific-PCR. RESULTS: Compared to normal tissue, a significant increase in expression of RUNX3 mRNA in 31/57 patient's tumor tissue (p < 0.04) was observed. The expression of EZH2 was found to be upregulated compared to normal, and a significant positive correlation between EZH2 and RUNX3 expression was observed (p = 0.002). 22 of the 27 unmethylated cases at the promoter region of the RUNX3 had elevated RUNX3 protein expression (p < 0.001). CONCLUSION: The data presented in this study provide new insights into the biology of RUNX3 and highlights the need to revisit our current understanding of the role of RUNX3 in cancer.

Pyruvate kinase M knockdown-induced signaling via AMP-activated protein kinase promotes mitochondrial biogenesis, autophagy, and cancer cell survival.

Preferential expression of the low-activity (dimeric) M2 isoform of pyruvate kinase (PK) over its constitutively active splice variant M1 isoform is considered critical for aerobic glycolysis in cancer cells. However, our results reported here indicate co-expression of PKM1 and PKM2 and their possible physical interaction in cancer cells. We show that knockdown of either PKM1 or PKM2 differentially affects net PK activity, viability, and cellular ATP levels of the lung carcinoma cell lines H1299 and A549. The stable knockdown of PK isoforms in A549 cells significantly reduced the cellular ATP level, whereas in H1299 cells the level of ATP was unaltered. Interestingly, the PKM1/2 knockdown in H1299 cells activated AMP-activated protein kinase (AMPK) signaling and stimulated mitochondrial biogenesis and autophagy to maintain energy homeostasis. In contrast, knocking down either of the PKM isoforms in A549 cells lacking LKB1, a serine/threonine protein kinase upstream of AMPK, failed to activate AMPK and sustain energy homeostasis and resulted in apoptosis. Moreover, in a similar genetic background of silenced PKM1 or PKM2, the knocking down of AMPKα1/2 catalytic subunit in H1299 cells induced apoptosis. Our findings help explain why previous targeting of PKM2 in cancer cells to control tumor growth has not met with the expected success. We suggest that this lack of success is because of AMPK-mediated energy metabolism rewiring, protecting cancer cell viability. On the basis of our observations, we propose an alternative therapeutic strategy of silencing either of the PKM isoforms along with AMPK in tumors.

WITHDRAWN: Regulation of Pyruvate Kinase M Switch towards PKM1 by LKB1-AMPK Axis Tolerates Hypoglycemic Stress in Cancer Cells.

This article has been withdrawn by the authors. The PKM2 immunoblot in Fig 2E was reused as part of the Caspase-3 immunoblot in Fig 9C. The PKM2 immunoblot from 5 mM Glu, fractions 1-10 was reused as the PKM2 immunoblot from 1 mM Glu, fractions 1-10. The actin immunoblot from A549 cells from Fig 5A was reused as the actin blot from Fig 7C.

Decreased expression of MGMT in correlation with aberrant DNA methylation in esophageal cancer patients from North India.

O-6-methylguanine-DNA methyltransferase, DNA repair gene, has been found to be involved with the pathogenesis of the esophageal cancer. DNA hypermethylation and other factors have been suggested to downregulate O-6-methylguanine-DNA methyltransferase. In this communication, the methylation status of O-6-methylguanine-DNA methyltransferase gene and the corresponding O-6-methylguanine-DNA methyltransferase protein expression in esophageal cancer from North India has been studied. In all, 80 samples of tumor tissue along with adjacent normal tissue as controls were analyzed for messenger RNA level of O-6-methylguanine-DNA methyltransferase gene, protein expression, and subcellular localization. The messenger RNA expression was studied using real-time quantitative polymerase chain reaction, protein expression, and its subcellular localization by Western blotting and immunohistochemistry. DNA methylation was assessed through methylation-specific polymerase chain reaction. Clinicopathological parameters were recorded and correlated with the O-6-methylguanine-DNA methyltransferase expression. O-6-methylguanine-DNA methyltransferase messenger RNA expression was found to be downregulated in 65% cases (52/80). The expression of O-6-methylguanine-DNA methyltransferase at the protein level was also found to be absent in 65% (52/80) cases. In all, 52 cases had low or no expression of the protein, whereas out of those 28 remaining cases, 11.25% (09/80) cases had high O-6-methylguanine-DNA methyltransferase protein expression. The absence of O-6-methylguanine-DNA methyltransferase protein coincided with the methylated cases in 84% (38/45), whereas in 07 cases, out of the 45 methylated, O-6-methylguanine-DNA methyltransferase protein was present. The aggressive esophageal cancer patients having methylated O-6-methylguanine-DNA methyltransferase had more than 50% cases with no/mild expression of the O-6-methylguanine-DNA methyltransferase protein ( p > 0.001). Loss of O-6-methylguanine-DNA methyltransferase protein was very frequent in the incidence of esophageal cancer from North Indian patients, and methylation of the promoter region of O-6-methylguanine-DNA methyltransferase was significantly associated in its downregulation.

ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells.

Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.

Protocol for performing metabolic pathway-based subtyping of breast tumors.

Here, we present a protocol for analyzing the global metabolic landscape in breast tumors for the purpose of metabolism-based patient stratification. We describe steps for analyzing 1,454 metabolic genes representing 90 metabolic pathways and subjecting them to an algorithm that calculates the deregulation score of 90 pathways in each tumor sample, thus converting gene-level information into pathway-level information. We then detail procedures for performing clustering analysis to identify metabolic subtypes and using machine learning to develop a signature representing each subtype. For complete details on the use and execution of this protocol, please refer to Iqbal et al.1.

HPV18 oncoproteins driven expression of PKM2 reprograms HeLa cell metabolism to maintain aerobic glycolysis and viability.

The molecular basis of human papillomavirus (HPV)-mediated cellular immortalization and malignant transformation has illustrated an indispensable role of viral E6/E7-oncoproteins. However, the impact of viral-oncoproteins on the metabolic phenotype of cancer cells remains ambiguous. We showed silencing of HPV18-encoded E6/E7-oncoprotein significantly reduced glucose consumption, lactate production, ATP level and viability. Silencing of HPV18-encoded E6/E7 in HeLa cells significantly down-regulated expression and activity of HK1, HK2, LDHA, and LDHB. Interestingly, there was an increased pyruvate kinase activity due to switch in expression from PKM2 isoform to PKM1. The switch in favor of alternatively spliced isoform PKM1, was regulated by viral-E6/E7-oncoprotein by inhibiting the c-Myc/hnRNP-axis. Further, the near absence of the PKM1 protein despite an adequate amount of PKM1 mRNA in HeLa cells was due to its proteasomal degradation. Our results suggests HPV18-encoded E6/E7 driven preferential expression of PKM2 is essential to support aerobic glycolysis and cell proliferation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00776-w.

Identification of plant-based hexokinase 2 inhibitors: combined molecular docking and dynamics simulation studies.

Cancer cells ferment glucose, even under aerobic conditions, following a phenomenon known as the 'Warburg effect.' Hexokinase 2 (HK2) catalyzes the crucial step of phosphorylation of glucose for subsequent utilization in glycolysis and other pathways. HK2 has been proposed as a potential therapeutic target for anti-cancer therapy because of its enhanced expression in glucose-dependent tumors. Here, we have employed structure-based virtual screening using in-house library to identify potential phytoconstituents which could inhibit the HK2 activity. The initial hits were selected based on their binding affinity towards HK2 using the molecular docking approach. Subsequently, the filters for physicochemical properties, PAINS patterns and PASS evaluation were applied to find potential hits against HK2. Finally, we have identified epigallocatechin gallate (EGCG) and quercitrin, two natural compounds with appreciable binding affinity, efficiency and specificity towards the HK2 binding pocket. Both compounds were found to be binding preferentially to the HK2 active site and showed a decent set of drug-like properties. All-atom molecular dynamics (MD) simulations for 100 ns were carried out to see the conformational dynamics, complexes stability and interaction mechanism of HK2 with EGCG and quercitrin. MD simulation results showed that HK2 forms stable protein-ligand complexes with EGCG and quercitrin with consistency throughout the trajectory. Overall, these findings suggest that EGCG and quercitrin might be further exploited as promising scaffolds in the drug development process against HK2..Communicated by Ramaswamy H. Sarma.

Dominant negative mutations affect oligomerization of human pyruvate kinase M2 isozyme and promote cellular growth and polyploidy.

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM(2)) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM(2) in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM(2) by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM(2) showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM(2) by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.

Phytocompounds Targeting Metabolic Reprogramming in Cancer: An Assessment of Role, Mechanisms, Pathways, and Therapeutic Relevance.

