Tobacco-related Oral Squamous Cell Carcinoma Research Articles

Association of DFNA5, SYK, and NELL1 variants along with HPV infection in oral cancer among the prolonged tobacco-chewers.

Southeast Asia, especially India, is well known for the highest use of smokeless tobacco. These products are known to induce oral squamous cell carcinoma. However, not all long-term tobacco-chewers develop oral squamous cell carcinoma. In addition, germline variants play a crucial role in susceptibility, prognosis, development, and progression of the disease. These prompted us to study the genetic susceptibility to oral squamous cell carcinoma among the long-term tobacco-chewers. Here, we presented a retrospective study on prolonged tobacco-chewers of Northeast India to identify the potential protective or risk-associated germline variants in tobacco-related oral squamous cell carcinoma along with HPV infection. Targeted re-sequencing (n = 60) of 170 genetic regions from 75 genes was carried out in Ion-PGM™ and validation (n = 116) of the observed variants was done using Sequenom iPLEX MassARRAY™ platform followed by polymerase chain reaction-based HPV genotyping and p16-immunohistochemistry study. Subsequently, estimation of population structure, different statistical and in silico approaches were undertaken. We identified one nonsense-mediated mRNA decay transcript variant in the DFNA5 region (rs2237306), associated with Benzo(a)pyrene, as a protective factor (odds ratio = 0.33; p = 0.009) and four harmful (odds ratio > 2.5; p < 0.05) intronic variants, rs182361, rs290974, and rs169724 in SYK and rs1670661 in NELL1 region, involved in genetic susceptibility to tobacco- and HPV-mediated oral oncogenesis. Among the oral squamous cell carcinoma patients, 12.6% (11/87) were HPV positive, out of which 45.5% (5/11) were HPV16-infected, 27.3% (3/11) were HPV18-infected, and 27.3% (3/11) had an infection of both subtypes. Multifactor dimensionality reduction analysis showed that the interactions among HPV and NELL1 variant rs1670661 with age and gender augmented the risk of both non-tobacco- and tobacco-related oral squamous cell carcinoma, respectively. These suggest that HPV infection may be one of the important risk factors for oral squamous cell carcinoma in this population. Finally, we newly report a DFNA5 variant probably conferring protection via nonsense-mediated mRNA decay pathway against tobacco-related oral squamous cell carcinoma. Thus, the analytical approach used here can be useful in predicting the population-specific significant variants associated with oral squamous cell carcinoma in any heterogeneous population.

Nicotinic acetylcholine receptor gene polymorphism is not associated with tobacco-related oral squamous cell carcinoma.

Nicotinic acetylcholine receptor is implicated in carcinogenesis indirectly through increasing nicotine dependence and directly through its impact on cell-cycle regulation. Functional polymorphism in nicotinic acetylcholine receptor alpha-5 subunit gene (CHRNA5 c.1192G>A; rs16969968) is associated with nicotine dependence and risk of lung cancer. The aim of this study was to evaluate the association of CHRNA5 c.1192G>A polymorphism with the risk of oral squamous cell carcinoma (OSCC). This was a rural teaching hospital-based case-control study. A total of 100 histopathologically confirmed cases of OSCC patients and 100 age- and gender-matched healthy individuals were genotyped for CHRNA5 c.1192G>A polymorphism by polymerase chain reaction-restriction fragment length polymorphism method. Allele and genotype frequencies among case and control groups were compared by Chi-squared test (Fisher's exact). The frequency of CHRNA5 c.1192A allele was 22% in OSCC patients and 26% in control individuals. The difference in the distribution of alleles and genotypes between case and control groups was not significant (P > 0.05). CHRNA5 c.1192G>A polymorphism is not associated with the risk of developing OSCC.

