Genetic Alterations in HSPA Family of Genes and their Putative Association with HNSCC

Introduction: Head and neck squamous cell carcinoma (HNSCC) is an aggressive life-threatening disease associated with high mortality rates. Several genes related to stress response have been studied extensively to reveal their possible association with HNSCC. Members of the HSPA (heat shock protein family A) family are exclusively expressed under stress conditions suggesting that these are specialised to function in the stress response. Aim: The aim of the present study is to demonstrate the genetic alterations in the HSPA gene family and their putative association with HNSCC. Materials and Methods: The source of the patient's data was procured from the cBioportal database. The TCGA Firehose Legacy comprised 528 head and neck squamous cell carcinoma cases. Oncoprint data analysis can be used to derive a putative association between the disease phenotype and genotype, to identify the variations and to identify any novel variations which can be associated with the disease phenotype. The gnomAD data analysis was used to compare between the observed variants documented in the present study with that of reported variants deposited in the gnomAD repository. Short Research Article Laksita et al.; JPRI, 33(47B): 364-375, 2021; Article no.JPRI.74358 365 Results and Discussion: The HSPA1A&HSPA1B genes harboured high frequency of amplification and deep deletions. The expression of two genes HSPA8 and HSPA13 was found to be up-regulated in the primary tumor sample in comparison to normal tissues. In Kaplan Meier analysis, HSPA8 gene expression was compared with gender, here, high expression in females shows low survival rate. Similarly, HSPA13 gene expression was compared with race, here low expression in africanamerican population shows low survival rate. Conclusion: The present study provides preliminary data on the involvement of the HSPA family of genes with HNSCC, which has to be validated using experimental evidence in other populations.


INTRODUCTION
The association between genetic alterations and human cancer was first observed decades ago [1]. Cytogenetic studies revealed that specific chromosomal abnormalities were linked to the development of certain cancers. In addition, substantial genetic disruption leads to chromosome aberrations, rearrangements, and aneuploidy and these are observed in tumour cells. However, it was not clear whether this widespread genetic instability was a cause or a consequence of the cancer phenotype [2]. An understanding of the role of genetic alterations in cancer development arose out of studies of oncogenic viruses and hereditary cancers.
Head and neck squamous cell carcinoma (HNSCC) is an aggressive life-threatening disease associated with high mortality rates. It is the sixth most common form of cancer as reported world-wide [3]. Most HNSCCs arise in the epithelial lining of the oral cavity, oropharynx, larynx and hypopharynx. About 600,000 new cases, including approximately 50,000 in the United States, are diagnosed each year. In males in their 50s or 60s, HNSCC occurs more frequently, but the occurrence in younger individuals is growing [4]. HNSCC follows a similar genetic pattern for progression in its development from premalignant lesions such as leukoplakia, dysplasia, erythroplakia and lichen planus [5]. These cancers are strongly associated with certain environmental and lifestyle risk factors like tobacco and alcohol consumption [6]. Several in silico studies have revealed novel variants which may act as putative drivers involved in transformation of normal cells to malignant ones [7]. In this respect, the therapeutic potential of targeting HSPA and modulating HSPA expression is attracting much interest [8].
All organisms, except some hyperthermophilic archaea, contain the family of HSPA chaperones [9]. The heat shock proteins function as molecular chaperones and are involved in the process of translation and transport of proteins across membranes. Members of the HSPA family are expressed under stress conditions indicating that they are specialized to function in the stress response [10]. Extracellular heatshock proteins are sensed by the immune system as damage-associated molecular patterns (DAMPs) [11]. The surge of HSPA in the peripheral circulation and serum is observed during different kinds of exercise, and excessive use of mobile phones. Concordant increase in the levels of serum C-reactive protein was also observed and can be used as potential biomarkers in systemic inflammation. Although the HSPA family of genes have been implicated in HNSCC, the underlying molecular mechanism is not clearly understood. In the essential gene families, the present research recognises genetic alterations. In laboratory work, any differences found in the sample can be repeated to arrive at definitive proof of the interaction of the variations/mutations with the phenotype of the disorder. The present in silico research was therefore intended to examine the genetic variations in the proposed gene family in order to establish the putative relation between the disease phenotype and genotype. Our team has extensive knowledge and research experience that has translated into high quality publications . Hence, the aim of the study is to demonstrate the genetic alterations in the HSPA gene family and their putative association with HNSCC.

