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.

Allicin Overcomes Hypoxia Mediated Cisplatin Resistance in Lung Cancer Cells through ROS Mediated Cell Death Pathway and by Suppressing Hypoxia Inducible Factors.

BACKGROUND/AIMS: The hypoxic microenvironment in NSCLC has been widely accepted as a contributor to both therapeutic resistance and tumor progression. In this study, we have explored Allicin, a key organosulfur compound present in garlic for its previously unreported effectiveness in the heterogeneous hypoxic tumor microenvironment of NSCLC. METHODS: The effect of Allicin on the viability of NSCLC cells was determined by MTT assay. To determine the migration rate of treated cells compared to the control, scratch and transwell migration assays were performed. Flowcytometry was done to explore cell cycle distribution, apoptosis and ROS production in cells. Fluorescence microscopy was used to examine autophagy and DNA damage in cells. Dot blot was done to check genome wide methylation. RNA expression was detected by RT-PCR and protein expression by western blotting. RESULTS: Allicin significantly decreases cell viability, proliferation and migration of NSCLC cells in both normoxia and hypoxia. It elicits both apoptosis and autophagy pathway in A549 cells by ROS accumulation and facilitating S/G2-M phase arrest in both normoxia as well as hypoxia. We suggest that ROS/MAPK and ROS/JNK signaling pathway together govern the cytotoxic effect of allicin in NSCLC cells. Notably, allicin suppresses the expression of HIF-1α and HIF-2α in hypoxic cells, pointing towards a mechanism of its effectiveness in hypoxia. A long term passive demethylation was observed, with decreased mC and no change in TET expression, thereby ruling out active demethylation by allicin. Furthermore, allicin synergistically enhances growth inhibitory activity of low dose cisplatin to effectively overcome hypoxia induced cisplatin resistance in A549 cells. CONCLUSION: Altogether, our results elucidate a potential use of allicin in sensitizing hypoxic and chemoresistant NSCLC to cisplatin-based chemotherapy and provide new, affordable therapeutic strategy with reduced side effects.

SNP rs16969968 as a Strong Predictor of Nicotine Dependence and Lung Cancer Risk in a North Indian Population

Background: The 15q24-25 loci contain genes (CHRNA5 and CHRNA3) encoding nicotinic acetylcholine receptor subunits. We here determined for the first time the association of genetic variants rs16969968 and rs3743074 in CHRNA5 and CHRNA3, respectively, on nicotine dependence and lung cancer risk in a North Indian population by a case-control approach. Methods: Venous blood samples were obtained from 324 participants (108 lung cancer patients and 216 healthy individuals). DNA was extracted and PCR amplified with primers flanking the SNPs rs16969968 and rs3743074. Amplicons were subjected to sequencing and logistic regression was used to analyze association between variables. Results: The risk variant SNP rs16969968 in both heterozygous and homozygous forms appeared to exert a significant effect on nicotine dependence [GA (OR=2.77) and AA (OR=2.53)]. As expected, smoking was strongly associated with lung cancer (OR= 2.62). Risk allele rs16969968 in CHRNA5 also showed a significant association with increased lung cancer risk in our cohort, alone (OR= 4.99) and with smoking as a co-variable (OR= 4.28). Comparison of our analysis with other populations suggested that individuals with rs16969968 risk allele in the Indian population are more susceptible to lung cancer. Conclusion: Overall, the results strongly indicated that, in our cohort North Indian population, the genetic variant rs16969968, but not rs3743074, is significantly associated with both nicotine dependence and increased risk of lung cancer. While the results are significant, there is further need to increase the sample size and improve precision of our risk prediction.

Scriptaid overcomes hypoxia-induced cisplatin resistance in both wild-type and mutant p53 lung cancer cells.

Non-small cell lung cancer (NSCLC), comprising 85% of lung cancer cases, has been associated with resistance to chemo/radiotherapy. The hypoxic tumor micro-environment, where insufficient vasculature results in poor drug penetrance and sub-optimal chemotherapy in the tumor interiors contributes heavily to this resistance. Additionally, epigenetic changes in tumorigenic cells also change their response to different forms of therapy. In our study, we have investigated the effectiveness of a combination of cisplatin with scriptaid [a pan-Histone Deacetylase inhibitor (HDACi)] in a model that mimics the tumor microenvironment of hypoxia and sub-lethal chemotherapy. Scriptaid synergistically increases the efficacy of cisplatin in normoxia as well as hypoxia, accompanied with reduced metastasis and enhanced DNA damage. Addition of scriptaid also overcomes the cisplatin resistance exhibited in lung cancer cells with stabilized hypoxia inducible factor 1 (HIF1)-α (mutant) and mutant p53. Molecular studies showed that the combination treatment increased apoptotic cell death in both normoxia and hypoxia with a dual role of p38MAPK. Together, our results suggest that the combination of low dose cisplatin and scriptaid is cytotoxic to NSCLC lines, can overcome hypoxia induced resistance and mutant p53- induced instability often associated with this cancer, and has the potential to be an effective therapeutic modality.

Transition metal oxide nanoparticles are effective in inhibiting lung cancer cell survival in the hypoxic tumor microenvironment.

BACKGROUND: The transition metal oxide nanoparticles are in focus for their anti-cancer potential. In this study we have synthesized and characterized CuO, NiO and Fe2O3 nanoparticles and, investigated their cytotoxic potential in the heterogeneous tumour microenvironment. METHODS: Nanoparticles were synthesized by aqueous precipitation method and characterized with UV-Visible spectrophotometer, Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) and X-ray diffraction (XRD). Cell viability of lung cancer cells (A549) grown in normoxia (18%O2) and hypoxia (1%O2) was determined for all nanoparticles. The mechanism of cell death was assessed by nuclear morphological analysis, flow cytometry analysis and western blotting. Generation of intracellular ROS in treated cells and its contribution to cell viability was determined. RESULTS: The synthesized metal oxide nanoparticles were successfully characterized with SEM, spectroscopy and X-ray diffraction patterns. Cell viability of lung cancer cells was compromised in both normoxia and hypoxia. ROS generation was shown to contribute to cellular toxicity in CuO, but not NiO and Fe2O3. CONCLUSION: We have shown the therapeutic potential of CuO, NiO and Fe2O3 nanoparticles in non small cell lung cancer cells cultured in hypoxia, a relevant feature of solid tumors along with normoxia. The newly synthesized nanoparticles showed efficacy in both conditions. GENERAL SIGNIFICANCE: Hypoxia drives metabolic alterations and epigenetic modifications in the tumor microenvironment. By using conditions that mimic tumour microenvironment, this study expands the possibility of using metal oxide nanoparticles as a therapeutic agent for lung cancer treatment.