The metabolism of cancer is remarkably different from that of normal cells and confers a variety of benefits, including the promotion of other cancer hallmarks. As the rewired metabolism is a near-universal property of cancer cells, efforts are underway to exploit metabolic vulnerabilities for therapeutic benefits. In the continued search for safer and effective ways of cancer treatment, structurally diverse plant-based compounds have gained substantial attention. Here, we present an extensive assessment of the role of phytocompounds in modulating cancer metabolism and attempt to make a case for the use of plant-based compounds in targeting metabolic vulnerabilities of cancer. We discuss the pharmacological interactions of phytocompounds with major metabolic pathways and evaluate the role of phytocompounds in the regulation of growth signaling and transcriptional programs involved in the metabolic transformation of cancer. Lastly, we examine the potential of these compounds in the clinical management of cancer along with limitations and challenges.

Silibinin induces metabolic crisis in triple-negative breast cancer cells by modulating EGFR-MYC-TXNIP axis: potential therapeutic implications.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with limited treatment modalities and poor prognosis. Metabolic reprogramming in cancer is considered a hallmark of therapeutic relevance. Here, we report disruption of metabolic reprogramming in TNBC cells by silibinin via modulation of EGFR-MYC-TXNIP signaling. Metabolic assays combined with LC-MS-based metabolomics revealed inhibition of glycolysis and other key biosynthetic pathways by silibinin, to induce metabolic catastrophe in TNBC cells. Silibinin-induced metabolic suppression resulted in decreased cell biomass, proliferation, and stem cell properties. Mechanistically, we identify EGFR-MYC-TXNIP as an important regulator of TNBC metabolism and mediator of inhibitory effects of silibinin. Highlighting the clinical relevance of our observations, the analysis of METABRIC dataset revealed deregulation of EGFR-MYC-TXNIP axis in TNBC and association of EGFRhigh -MYChigh -TXNIPlow signature with aggressive glycolytic metabolism and poor disease-specific and metastasis-free survival. Importantly, combination treatment of silibinin or 2-deoxyglucose (glycolysis inhibitor) with paclitaxel synergistically inhibited proliferation of TNBC cells. Together, our results highlight the importance of EGFR-MYC-TXNIP axis in regulating TNBC metabolism, demonstrate the anti-TNBC activity of silibinin, and argue in favor of targeting metabolic vulnerabilities of TNBC, at least in combination with mainstay chemotherapeutic drugs, to effectively treat TNBC patients.

Multiomics integrative analysis reveals antagonistic roles of CBX2 and CBX7 in metabolic reprogramming of breast cancer.

Striking similarity exists between metabolic changes associated with embryogenesis and tumorigenesis. Chromobox proteins-CBX2/4/6/7/8, core components of canonical polycomb repressor complex 1, play essential roles in embryonic development and aberrantly expressed in breast cancer. Understanding how altered CBX expression relates to metabolic reprogramming in breast cancer may reveal vulnerabilities of therapeutic pertinence. Using transcriptomic and metabolomic data from breast cancer patients (N > 3000 combined), we performed pathway-based analysis and identified outstanding roles of CBX2 and CBX7 in positive and negative regulation of glucose metabolism, respectively. Genetic ablation experiments validated the contrasting roles of two isoforms in cancer metabolism and cell growth. Furthermore, we provide evidence for the role of mammalian target of rapamycin complex 1 signaling in mediating contrary effects of CBX2 and CBX7 on breast cancer metabolism. Underpinning the biological significance of metabolic roles, CBX2 and CBX7 were found to be the most up- and downregulated isoforms, respectively, in breast tumors compared with normal tissues. Moreover, CBX2 and CBX7 expression (not other isoforms) correlated strongly, but oppositely, with breast tumor subtype aggressiveness and the proliferation markers. Consistently, genomic data also showed higher amplification frequency of CBX2, not CBX7, in breast tumors. Highlighting the clinical significance of findings, disease-specific survival and drug sensitivity analysis revealed that CBX2 and CBX7 predicted patient outcome and sensitivity to FDA-approved/investigational drugs. In summary, this work identifies novel cross talk between CBX2/7 and breast tumor metabolism, and the results presented may have implications in strategies targeting breast cancer.

MicroRNA in lung cancer: role, mechanisms, pathways and therapeutic relevance.

