Comparative study of TNF-α and vitamin D reveals a significant role of TNF-α in NSCLC in an ethnically conserved vitamin D deficient population

Abstract

Growing evidence has implicated tumor necrosis factor-alpha (TNF-α) as an important regulator of the tumor microenvironment. Moreover, various molecular epidemiological studies have proposed vitamin D deficiency to be a mediator of cancer progression. Here we comparatively analyzed the role of TNF-α and vitamin D in non-small cell lung cancer (NSCLC) in an ethnically conserved vitamin D deficient population. Confirmed NSCLC cases (n = 190) matchedfor age, gender, dwelling, and smoking against cancer-free healthy controls ((n = 200) were genotyped for TNF-α promoter polymorphisms (rs361525 and rs1800629) by PCR-RFLP. 48 NSCLC tumor and adjacent normal tissues were quantified for TNF-α mRNA expression by RT-qPCR. 48 NSCLC cases and 60 healthy controls were analyzed for TNF-α and vitamin D serum levels by ELISA and chemiluminescence respectively. Our study indicates thatrs361525 and rs1800629 bear a significant risk towards NSCLC. Both mutant genotype and mutant allele of rs361525 elicit a likelihood of NSCLC reflected by their odds ratio (OR) of 3.16 and 1.81 respectively. In case of rs1800629, the heterogeneous genotype (GA) showed two fold higher risk for NSCLC (OR-2.07, P = 0.006), which could be attributed to the presence of the mutant allele as reflected by overall frequency of mutant A allele vs wild G allele (OR-1.92, P = 0.01). A combined effect of genotypes for rs361525 and rs1800629 revealed a 3.7 fold higher risk towards NSCLC for the presence of heterozygous genotype at both loci. Our preliminary expression results showed significant increase of TNF-α mRNA expression in tumor tissues of NSCLC as compared to adjacent normal tissues [cases- 8.56 ± 3.90vs controls-4.88 ± 2.96, P < 0.0001)] which was further affirmed by extrapolation of TNF-α expression in serum (Cases- 55.75 ± 22.50vs controls- 21.46 ± 27.75, P < 0.0001). Multivariate regression analyses revealed TNF-α mRNA expression to be significantly associated with NSCLC cases less than 50 years of age (P < 0.05). In comparison to the putative role of TNF-α in NSCLC as suggested by the results observed, vitamin D showed no significance towards any of the analyzed parameters or with the risk of NSCLC. This study suggests that TNF-α could be a potential mediator of NSCLC which bears important clinical implications and could be an important therapeutic marker in NSCLC.

Keywords: Chemiluminescence; ELISA; Multivariate Logistic Regression; NSCLC; PCR-RFLP; TNF-α polymorphism; TNF-α serum levels; Vitamin D serum levels; mRNA expression.