Rameshwar N. K. Bamezai

Identifying Recent Adaptations in Large-Scale Genomic Data

Although several hundred regions of the human genome harbor signals of positive natural selection, few of the relevant adaptive traits and variants have been elucidated. Using full-genome sequence variation from the 1000 Genomes (1000G) Project and the composite of multiple signals (CMS) test, we investigated 412 candidate signals and leveraged functional annotation, protein structure modeling, epigenetics, and association studies to identify and extensively annotate candidate causal variants. The resulting catalog provides a tractable list for experimental follow-up; it includes 35 high-scoring nonsynonymous variants, 59 variants associated with expression levels of a nearby coding gene or lincRNA, and numerous variants associated with susceptibility to infectious disease and other phenotypes. We experimentally characterized one candidate nonsynonymous variant in Toll-like receptor 5 (TLR5) and show that it leads to altered NF-κB signaling in response to bacterial flagellin. PAPERFLICK:

Human pyruvate kinase M2: A multifunctional protein

Glycolysis, a central metabolic pathway, harbors evolutionary conserved enzymes that modulate and potentially shift the cellular metabolism on requirement. Pyruvate kinase, which catalyzes the last but rate‐limiting step of glycolysis, is expressed in four isozymic forms, depending on the tissue requirement. M2 isoform (PKM2) is exclusively expressed in embryonic and adult dividing/tumor cells. This tetrameric allosterically regulated isoform is intrinsically designed to downregulate its activity by subunit dissociation (into dimer), which results in partial inhibition of glycolysis at the last step. This accumulates all upstream glycolytic intermediates as an anabolic feed for synthesis of lipids and nucleic acids, whereas reassociation of PKM2 into active tetramer replenishes the normal catabolism as a feedback after cell division. In addition, involvement of this enzyme in a variety of pathways, protein–protein interactions, and nuclear transport suggests its potential to perform multiple nonglycolytic functions with diverse implications, although multidimensional role of this protein is as yet not fully explored. This review aims to provide an overview of the involvement of PKM2 in various physiological pathways with possible functional implications.

Leprosy and the adaptation of human toll-like receptor 1.

Leprosy is an infectious disease caused by the obligate intracellular pathogen Mycobacterium leprae and remains endemic in many parts of the world. Despite several major studies on susceptibility to leprosy, few genomic loci have been replicated independently. We have conducted an association analysis of more than 1,500 individuals from different case-control and family studies, and observed consistent associations between genetic variants in both TLR1 and the HLA-DRB1/DQA1 regions with susceptibility to leprosy (TLR1 I602S, case-control P = 5.7×10−8, OR = 0.31, 95% CI = 0.20–0.48, and HLA-DQA1 rs1071630, case-control P = 4.9×10−14, OR = 0.43, 95% CI = 0.35–0.54). The effect sizes of these associations suggest that TLR1 and HLA-DRB1/DQA1 are major susceptibility genes in susceptibility to leprosy. Further population differentiation analysis shows that the TLR1 locus is extremely differentiated. The protective dysfunctional 602S allele is rare in Africa but expands to become the dominant allele among individuals of European descent. This supports the hypothesis that this locus may be under selection from mycobacteria or other pathogens that are recognized by TLR1 and its co-receptors. These observations provide insight into the long standing host-pathogen relationship between human and mycobacteria and highlight the key role of the TLR pathway in infectious diseases.

Pyruvate kinase M2 and cancer: an updated assessment

Cancer cells are characterized by high glycolytic rates to support energy regeneration and anabolic metabolism, along with the expression of pyruvate kinase isoenzyme M2 (PKM2). The latter catalyzes the last step of glycolysis and reprograms the glycolytic flux to feed the special metabolic demands of proliferating cells. Besides, PKM2 has moonlight functions, such as gene transcription, favoring cancer. Accumulating evidence suggests a critical role played by the low‐activity‐dimeric PKM2 in tumor progression, supported by the identification of mutations which result in the down‐regulation of its activity and tumorigenesis in a nude mouse model. This review discusses PKM2 regulation and the benefits it confers to cancer cells. Further, conflicting views on PKM2s role in cancer, its therapeutic relevance and future directions in the field are also discussed.

Role of H2AX in DNA damage response and human cancers.

H2AX, the evolutionarily conserved variant of histone H2A, has been identified as one of the key histones to undergo various post-translational modifications in response to DNA double-strand breaks (DSBs). By virtue of these modifications, that include acetylation, phosphorylation and ubiquitination, H2AX marks the damaged DNA double helix, facilitating local recruitment and retention of DNA repair and chromatin remodeling factors to restore genomic integrity. These modifications are essential for effective DSB repair, so is their removal for cell, to recover from checkpoint arrest. Because of these vital roles during DSB signaling and also its activation during early cancer stages, H2AX is emerging as an intriguing gene in tumor biology, supported further by frequent deletion of the region harboring this gene. This review focuses on the insights gained from recent studies on dynamic regulation of H2AX in DSB repair. Also, posing future challenges in the area of chromatin reorganization and retention of epigenetic signature post-DSB-repair with implication of its haploinsufficiency in human cancers.

