Genetic Analysis of NF-Kappa B Pathway in Head and Neck Cancer

Abstract

Head and neck cancer (HNC) is a subtype of cancers that affects the respiratory and upper digestive tracts, as well as the oral cavity, lips, oro-hypo-nasopharyngeal regions, and larynx at various anatomic sites. It is a common form of cancer that causes 330,000 fatalities and 650,000 new cases each year. According to the International Agency for Research on Cancer's (IARC) Global Cancer Statistics 2020, Around the world, 1.8% of all cancer deaths and 2% of all cancer diagnoses are caused by oral cancer. According to the Karachi Cancer Registry, the incidence of HNC in Pakistan in 2008 was 22.5% for men and 11.4% for women per 100,000 people (KCR, Pakistan). In Pakistan, there are 12.3 cases of head and neck cancer for every 100,000 cancer cases treated. The peak occurrence is, 22.6% in Sindh province, from there on Punjab, Islamabad, Baluchistan, and Khyber Pakhtunkhwa, respectively. Head and neck carcinomas (HNC), are a divergent, set of tumors, proceeding, as a consequence of exposure to speckled risk factors, both environmental and genetic. The aggressive potential of viral infections makes the viral genome adept at controlling and modifying, nuclear factor kappa B (NF-B) and other transcription factors that are expressed in cells. With the progression of the malignancy, NF-kappa B Inhibitor alpha (IκB) dysregulation is also observed.

NF-κB, a transcription factor that promotes inflammation, has been documented as an imperative contributor, considering the growth and advancement of malignant carcinomas. NF-κB targets downstream genes which are important promoters of tumor cell features like; proliferation, survival, metastasis, inflammation, invasion, and angiogenesis. Many human tumors display an atypical stimulation of NF-κB, which is associated, with a resilient phenotype and indigent prognosis. This insistent NF-κB stimulation might include signalling pathway defects, mutations, or chromosomal reshuffles. The principal objective, of the current study, was to provide insight, into the presence of selected, SNPs in NF-κB genes. Further, to understand the association of these SNPs, with NF-κB gene activity, if it is, contributing to cancer progression, and to explore, its role as DRSML QAU xi an effective molecular target, for detection and prospective inhibition of HNC. The third goal, of this study, was to conduct, predictive modeling of HNC, with variables, to scrutinize, the fit model for the data. The fourth objective was, to spot the NF-B signaling pathway protein’s expression and high-risk human papillomavirus (HR-HPV) variations in HNC patients with an HPV infection. The study's goal was to identify p50 polymorphisms as risk factors for both HNC cases with and without an HPV connection. Analysis of NF-κB pathway genes polymorphism in head and neck cancer was carried out by blood sampling, from different hospitals of Rawalpindi/Islamabad and Peshawar, with the informed consent of the patients and volunteers, in line with the guidelines of Declaration of Helsinki (II). From these blood samples, DNA was extracted, followed by PCR for the amplification of the study-relevant genes (P50, P56, and NF-κBIA), Single Standard Conformation Polymorphism (SSCP), and DNA sequencing for the investigation of SNPs. To understand, the genetic association of SNPs with HNC, PLINK v1.09 was used. The data was used, to make predictive models, for classifying the unseen case, as HNC, using packages in Python language. After gel electrophoresis, PCR was employed to genotype the HPV in blood samples from patients with head and neck cancer. Using the Exgene SV DNA extraction Kit, genomic DNA was also extracted from HNC tumour tissues before being subjected to PCR and gel electrophoresis. Later, for Immunohistochemistry (IHC), several antibodies were applied. Additionally, using specific antibodies and immunohistochemistry, the expression levels of the NF- κB pathway proteins P50, P65, and NF- κB IA in tissue samples from head and neck cancer were evaluated. For HNC samples that were HPV positive, these proteins were examined. The data had 470 controls and 471 cases, of HNC patients, which were included in SNP association analyses. SNP1(P50) on Chr:4 rs1598858 AG AA GG (A>G), compared to the controls, the frequency of the AG allele was greater in cases while AA was higher in controls. Similarly, in SNP2 (P50) detected on Chr:4, rs4648068 AG AA GG (A>G), AG allele frequency, was higher compared to the control group. SNP4(P50), detected in the current study on Chr:4 rs28362491 ATTG- ATTGATTG-- (ATTGATTG>ATTG-) insertion mutation, was detected and its frequency in cases was observed higher as compared to the control group. Frequency of SNP6(P65), detected on Chr:11 rs7101916 CC TT CT (C>T), allele CT was higher in cases as compared to control. The studied SNPs showed statistically significant at p<0.05. SNP on chromosome 4, DRSML QAU xii rs1598858, and rs4648068 showed an odd ratio higher than 1, and SNP rs7101916 on chromosome 11, also displayed, an odd ratio greater than 1. SNP8 (NF-κB IA) and SNP10 (NF-κB IA) on chromosome 14, rs696, and rs1957106 had an odd ratio of 3.036 and 2.066 respectively. OR greater than 1 predicts that these SNPs have a greater association with the disease and are directly associated with the occurrence of HNC in the subject. Model tests, for each SNP, in the studied population were performed, and the allelic model, finds an association of individual alleles, with the outcome. The genetic model, finds the association of the genotype, with the outcome. All the SNPs displayed, an association with the disease outcome, in a dominant manner. Expressional analysis, PCR, and IHC were carried out for HPV genotyping, NF-κB (P50, P65), and NF-B IA proteins. 37.3% (152) of the HNC patients were positive for HPV based on the PCR results. 66.9% of those who were HPV-negative and 86.5% of those who were HPV-positive smoked. HPV positivity and tobacco use were significantly associated, according to a univariate analysis [P = 0.01, odds ratio (OR) = 0.310, 95% CI = 0.110 to 0.870]. A higher percentage of HPV-positive patients (77.3%) had poor oral hygiene compared to those who practised good oral hygiene (21.6%) [P 0.03, OR: 2.440, 95% CI: 1.650 to 3.600]. The results of the logistic regression analysis show that age, smoking, and oral hygiene are all significant predictors of oral health (P 0.02). The results of PCR and IHC staining showed that HPV16 was more common in HNC cases (64.8%) than HPV18 (35.2%). In HPV- and non-HPV infected HNC tissues, expression of NF-B proteins (p50, p65, and IB inhibitor) was also noted. Contrary to the IkBa inhibitor, NF-κB, p50, and p65 expression during IHC showed nuclear staining. Protein expression was higher in HPV cases compared to non-HPV cases (P 0.05). From the study, it may be deduced that NF-κB genetic variations affect Pakistani population’s susceptibility to head and neck cancers. According to the study, environmental variables, NF-κB pathway protein expression, and poor hygiene are all linked to the use of tobacco, poor hygiene, and HPV infection. The activation of the NF κB signalling pathway caused by HPV infection, which results in HNC carcinogenesis, is still not fully understood, and more study with a larger sample size is needed to develop straightforward, trustworthy, and highly specific molecular biomarkers. HNC is Pakistan's next great health challenge, there is an immediate need, to introduce population-based cancer registration and methods for early detection. The disease can be controlled by mobilising the health-services infrastructure, preserving hygienic conditions, and human resources to hold disease awareness seminars that can lower the risk of developing HNC in the local community