Data Source
Retrospective study design has been followed in the present study. The source of the patient's data is procured from the cBioportal database [34]. This database includes an extensive array of patient information from different cohorts. 528 HNSCC cases were found in 'The Cancer Genome Atlas' (TCGA) where sequencing and the copy number alteration data are available for tumour samples. The list of vital genes areobtained from literature search. The genes includes, HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA4, HSPA4L, HSPA5, HSPA6, HSPA7, HSPA8, HSPA9, HSPA12A, HSPA12B, HSPA13, HSPA14, HSPH1 and HYOU1. The user described queries based on these were submitted to the cBioportal database, and the resulting oncoprint data are used for further analysis.

Oncoprint Data Analysis
Oncoprint data analysis provides information on frequency distribution, variations in each of selected genes, type of variation, changes in protein coding amino acids, gene amplification, deletions, insertions, frameshifts, splice site mutations, etc. These information are used for possible correlation between genotype and phenotype, to identify the differences of less known mechanisms/genes and to know the novel variations that may be associated with the phenotype of the disease.

gnomAD Data Analysis
The data compares the observed variants documented in this study with the reported variants deposited in gnomAD repository [31].

Gene Expression and Survival Analysis
The expression of the gene presenting with highest frequency of gene alteration in HNSCC was analysed using the UALCAN ( http://ualcan.path.uab.edu/cgi-bin/TCGAsurvival) database. Survival curve analysis based on the tumor grade and expression profile was performed to demonstrate the putative role of heat shock protein family of genes with HNSCC. Combined survival effect analysis of gene expression and other clinical parameters such as race, gender, tumor grade, cancer subtypes were assessed using log-rank test that generated a p value which was further used to indicate statistical significance of survival correlation between groups [35].

RESULTS
The primary database, cBioportal and several other datasets are selected for the present study.
The TCGA dataset contained information of 528 HNSCC patients. The demographic details of the patients are given in Table 1. Oncoprint data analysis is performed to analyse the genetic alterations and the variations seen in the gene family. Here, HSPA8 and HSPA13 genes were observed with high levels of variation (Fig. 1). The gene amplification was seen in 2 genes, where, both HSPA1A and HSPA1Bharboured equal frequency of gene amplification. Two genes HSPA1A and HSPA1B demonstrated deep deletions. HSPA8 and HSPA13 genes showed the highest number of variations/ mutations among all the genes identified with alterations ( Table 2). Various truncating and missense variants with unknown significance are documented in the present study ( Fig. 1). Genetic alterations of the genes associated with the HSPA family are documented. Several novel variants were identified, along with a few reported variants in HSPA4 (rs144576995, rs377082440), HSPA4L (rs773358656), HSPA6 (rs1454026580), and HSPA12A (rs782770261, rs1161715269, rs781956168).
The primary tumour in both the genes showed higher expression than the normal tumour. This implies the fact that these rare variants are associated with the risk of particular disease. The gene expression profile of HSPA8 and HSPA13 showed significant differences as compared to paired normal tissuesfrom patients with a value of 8.36 x 10 -4 and 10 -12 (Fig. 2) respectively. The Kaplan -Meier plot shows the effect of gene expression level classified based on gender and race on HNSCC patient survival. The patients were stratified based on gender, race, grade of tumor to correlate with the expression profile (high, low or medium). Fig. 3 shows the expression level of HSPA8 based on gender. A significant difference observed between the four groups is 0.0042 (p-value), wherein females showing higher expression of HSPA8 were accompanied by low survival rate. Further, Fig. 4 showed that the expression level of HSPA13 in African American 0.026 (p-value) is related to low survival rate.