Abstract A67: Phenotypic and functional dynamics of CD4+CD25+FOXP3+ regulatory T cells in patients with tobacco-related oral squamous cell carcinoma

Abstract Background: Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers worldwide. Dysregulation of immune response plays a major role in establishment and development of cancer. CD4+CD25+FoxP3+ regulatory T cells (Tregs) have been reported to downregulate effector immune functions and its inactivation have been associated with development of autoimmune diseases. We therefore investigated the phenotypic and functional dynamics of CD4+CD25+FoxP3+ (Tregs) cells in tobacco-related OSCC patients. Aim: Aim of the study was to investigate the phenotypic and functional characteristics of Tregs cells in OSCC patients in terms of their subsets, expression of functional markers and cytokine profile etc. Methods: The phenotypic and functional characteristics of Treg subsets in patients with OSCC were assessed by multicolour flowcytometry. The levels of TGFβ were assessed by realtime PCR and western blot. Results: An increased (p&amp;lt;0.05) prevalence of Tregs phenotypes (CD4+CD25+, CD4+FoxP3+, CD8+FoxP3+, CD4+CD25+FoxP3+) was observed in the peripheral circulation of OSCC patients that positively correlated with clinicopathological features. The increased frequency of CD4+CD8+CD25+FoxP3+, a unique T cells subset also showed significantly higher prevalence in OSCC patients. Functionally CD4+FoxP3+ Tregs showed skewed expression of IL-2, IL-10 and IL-35 in patients as compared with the normal controls. Higher expression of TGF-β in tumour tissues suggests their dominant role in upregulation of differentiation of Tregs from naive T cells in tumour bearing host. Further enhanced expression of CCR5 and CCR7 on Tregs with upregulation of their ligands (CCL5, CCL19 and CCL21) in tumour cells indicate efficient recruitment and trafficking of Tregs to the tumour site. Conclusions: Accumulation of heterogeneous subsets of Tregs in the peripheral circulation indicates their suggestive role in immuno-suppression leading to growth and proliferation of OSCC. Therefore manipulation of Tregs can be added to the therapeutic armamentarium for enhancing anti-tumour immunity in OSCC patients. Note: This abstract was not presented at the conference. Citation Format: Satya N. Das, Sadhna Aggarwal, Suresh C. Sharma. Phenotypic and functional dynamics of CD4+CD25+FOXP3+ regulatory T cells in patients with tobacco-related oral squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A67.

Peroxiredoxin 1 promotes invasion and migration by regulating epithelial-to-mesenchymal transition during oral carcinogenesis.

Tobacco smoking is the major risk factor for oral squamous cell carcinoma (OSCC). Previously, we found that nicotine up-regulates peroxiredoxin 1 (Prx1), an important antioxidant enzyme, and nuclear factor kappa B (NFκB) in OSCC cells. However, the molecular mechanism of Prx1 in oral carcinogenesis remains obscure. To improve our understanding of the functional role of Prx1 during the cascade of tobacco-associated oral carcinogenesis, we characterized Prx1, NFκB, and epithelial-to-mesenchymal transition (EMT) markers including E-cadherin, vimentin and Snail in 30 primary oral tumors (15 from smokers with OSCC and 15 from non-smokers with OSCC) and 10 normal oral mucosa specimens from healthy individuals. The expression levels of Prx1, nuclear NFκB, vimentin and Snail were higher in the tumors from smokers with OSCC than in those from non-smokers with OSCC or the healthy controls. The expression levels of E-cadherin showed an opposite trend. Prx1 silencing suppressed the nicotine-induced EMT, cell invasion and migration in SCC15 cells in vitro. Furthermore, Prx1 activated the NFκB pathway in SCC15 cells. Prx1 might therefore play an oncogenic role in tobacco-related OSCC and thus serve as a target for chemopreventive and therapeutic interventions.

