Increased Expression of ITGA6 as a Predictor for Poor Prognosis in Head and Neck Squamous Cell Carcinoma

Keywords

ITGA6; mRNA Expression; HNSCC; Prognostic Value; TCGA Database.

Introduction

Head and neck cancer is the 6th most common cancer worldwide with an annual increase of approximately 6,30,000 patients and a mortality rate of 3,50,000 deaths every year [1, 2]. The head and neck cancer include malignant tumours arising from various sites in the upper aerodigestive tract [3, 4]. Among head and neck cancer types, head and neck squamous cell carcinoma (HNSCC) is the most common variety and accounts for 90% of the head and neck cancers [3, 5, 6].

Progression of a suspicious lesion into cancer depends on the progression of epithelial dysplasia the which does not follow a predictable sequential progression from mild to moderate to severe dysplasia and in rare cases may revert to normal [7, 8]. The etiological factors for HNSCC include tobacco chewing, smoking, alcohol consumption, virus etiologies like HPV and genetic factors [9-11]. Recent molecular genetic studies provided evidence that the majority of head and neck squamous cell carcinomas (HNSCCs) develop within a contiguous field of preneoplastic cells [12]. Based on several studies conducted, it can be inferred that these alterations in several cellular molecules including DNA, RNA, and proteins play a significant role in tumor progression and the overall survival of the malignant cells [13]. Hence these markers can assist in early diagnosis and prediction of prognosis. The diagnosis of carcinoma at an early stage can prevent extensive treatment and thus biomarkers can serve as a tool for diagnosis [14-16].

The ITGAG gene is located on chromosome 2q.31.1, codes for Integrins [17]. Integrins are a family of transmebrane heterodimeric glycoprotein receptors which mainly functions in cell to cell adhesion [17]. Integrins are made of alpha and beta subunits bound together by Covalent bond [17, 18]. 18 alpha and 8 beta subunits are known till now which can form 24 distinct integrin heterodimers [19]. Integrins can bind to extracellular materials like collagens, fibronectins, laminins, receptors such as vascular cell adhesion molecules (VCAM-1) and intercellular cell adhesion molecules [20, 21]. Integrins are bidirectional in signalling functions, which transmit Signals from extracellular to intracellular and vice versa [17]. This indicates their major role in Immune response, homeostasis and overall cell development [17, 22]. Through various signalling pathways integrins can cross talk with growth factor receptors and it is required for many growth factor receptors to function [23].

ITGAG expression has significant impact on angiogenesis, tumor metastasis and self renewal and other properties of cancer stem cells [23, 24]. There are several studies showing increased levels of ITGA6 can promote tumour susceptibility and progression, or SNP/ mutations in ITGA6 genes may be responsible for cancer progression [25, 26]. But some of the researchers also stated that decreased levels of ITGA6 can also lead to cancer progression [27]. The expression of ITGA6 and its association with cancer susceptibility and progression is a controversial topic. Hence more research in the area of ITGA6 gene expression and analysis of association between this ITGA6 gene expression and tumour susceptibility progression of cancer should be promoted. Aim of this present study was to analyse the ITGA6 gene expression in HNSCC which is an aggressive malignancy with high morbidity and mortality rates.

Materials and Methods

Gene Expression Analysis

The present study initially analyzed the ITGA6 expression in HNSCC (n=520) and normal tissues (n=44) using data from TCGA dataset. The data regarding the samples were collected and analysed during April 2020. We used the ONCOMINE (https:// www.oncomine.org/) and GEPIA (http://gepia.cancer-pku.cn/) used to analyse the ITGA6 expression in primary HNSCC and normal tissues.

Survival analysis by Kaplan-Meier plotter

In the present study, the prognostic values of ITGA6 at mRNA level in HNSCC was analyzed using Kaplan-Meier Plotter (http:// kmplot.com/analysis/) is an online database containing gene expression profiles and survival information of cancer patients.