IL-10 promoter single nucleotide polymorphisms are significantly associated with resistance to leprosy

The minor haplotype −3575A/-2849G/-2763C in IL-10 promoter has been defined as a marker of disease resistance to leprosy and its severity in Brazilian population. Our investigation of six single-nucleotide polymorphisms (SNPs) in IL-10 promoter in 282 Indian leprosy patients and 266 healthy controls by direct PCR sequencing, however, showed that the extended haplotype: −3575T/-2849G/-2763C/-1082A/-819C/-592C was associated with resistance to leprosy per se and to the development of severe form of leprosy, using either a binomial (controls vs cases, P=0.01, OR=0.58, CI=0.37–0.89) or ordinal (controls vs paucibacillary vs multibacillary, P=0.004) model. Whereas, IL-10 haplotype −3575T/-2849G/-2763C/-1082A/-819T/-592A was associated with the risk of development of severe form of leprosy (P=0.0002) in contrast to the minor risk haplotype −3575T/-2849A/-2763C in the Brazilian population. The role of IL-10 promoter SNPs in Brazilian and Indian population strongly suggests the involvement of IL-10 locus in the outcome of leprosy.

miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2 : a potential for therapeutic intervention

IntroductionNew levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored.MethodsChanges in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2.ResultsIt was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2.Conclusionsmir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.

p53 mutation profile of squamous cell carcinomas of the esophagus in Kashmir (India): a high-incidence area.

Esophageal squamous cell carcinoma (ESCC) has been reported to show geographical variation in its incidence, even within areas of ethnic homogeneity. Kashmir valley, in north of India, has been described as a high‐risk area for ESCC. Here, we make a preliminary attempt to study mutations in exons 5–8 (the DNA binding domain) of the tumor suppressor gene, p53, in 55 ESCC patients from Kashmir. Polymerase chain reaction followed by direct sequencing analysis revealed the presence of mutations in 36.36% (20/55) tumors, assessed for the extent of allelic instability. The 20 mutations, found in 20 patients, comprised of 17 single‐base substitutions (11 transitions + 6 transversions) and 3 deletions. The 17 single‐base variations represented 12 missense mutations, 2 nonsense mutations and 3 variations located in intron 6, 1 of which resulted in a splicing variant. The patients when compared for the incidence of p53 mutation with various demographic features revealed females to be at increased risk (p = 0.016; OR = 4.13; 95% CI = 1.26–13.46). Comparison of mutation profile with other high‐risk areas reflected both differences and similarities indicating coexposure to a unique set of risk factors. This might be due to the special dietary and cultural practices of Kashmir that needs validation, as does the gender‐based difference in the incidence of p53 mutation observed in this study.

Resveratrol Inhibits Cancer Cell Metabolism by Down Regulating Pyruvate Kinase M2 via Inhibition of Mammalian Target of Rapamycin

Metabolism of cancer cells with pyruvate kinase M2 (PKM2) at its centre stage has assumed a prime significance in cancer research in recent times. Cancer cell metabolism, characterized by enhanced glucose uptake, production of lactate and anabolism is considered an ideal target for therapeutic interventions. Expression of PKM2 switches metabolism in favor of cancer cells, therefore, the present study was designed to investigate the hitherto unknown effect of resveratrol, a phytoalexin, on PKM2 expression and resultant implications on cancer metabolism. We observed that resveratrol down-regulated PKM2 expression by inhibiting mTOR signaling and suppressed cancer metabolism, adjudged by decreased glucose uptake, lactate production (aerobic glycolysis) and reduced anabolism (macromolecule synthesis) in various cancer cell lines. A contingent decrease in intracellular levels of ribose-5-phosphate (R5P), a critical intermediate of pentose phosphate pathway, accounted for a reduced anabolism. Consequently, the state of suppressed cancer metabolism resulted in decreased cellular proliferation. Interestingly, shRNA-mediated silencing of PKM2 inhibited glucose uptake and lactate production, providing evidence for the critical role of PKM2 and its mediation in the observed effects of resveratrol on cancer metabolism. Further, an over-expression of PKM2 abolished the observed effects of resveratrol, signifying the role of PKM2 downregulation as a critical function of resveratrol. The study reports a novel PKM2-mediated effect of resveratrol on cancer metabolism and provides a new dimension to its therapeutic potential.

Insulin enhances metabolic capacities of cancer cells by dual regulation of glycolytic enzyme pyruvate kinase M2

BackgroundInsulin is tightly associated with cancer progression; however, mechanistic insights into such observations are poorly understood. Recent studies show that metabolic transformation is critical to cancer cell proliferation. Here, we attempt to understand the role of insulin in promotion of cancer metabolism. To this end, the role of insulin in regulating glycolytic enzyme pyruvate kinase M2 (PKM2) was examined.ResultsWe observed that insulin up-regulated PKM2 expression, through PI3K/mTOR mediated HIF1α induction, but significantly reduced PKM2 activity independent of this pathway. Drop in PKM2 activity was attributed to subunit dissociation leading to formation of low activity PKM2 oligomers, as assessed by density gradient centrifugation. However, tyrosine 105 phosphorylation of PKM2, known for inhibiting PKM2 activity, remained unaffected on insulin treatment. Interestingly, insulin-induced ROS was found responsible for PKM2 activity reduction. The observed changes in PKM2 status led to augmented cancer metabolism. Insulin-induced PKM2 up-regulation resulted in enhanced aerobic glycolysis as confirmed by PKM2 knockdown studies. Further, PKM2 activity reduction led to characteristic pooling of glycolytic intermediates and increased accumulation of NADPH; suggesting diversion of glucose flux towards macromolecular synthesis, necessary for cancer cell growth.ConclusionThe study identifies new PKM2-mediated effects of insulin on cancer metabolism, thus, advancing the understanding of insulin’s role in cancer.