Single nucleotide polymorphism in hMLH1 promoter and risk of tobacco-related oral carcinoma in high-risk Asian Indians

hMLH1 is a member of mismatch repair genes (MMR) that plays a crucial role in correcting replication errors, cell cycle arrest, apoptosis and oxidative stress. We explored the risk associated with hMLH1 −93 A>G (rs 1800734) single nucleotide polymorphism (SNP) with the oral squamous cell carcinoma (OSCC) in Asian Indians. We genotyped 242 patients with tobacco-related OSCC and 205 healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The frequency of AA genotype was found to be significantly (Pc<0.0006) lower in patients as compared to the controls (21.49% vs. 47.8%) while GG genotype showed significantly higher (Pc<0.0006) prevalence in patients as compared to the healthy controls (41.32% vs. 13.66%). In logistic regression analysis AG (adjusted OR=1.95, 95% CI=0.72–5.26) and GG genotype (adjusted OR=4.5, 95% CI=1.54–13.16, P=0.006) appeared susceptible when compared with the wild-type AA genotype. The allelic distribution showed that variant G allele is significantly higher (Pc<0.0004) in patients and associated with increased risk (adjusted OR=2.36, 95% CI=1.33–4.19, P=0.003) as compared to the wild-type A allele. Altogether, our results suggest that the hMLH1 −93 A>G polymorphism is associated with the higher risk of tobacco-related OSCC in Asian Indians and could be useful in screening population at a higher risk.

Functional genetic variants of CTLA-4 and risk of tobacco-related oral carcinoma in high-risk North Indian population

Single nucleotide polymorphisms (SNPs) of the CTLA-4 gene have been implicated in susceptibility to different cancer in different ethnic populations. We assessed the association of five SNPs [-1722C/T, -1661A/G and -318C/T in the promoter region49A/G in exon 1 and CT60A/G in the 3'untranslated region (UTR)] with tobacco-related oral squamous cell carcinoma (OSCC) in North Indian subjects. We genotyped 130 OSCC patients and 180 normal subjects by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) using BbvI, MseI, NcoI and BstEII restriction endonucleases. Among these SNPs, -1722CC, -1661AG and CT60AA genotypes were more prevalent in OSCC patients as compared to controls and in the logistic regression analysis with odd ratio (OR) 2.85, 95% CI (0.69-11.68); OR 2.48, 95% CI (1.29-4.78) and OR 3.0, 95% CI (1.43-6.28) respectively, these genotypes showed strong association with OSCC risk. With higher prevalence in controls 49GG genotype and G allele (OR 0.57, 95% CI 0.40-0.81) appeared to be protective. Moreover, TACAG, TACGA and TATAG appeared as susceptible while TACGG and CACGG appeared as protective haplotypes. These results suggest significant risk modifying effects of CTLA-4 -1722C/T, -1661A/G, -318T/C, CT60 A/G and 49A/G SNPs in tobacco-related OSCC in North Indian population.

Abnormal expression of PI3K isoforms in patients with tobacco-related oral squamous cell carcinoma

BackgroundThe phosphatidylinositol 3-kinase (PI3K) signaling regulates several cellular functions such as motility, proliferation, angiogenesis and survival. MethodsSince there is no information on expression of PI3K isoforms in oral cancer, we studied the expression of different isoforms of PI3K (p110α, p110γ, PI3K-C2, Vps34p and p85α) in tumor samples and PBMC by RT and q-RTPCR and serum levels of PI3K p110α by SPR and ELISA techniques in 108 patients with tobacco-related oral squamous cell carcinoma (OSCC) and 46 normal subjects. ResultsWe observed significantly higher PI3K p110α (p<0.0001) and lower (p<0.0001) vesicular sorting protein 34p (Vps34p) mRNA both in PBMC and tissue samples of oral cancer patients as compared to the normal controls. Other PI3K isoforms did not show such change. Circulating PI3K p110α levels were higher in patients (p<0.0001) as compared to healthy subjects, the SPR data showed direct correlation with advancing stage of the disease. PI3K p110α was overexpressed in tumor samples but not in the normal buccal mucosa. ConclusionsUpregulation of circulating PI3K p110α isoform and its direct correlation with increasing tumor load in OSCC patients indicates that it may be a significant prognostic indicator and a suitable target for therapeutic/chemo-preventive strategies for tobacco-related OSCC.