DISCUSSION
It has been postulated that the minimum constellation of mutations required for oncogenic transformation in humans includes inactivation of TP53 and RB, activation of RAS (or other members of that pathway), and constitutive expression of hTERT. More than five million people are affected and 379,000 deaths have occurred globally due to head and neck cancer [36]. Recently, our team has identified a prognostic marker associated with HNSCC (34). The HPV, i.e., the human papilloma virus has emerged as a major cause of HNSCC among non-smokers and light drinkers [37]. According to the GLOBOCAN survey, 2018, the incidence of head and neck squamous cell carcinoma was found to be clustered in specific regions worldwide with a high incidence rate recorded in the south asian countries [38,39]. A large number of tumor suppressor genes and oncogenes have now been identified and characterized through the analysis of tumor cell DNA [40]. HSPA is expressed in very low levels in non-stressed cells and is dramatically induced in hyperthermia [41]. HSPA is the most conserved hsp throughout the evolution of prokaryotes and eukaryotes [42]. Several studies have shown that the thermosensitivity of cells is altered if the expression of HSPA is enhanced or reduced [43]. The expression of HSPAs can be induced by insults other than thermal stress, including ischemia, heavy metals, nutrient deprivation, irradiation, infections, inflammation, and exposure to organics and oxidants [44]. Members of the HSPA family are known to control all aspects of cellular protestations such as nascent protein chain folding, protein import into organelles, recovering of proteins from aggregation, and assembly of multi-protein complexes [45]. Extracellular HSPAs are stimulators of innate immune responses [46]. Studies have linked the expression of HSPAs to several phenotypes of carcinoma, viz., therapeutic resistance, metastasis, and poor clinical outcome. In malignant cells, HSPA protects cells from the stress associated with rapid proliferation, suppress cellular senescence, and resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy [47].
HSPA1 is among the best characterized cancerrelated chaperones, while the significance of HSPA2 for cancer remains poorly understood. Previously researchers demonstrated that in primary NSCLC, HSPA1 was associated with good prognosis while HSPA2 correlated with bad prognosis, suggesting different roles of these proteins in cancer [48]. The changes in the induction of HSPA transcription are closely associated with the aging process. Various authors have studied the heat inductibility of different genes in HSPA family, such as, HSPA1A/1B and HSPA6, following the stress conditions [49]. Vidyashri et.al., has done a similar research based on genetic alterations of SPARC gene family and its association with HNSCC. Longxiang et.al., showed that downregulation of HSPA2 inhibits proliferation via ERK ½ pathway and endoplasmic reticular stress in lung adenocarcinoma [50]. In present study, the expression of the primary tumour of 2 genes, i.e., HSPA8 and HSPA13 has shown upregulation and among them, the highly significant one is the expression of HSPA13 gene which shows the p value as 10 -12 . Shan Lu et.al has studied the regulation of HSPA gene and have concluded that androgen receptor and its signaling regulates HSPA expression in prostate cancer cells and that HSPA1B could be an androgen receptor target gene [51]. Dorota et.al., has discussed the expression, function and regulation of HSPA2 protein in spermatogenic, somatic and cancer cells [52]. JG-98, a novel class of allosteric inhibitors of HSPAs, causes effective disruption of HSPAs-BAG3 interaction. Hence, HSPA-BAG3 complexes can be considered as broad-acting regulators of cancer cell signaling and a promising anticancer target [53]. With an ever increasing trend of head and neck cancers in Asian population [54], it has become a crucial point to identify the molecular targets involved in the pathogenesis of the disease. Accumulating evidences have proved the effect of several markers [55] in different types of patients with [56] and without habits. An extensive probing into each of these genomic signatures would aid in limiting the number of markers directly associated with the disease phenotype.
The present study demonstrated the effect of highly expressed genes of the HSPA family i.e., HSPA8 and HSPA13 under gender and race. Under gender, high expression in females has shown low survival rate and under race, low

CONCLUSION
The present study has discussed the relationship between the various HSPA genes about the expression, regulation, mutations and variations. It also provides preliminary data on the involvement of the HSPA family of genes with Head and neck squamous cell carcinoma (HNSCC), which has to be validated using experimental evidence.

FUNDING
With an ever increasing trend of head and neck cancers in Asian population [54], it has become a crucial point to identify the molecular targets involved in the pathogenesis of the disease. Accumulating evidences have proved the effect of several markers [55] in different types of patients with [56] and without habits. An extensive probing into each of these genomic signatures would aid in limiting the number of markers directly associated with the disease phenotype.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
We conducted our research after obtaining proper IEC approval.