Hypoxia-Mediated Epigenetic Regulation of Stemness in Brain Tumor Cells.

Activation of pluripotency regulatory circuit is an important event in solid tumor progression and the hypoxic microenvironment is known to enhance the stemness feature of some cells. The distinct population of cancer stem cells (CSCs)/tumor initiating cells exist in a niche and augment invasion, metastasis, and drug resistance. Previously, studies have reported global hypomethylation and site-specific aberrant methylation in gliomas along with other epigenetic modifications as important contributors to genomic instability during glioma progression. Here, we have demonstrated the role of hypoxia-mediated epigenetic modifications in regulating expression of core pluripotency factors, OCT4 and NANOG, in glioma cells. We observe hypoxia-mediated induction of demethylases, ten-eleven-translocation (TET) 1 and 3, but not TET2 in our cell-line model. Immunoprecipitation studies reveal active demethylation and direct binding of TET1 and 3 at the Oct4 and Nanog regulatory regions. Tet1 and 3 silencing assays further confirmed induction of the pluripotency pathway involving Oct4, Nanog, and Stat3, by these paralogues, although with varying degrees. Knockdown of Tet1 and Tet3 inhibited the formation of neurospheres in hypoxic conditions. We observed independent roles of TET1 and TET3 in differentially regulating pluripotency and differentiation associated genes in hypoxia. Overall, this study demonstrates an active demethylation in hypoxia by TET1 and 3 as a mechanism of Oct4 and Nanog overexpression thus contributing to the formation of CSCs in gliomas. Stem Cells 2017;35:1468-1478.

When "No-Smoking" is not enough: Hypoxia and nicotine acetylcholine receptor signaling may drive lung adenocarcinoma progression in never-smokers.

The question of how lung cancer progresses in never-smokers remains largely unanswered. In our analysis of data from 1727 lung cancer patients, we observed a difference of only 47 days in the overall survival between lung adenocarcinoma patients who were smokers vis-a-vis never-smokers - the disease has a poor prognosis irrespective of the smoking status, or gender. We have investigated the possible collaboration between the nAChR and hypoxia signaling pathway to explicate a mechanism of disease progression in never-smokers using patient-derived tumor cells. We found a previously unidentified increase in both acetylcholine and nAChR-α7 levels in non-small cell lung cancer cells in hypoxia. A similar increase in ubiquitously expressed nAChR-α7 transcripts was also observed in other cancer lines and primary tumor tissues. A direct binding of HIF-1α with the hypoxia-response element (HRE) present at -48 position preceding the transcriptional start site in nAChR-α7 promoter region was established. Crucially, the increased acetylcholine levels in hypoxia drove a feedback loop via modulation of PI3K/AKT pathway to stabilize HIF-1α in hypoxia. Further, hypoxia-mediated metastasis and induction of HIF-1α in these cells was significantly reversed by bungarotoxin, an antagonist of nAChR-α7. The nAChR-AKT-HIF network needs to be further investigated to conclusively prove its mechanism and to explore its therapeutic potential. Our study gives a plausible explanation for the equally worse prognosis of lung adenocarcinoma in never-smokers wherein the nAChR signaling is enhanced in hypoxia by acetylcholine in the absence of nicotine.

SNP rs9387478 at ROS1-DCBLD1 Locus is Significantly Associated with Lung Cancer Risk and Poor Survival in Indian Population.

OBJECTIVE: Receptor tyrosine kinases (RTK) are relevant therapeutic targets in the treatment of lung cancer. Germline susceptibility variants that influence these RTKs may provide new insights into their regulation.  rs9387478 is located in the genomic interval between two RTK-genes ROS1/DCBLD1, of which ROS1 alterations are implicated in lung carcinogenesis and treatment response while the latter remains poorly understood. MATERIALS AND METHODS: Venous blood was drawn from 100 control and 231 case subjects. Genotype was scored by restriction fragment length polymorphism (RFLP), PCR amplification followed by HindIII digestion. Logistic regression was applied to compare the association between variables. Survival curve was plotted to draw a correlation between the genotype and overall survival. Also, eQTL and chromatin state changes were analyzed and correlated with the survival of patients using available datasets. RESULTS: In our population smoking correlated significantly with lung cancer [OR= 2.607] with the presence of the minor allele 'A' enhancing the nicotine dependence [CA (OR=3.23)]. Individuals with homozygous risk allele 'A' had a higher chance of developing lung cancer [OR=2.65] than individuals with CA/CC implying a recessive model of association. Patients with CC/CA genotype had better overall survival than patients with AA genotype [161 days/142 days vs 54 days, p=0.005]. The homozygous risk allele was significantly associated with increased DCBLD1 and ROS1 expression in lung cancer, with enriched active histone marks due to the polymorphism. Interestingly, increased DCBLD1 expression was associated with poor outcomes in lung cancer. CONCLUSION: Overall, our study provides strong evidence that rs9387478 is significantly associated with both nicotine dependence and lung cancer in our North Indian cohort. The association of the SNP with prognostic genes, DCBLD1 and ROS1 make rs9387478 a promising prognostic marker in the North Indian population. The results obtained are significant, however, the study needs to be performed in a larger sample size.