Deregulation of Beclin 1 in patients with tobacco-related oral squamous cell carcinoma

Autophagy is a physiologically regulated and evolutionary conserved process that plays a critical role in degradation of cytoplasmic proteins and other macromolecules within the lysosomes. Beclin-1, the mammalian orthologue of yeast Atg6, is an important mediator of autophagy that has been studied in many human cancers. However, the expression of Beclin-1 has not yet been investigated in oral cancer. We for the first time investigated the expression of Beclin-1 in serum and tissues and correlated it with the clinic-pathological features of oral cancer patients. m-RNA expression of Beclin-1 was evaluated in tumor and normal areas of surgical specimens from 10 oral cancer patients by real-time PCR. Approximately, 8-fold lower expression (p<0.001) of Beclin-1 mRNA was observed in tumor tissue as compared to the normal tissue. Serum levels of Beclin-1 were evaluated by SPR and ELISA. No significant difference was observed in serum Beclin-1 levels in patients as compared to healthy subjects, similarly no correlation was found between serum levels and clinic-pathological parameters such as stage, lymph node involvement and tumor size. Our results demonstrate that down-regulation of Beclin-1 may play an important role in the development and progression of oral cancer possibly by dysregulation of autophagy in tumor cells.

Poster #109 NEURO-IMMUNE CROSSTALK: GENETIC UNDERPINNINGS OF IMMUNE SYSTEM ACTIVATION WITH RESPECT TO FRONTAL LOBE COGNITIVE FUNCTIONS IN SCHIZOPHRENIA

Single nucleotide polymorphisms (SNPs) of the CTLA-4 gene have been implicated in susceptibility to different cancer in different ethnic populations. We assessed the association of five SNPs [−1722C/T, −1661A/G and −318C/T in the promoter region49A/G in exon 1 and CT60A/G in the 3′untranslated region (UTR)] with tobacco-related oral squamous cell carcinoma (OSCC) in North Indian subjects. We genotyped 130 OSCC patients and 180 normal subjects by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) using BbvI, MseI, NcoI and BstEII restriction endonucleases. Among these SNPs, −1722CC, −1661AG and CT60AA genotypes were more prevalent in OSCC patients as compared to controls and in the logistic regression analysis with odd ratio (OR) 2.85, 95% CI (0.69–11.68); OR 2.48, 95% CI (1.29–4.78) and OR 3.0, 95% CI (1.43–6.28) respectively, these genotypes showed strong association with OSCC risk. With higher prevalence in controls 49GG genotype and G allele (OR 0.57, 95% CI 0.40–0.81) appeared to be protective. Moreover, TACAG, TACGA and TATAG appeared as susceptible while TACGG and CACGG appeared as protective haplotypes. These results suggest significant risk modifying effects of CTLA-4 −1722C/T, −1661A/G, −318T/C, CT60 A/G and 49A/G SNPs in tobacco-related OSCC in North Indian population.

Functional genetic variants of TGF-β1 and risk of tobacco-related oral carcinoma in high-risk Asian Indians

Transforming growth factor (TGF)-β1, the most abundant isoform of TGF-β have been implicated in various stages of carcinogenesis such as epithelial to mesenchymal transition, enhanced expression of metalloproteases, down-regulation of cellular adhesion molecule, increased tumor motility and angiogenesis as well as local and systemic immunosuppression leading to a more aggressive and metastatic behavior. We assessed the association of TGF-β1 functional genetic polymorphisms at codon 10 (869 T>C) and 25 (915 G>C) of exon 1 in 140 patients with tobacco-related oral squamous cell carcinoma (OSCC) and 120 normal subjects by PCR-RFLP. The frequency of 869 CC genotype and C allele were significantly higher in patients as compared to controls (P(c), 0.024 and 0.0004, respectively) while no significant difference was observed in the frequency of 915 CC genotype and C allele. In logistic regression analysis CC genotype (OR, 3.87; 95% CI, 1.78-8.41) and C allele (OR, 2.20; 95% CI 1.51-3.20) appeared as susceptible while TT genotype and T allele as protective. In addition C(869)-C(915) haplotype with OR of 2.48 at 95% CI, 1.51-4.06 significantly (P=0.0003) increased the risk of tobacco-related OSCC in Asian Indians.