Lung cancer is the cardinal cause of cancer-related deaths with restricted recourse of therapy throughout the world. Clinical success of therapies is not very promising due to - late diagnosis, limited therapeutic tools, relapse and the development of drug resistance. Recently, small ∼20-24 nucleotides molecules called microRNAs (miRNAs) have come into the limelight as they play outstanding role in the process of tumorigenesis by regulating cell cycle, metastasis, angiogenesis, metabolism and apoptosis. miRNAs essentially regulate gene expression via post-transcriptional regulation of mRNA. Nevertheless, few studies have conceded the role of miRNAs in activation of gene expression. A large body of data generated by numerous studies is suggestive of their tumor-suppressing, oncogenic, diagnostic and prognostic biomarker roles in lung cancer. They have also been implicated in regulating cancer cell metabolism and resistance or sensitivity towards chemotherapy and radiotherapy. Further, miRNAs have also been convoluted in regulation of immune checkpoints - Programmed death 1 (PD-1) and its ligand (PD-L1). These molecules play a significant role in tumor immune escape leading to the generation of a microenvironment favouring tumor growth and progression. Therefore, it is imperative to explore the expression of miRNA and understand its relevance in lung cancer and development of anti-cancer strategies (anti - miRs, miR mimics and micro RNA sponges). In view of the above, the role of miRNA in lung cancer has been dissected and the associated mechanisms and pathways are discussed in this review.

Posttranslational Modifications of Pyruvate Kinase M2: Tweaks that Benefit Cancer.

Cancer cells rewire metabolism to meet biosynthetic and energetic demands. The characteristic increase in glycolysis, i.e., Warburg effect, now considered as a hallmark, supports cancer in various ways. To attain such metabolic reshuffle, cancer cells preferentially re-express the M2 isoform of pyruvate kinase (PKM2, M2-PK) and alter its quaternary structure to generate less-active PKM2 dimers. The relatively inactive dimers cause the accumulation of glycolytic intermediates that are redirected into anabolic pathways. In addition, dimeric PKM2 also benefits cancer cells through various non-glycolytic moonlight functions, such as gene transcription, protein kinase activity, and redox balance. A large body of data have shown that several distinct posttranslation modifications (PTMs) regulate PKM2 in a way that benefits cancer growth, e.g., formation of PKM2 dimers. This review discusses the recent advancements in our understanding of various PTMs and the benefits they impart to the sustenance of cancer. Understanding the PTMs in PKM2 is crucial to assess their therapeutic potential and to design novel anticancer strategies.

Curcumin decreases Warburg effect in cancer cells by down-regulating pyruvate kinase M2 via mTOR-HIF1α inhibition.

Warburg effect is an emerging hallmark of cancer cells with pyruvate kinase M2 (PKM2) as its key regulator. Curcumin is an extensively-studied anti-cancer compound, however, its role in affecting cancer metabolism remains poorly understood. Herein, we show that curcumin inhibits glucose uptake and lactate production (Warburg effect) in a variety of cancer cell lines by down-regulating PKM2 expression, via inhibition of mTOR-HIF1α axis. Stable PKM2 silencing revealed that PKM2 is required for Warburg effect and proliferation of cancer cells. PKM2 over-expression abrogated the effects of curcumin, demonstrating that inhibition of Warburg effect by curcumin is PKM2-mediated. High PKM2 expression correlated strongly with poor overall survival in cancer, suggesting the requirement of PKM2 in cancer progression. The study unravels novel PKM2-mediated inhibitory effect of curcumin on metabolic capacities of cancer cells. To the best of our knowledge, this is the first study linking curcumin with PKM2-driven cancer glycolysis, thus, providing new perspectives into the mechanism of its anticancer activity.

Resveratrol inhibits TIGAR to promote ROS induced apoptosis and autophagy.

Resveratrol has been shown to exhibit its anti-cancer effect through a variety of mechanisms. Here, TIGAR (TP53-Induced Glycolysis and Apoptosis Regulator) was identified as an important target of resveratrol for exhibiting ROS-dependent-consequences on apoptosis and autophagy. Resveratrol treatment decreased TIGAR protein irrespective of cell line used. Down-regulated TIGAR protein triggered a drop in reduced-glutathione levels which resulted in sustained ROS, responsible for apoptosis and autophagy. Over-expression and silencing experiments demonstrated the importance of TIGAR in affecting the ROS-dependent anti-cancer effects of resveratrol. Resveratrol treated cells exhibited autophagy to escape apoptosis, however, chloroquine treatment along with resveratrol, blocked protective autophagy and facilitated apoptosis. Collectively, results unravel the effects of resveratrol on TIGAR in mediating its ROS dependent influence and suggest a better combination therapy of resveratrol and chloroquine for probable cancer treatment.