OSCC remains one of the deadliest malignancies in the past couple of decades possibly due to paucity of appropriate diagnostic and treatment modalities.Proteomic analyses with advances in mass spectrometry has paved the way by exploring novel biomarkers that could be used to timely and accurately diagnose OSCC. The aim of this study is to identify altered expression of proteins (that could be clinically useful) in OSCC upon varlitinib treatment. Cytotoxicity assessment of varlitinib showed dose and time dependent inhibitory action on cell proliferation. Growth inhibitory potential (in terms of reduced cell survival) was attained at 24 hours of treatment.
To determine the effects of varlitinib on protein expression levels in SCC-25 cells, we performed 2D-GE (two-dimensional gel electrophoresis) followed by Mass spectrometric analysis to identify differentially expressed proteins. The study exhibited 6 different proteinspots in total from varlitinib treated as well as control group of SCC-25 cells.We observed significant fold changes in protein expression levels for 6 protein spotsafter 24 hours of treatment. The catalogue of these identified proteins using mass-spectrometry after treatment is shown in Table 1. Each protein is briefly described in the subsequent paragraphs.
We identified cytoskeletal protein (actin), extracellular matrix protein (keratin), heat shock proteins and molecular chaperones (Bip and HSP7C respectively).
The first group includes proteins that are correlated to evasion of apoptosis such asBiP, HSP7C and PDIA1. Binding immunoglobulin protein (BiP, also known as Glucose Regulated Protein-78; GRP-78) is a member of HSP 70 family hence encoded by HSPA5 gene [13]. BiP is an Endoplasmic Reticulum (ER) residing molecular chaperone protein. It is responsible for mediating folding, translocation of proteins, initiating unfolded protein response (UPR) and ER-associated degradation (ERAD).BiP is of utmost importance for regulating homeostasis[13]. Under stress condition(s), BiP undergoes several changes in terms of function, expression and activation which blocks translocation of proteins to ER. Subsequently causing impaired degradation resulting in pathological conditions such as cancers, cardiovascular as well as neurodegenerative disorders [13]. BiP is an established master regulator of cancer and has been found to be over-expressed in wide variety of cancers including lung cancer, liver cancer, breast cancer etc.[13, 14]. Increased expression of BiP inactivatespro-apoptotic markers such as BIK and BAX andinhibits apoptosis [13, 15]. BiP, through signal transducer, activates intracellular kinases thus activating cellular pathways that promote cell proliferation and survival [16]. Moreover, BiP plays a critical role in OSCC progression by inducing enhanced proliferation, chemoresistance and metastasis[17].BiP implicitly activates MAPKand PI3Ksignaling cascades facilitating cancer cell progression. [18]. BiP pathogenesis is evidenced through different studies [16–19]. It has been suggested that downregulation of BiP can be potentially linked with impeded tumor formation and growth resulting in apoptotic induction and improved survival. Our study shows down-regulation of BiP in response to varlitinib treatment suggesting therapeutic potential of the drug.
Protein disulfide isomerase 1 (PDIA1) - a dithiol-disulfide oxidoreductase is also an ER residing molecular chaperone [20, 21]. PDIA1 primarily catalyzes disulfide bond formation via its oxidizing, reducing and isomerizing capabilities thusmediating proteinfolding, translocation to ER and degradation thus contributing to maintain cellular homeostasis[20, 21]. Deregulated PDIA1 expression and/or enzyme activity is often associated with different human diseases such as CVDs, neurodegenerative disorders, and different cancer types [21]. Higher expressions of PDIA1 have been demonstrated in brain cancer [22], lymphoma [23], colorectal cancer[24], breast cancer [25], often conferring metastasis, invasiveness and chemoresistance. The mechanisms causing increased expression of PDIA1 and associated pathological outcomes are poorly understood [24]. However, PDIA1 is considered as an upstream regulator for balancing Reactive Oxygen Species (ROS) as well as controlling the activity of a metalloprotease ADAM17 which in turn acts as an intermediate in EGFR signaling[25]. ADAM17 activation is based on change in redox balance – an effect which is important for growth factor dependent signaling in cancer cells. It is reported that gene silencing or knocking out PDIA1 results in induction of apoptosis and decreased cell proliferation [24]). Ourproteomics data revealed upregulation of PDIA1 in control SCC-25 cellswhile varlitinib treated group showed reduction in PDIA1 expression levels. The results remainedconsistent with previously conducted studies.