Functional variants of IL4 and IL6 genes and risk of tobacco‐related oral carcinoma in high‐risk Asian Indians

Tobacco-related oral squamous cell carcinoma (OSCC) is one of the most common cancers involving Indian males. We assessed the association of IL4 promoter -589 T>C, -33 T>C, and IL6-174 G>C functional genetic polymorphisms with tobacco-related OSCC in Asian Indians. The IL4 and IL6 promoter polymorphisms were assessed in 140 patients with OSCC and 120 normal subjects by PCR-RFLP technique, and significance of the data was determined using chi-square test. The frequency of TC, CC genotype, and C allele at IL4 promoter sites -589 and -33 were higher in patients when compared with controls. Consequently, TC/CC genotypes and C allele at both sites appeared as susceptible. However, IL6-174 G>C single-nucleotide polymorphisms (SNP) appeared to be protective in patients with OSCC. Of eight haplotypes, five were associated with two- to seven-fold increased risk of tobacco-related OSCC. These SNPs further showed heterogeneity among different ethnic population, but their distribution in Asian Indians stand closer to other Asian populations. In this study, IL4-589 CC, -33 CC genotype, and *C allele at both sites appeared to be susceptible, while IL6-174 CC genotype and *C allele appeared to be protective in patients with OSCC; hence, these SNPs may be a potential prognostic markers for tobacco-related OSCC in Asian Indians.

Functional variants of COX-2 and risk of tobacco-related oral squamous cell carcinoma in high-risk Asian Indians

We assessed the association of COX-2 polymorphisms at promoter sites -1195, -765 and at 3'UTR (8473) in 193 patients with oral squamous cell carcinoma (OSCC) and 137 normal subjects by PCR-RFLP. Although no significant difference was observed in the frequency of COX-2 -1195G>A, -765G>C and 8473C>T single nucleotide polymorphisms (SNPs) between patients and controls, COX-2 -1195G/A genotype showed higher while -765G/C and 8473C/T showed lower prevalence in patients as compared to normal subjects. Logistic regression analysis of these three polymorphisms revealed a quantitative risk associated with -1195G>A SNP (OR 3.07 at 95%CI) while -765G>C and 8473C>T appeared to be protective. Results of the present study indicate that these three functional variants in the COX-2 regulatory region may contribute to risk modification of tobacco-related oral squamous cell carcinoma in Asian Indians.

Association of TNF-α and TNFR1 promoters and 3′ UTR region of TNFR2 gene polymorphisms with genetic susceptibility to tobacco-related oral carcinoma in Asian Indians

Tobacco-related oral squamous cell carcinoma is a common malignancy in Asian people. It accounts for almost 40% of cancers among Indian men and 3% in the Western world. Smokeless tobacco has been shown to induce tumor necrosis factor-alpha (TNF-alpha), which, along with its receptors, is over-expressed in people with oral carcinoma. Single nucleotide polymorphisms (SNPs) in TNF-alpha and TNF receptor genes may affect their expression and may be a potential determinant of susceptibility to tobacco-related oral carcinomas. We assessed SNPs in TNF-alpha(-308, -238) and TNF receptor 1 (TNFR1; -609) promoters by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and at four sites of TNF receptor 2 gene (TNFR2; exon 9 site 1176; exon 10 sites 1663, 1668 and 1690) by PCR-sequence-specific primers (PCR-SSP) techniques, respectively, in 94 patients and 130 healthy controls. TNF-alpha-308 G allele was significantly lower (Pc=0.004; OR=3.85), whereas A allele was significantly higher (Pc=0.004; OR=0.25) in patients compared with controls. No significant change was observed at -238 promoter site between the two groups. In the case of TNF receptors, both TNFR1 -609 TT (Pc=0.006; OR=15.3) and TNFR2 1690 CT (Pc=0.018; OR=5.6) genotypes were significantly lower in patients compared with controls. It seems that TNF-alpha-308 G/A may be related to susceptibility, whereas -609 TT TNFR1 and 1690 C/T TNFR2 SNPs may be protective to tobacco-related oral squamous cell carcinoma. These SNPs may be useful as a marker for high-risk groups among Asian Indians.