Results and Discussion

The ITGA6 gene encodes the integrin alpha Chain family of proteins which are proteolytically processed to form light and heavy chains that comprises alpha 6 subunits of integrin [26]. The functions of ITGA6 include integrin regulate and cell survival and migration, roles in cell survival, migration, and angiogenesis. This integrin also plays a role in tumour invasion and cell invasion and metastases [23]. Integrins can interact with extracellular materials. The lack of cell adhesion leads to disordered integrin signalling pathways including PI3K/AKT, MEK/ERK, FAK and NF-KB [24]. Through these signalling pathways integrins can cross talk with growth factor receptors and it is required for many growth factor receptors to function [21]. Integrin ligation was found to suppress apoptosis by activating suppressors of apoptosis [27, 28]. Stupack et al., in 2001, Kim et al. in 2002 found that integrin can inhibit caspase activation [29, 30]. Integrins also stimulate Cell migration by activating Rho and Rac GTPase [31]. Integrins promote Cell cycle entry by stimulating cyclins expression [32].

Predilection of epidemiological and clinical factors are also very important in pathologies [28]. In the present study, the ITGA6 expressionin HNSCC was first determined using the Oncomine and GEPIA database. We found that ITGA6 was highly expressed in various types of cancer including HNSCC (Figure-1A,1B). In addition, the GEPIA database used to evaluate the exact ITGA6 mRNA expressionin HNSCC and normal tissues. We found that the mRNA level of ITGA6 was significantly up-regulated in HNSCC compared to normal tissues (p<0.01) (Figure-2A). Among the selected samples of HNSCC, males were found to be more affected than females. This was also in accordance with an epidemiological study by Nadarajah Vigneswaran et al, 2015. Males were found to be more affected because the HNSCC development can be associated with habit history and habits are more prevalent in male population [29]. Hence HNSCC can be prevalent in males (67.3%).

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The age group most affected in our study was 21-40 years and 81- 100 years. Many studies showed that HNSCC was also prevalent in younger age groups [30]. Several studies showed that there is an increase in incidence of HNSCC after 50 years [21, 22] This can be in concordance with our study, even though some variation from the most prevalent age group was also observed by some researchers [Muir et al – 24% of HNSCC are found in patients older than 70 years].

Most of the HNSCC patients were of grade 2 tumour and stage 4 cancer and most of the affected population showed no nodal metastasis Our result also showed that ITGA6 was highly expressed HPV negative patients compared with HPV positive patients. This is in line with study by Bratman SV et al, identified the presence of HPV transcripts in 14% of HNSCC samples [23].

ITGA6 expression analysis using the GEPIA and Oncomine datasets showed the overexpression of ITGA6 in various types of cancer including HNSCC than normal (p <0.01). Similar results were observed in studies by Shauntell N Luke MS and in his study he concluded that increased ITGA6 expression especially the cleaved ITGA6 expression allows cells to develop cancerous abilities such as aggressiveness, motility through ECM, invasion and metastasis. Shauntell N Luke MS stated in his study that ITGA6 gene expression can be used as a biomarker in HNSCC. Bo Yang et al., in 2017 also showed that one of the 4 highly expressed genes in HNSCC cases was ITGA6 and It can be used as a biomarker in HNSCC [31, 32]. Apart from this Ting hu et al in 2016 also showed that ITGA6 gene expression is increased in breast cancer cell lines and causes radiation resistance in vitro and interferes with radiation induced cell apoptosis [33].

In the present study, high ITGA6 expression was found related to poor survival rate in HNSCC patients (p=1e-04, Figure 2B). Yang et al., in 2017 stated that high expression of ITGA6 gene can be associated with poor overall survival in HNSCC patients [32]. Apart from HNSCC, gall bladder carcinoma, breast cancer also showed decreased overall survival rate associated with increased expression of ITGA6 gene [34, 35]. The present study results were also in accordance with previous literature in the case of increased ITGA6 expression and decreased overall survival rate of HNSCC patients. However, large scale studies are required to substantiate the findings obtained in this study [36].

Conclusion

In conclusion, ITGA6 mRNA level was overexpressed in HNSCC. In addition, high ITGA6 expression was significantly related to poor survival in HNSCC patients. Hence ITGA6 can be used as a potential prognostic biomarker for HNSCC.

Acknowledgement

The authors would like to acknowledge the help and support rendered by the department of Dental research cell and the department of Oral pathology of Saveetha Dental College for their constant assistance with the research.