Another interesting protein found to be decreased in varlitinib treated cells was HSP7C. HSP7C is a 71 KDaheat shock related proteinwhich is constitutively expressed in cells and regulates trafficking and folding of nascent proteins. Ithas been frequently observed in human breast cancer [26] autoimmune retinopathy [27] etc.Increased expression of HSP7C due to heat, oxidative stress and hypoxia promotes cell survival, apoptotic evasion from proteolytic stress related rapid and abnormal cell growth[28]. Our proteomics data showed increased expression of HSP7C among SCC-25 cells which is in consensus with the already performed studies [28, 29]. The expression was found to be decreased following 24 hours varlitinib treatment of SCC-25 cells confirming its oncogenic role.
The second group of proteins belong to the family of intermediate filament proteins including Vimentin and cytoskeletal Keratin Type 14. Vimentin plays key role in cellular, physiological,structural, mechanical, biological, and developmental processes. Vimentin is known to provide phosphorylation site(s) for kinases thereby regulating signal transduction[30]. Vimentin has been found to be present in wide range of cells including mesenchymal cells and is typically associated with Epithelial to mesenchymal transition - a prominent feature associated with tumor progression [31]. Increased vimentin expression is often associated with breast cancers [31], gastrointestinal [31] and prostate cancer [32].Knock down clones of these proteins have shown decline in carcinoma cell proliferation and decreased vimentin expression could likely lead to anti-neoplastic behavior of cells by driving them less aggressive and controlled proliferation [33–35]. We also observed reduced expression of vimentin in varlitinib treated SCC-25 cell line in comparison to control cells. Second protein, Keratin is also a member of the family of intermediate filament and is reported as protein marker of epithelial differentiation generating polarity which plays a significantly importantrole in providing mechanical strength and integrity to epithelial cells [36, 37]Besides mechanical support and stability, keratins contribute in intracellular signal transduction [36, 37]. We identified Type I Keratin cytoskeletal 14 which is typically expressed in basal layer cells of non-keratinized stratified squamous epithelial lining [36]. K1C14 has been associated with carcinogenesis and it is usually overexpressed in different cancers[37, 38]. However, we found up-regulated expression of K1C14 among varlitinib treated group of SCC-25 cellswith respect to their counter control. Our results are contradictory to previous reports and needs to be explored further to identify the exact pattern of K1C14 in response to TKI mediated treatment.A possible reason for thiscontradiction in expression pattern of keratins could be modified expression of genes and proteins (such as cell cycle regulator biomarker p21), associated with carcinogenesis[36]. This, in turn, may result in responding differently to varlitinib mediated targeted therapy. However, alterations at genetic or protein level are key players behind progression / pathogenesis of oral carcinogenesis [36] resulting in apoptotic resistance [38, 39].
Lastly, ACTB Human Actin, cytoplasmic 1 is one of the six isoforms of actin protein, which is considered a highly conserved and ubiquitously expressed [40] protein. Actin is essentially involved in cellular activities such as motility, development etc.[40].Actin has integral part in survival for most of the cells since it provides mechanical support and force to drive cellular activities as well as motility. Cancer cells also undergo locomotion by moving from one location in the body to another via making contacts with neighboring cells[41], resulting in migration. ACTB has been found to be upregulated during different cancers including prostate, breast, and colorectal cancers [42].In the present study, ACTB showedincreased expressionin control SCC-25 cells whereas 50 µM 24 hours prolonged treatment resulted in downregulation of ACTB. This agreeswith previous reports [43, 44]. Protein-protein interaction via STRING 9.1 with experimental databases (including GO, KEGG etc.) and text-mining as active methods of predicting protein associations indicated that all identified proteins interacted with each with an exception of KR1T14.