Overexpression of EGFR in Head and Neck Squamous Cell Carcinoma Is Associated with Inactivation of SH3GL2 and CDC25A Genes

Guru Prasad Maiti,Susanta Roychoudhury,Sanjib Dey,Anup Roy,Amlan Ghosh,Nupur Mukherjee,Susmita Ghosh,Pinaki Mondal,Jayanta Chakrabarty,Chinmay Kumar Panda,Jaydip Biswas,Hiromu Suzuki

doi:10.1371/journal.pone.0063440

Abstract

The aim of this study is to understand the mechanism of EGFR overexpression in head and neck squamous cell carcinoma (HNSCC). For this reason, expression/mutation of EGFR were analyzed in 30 dysplastic head and neck lesions and 148 HNSCC samples of Indian patients along with 3 HNSCC cell lines. In addition, deletion/methylation/mutation/expression of SH3GL2 (associated with EGFR degradation) and CDC25A (associated with dephosphorylation of EGFR) were analyzed in the same set of samples. Our study revealed high frequency of EGFR overexpression (66–84%), low frequency of gene amplification (10–32.5%) and absence of functional mutation in the dysplastic lesions and HNSCC samples. No correlation was found between protein overexpression and mRNA expression/gene amplification status of EGFR. On the other hand, frequent alterations (deletion/methylation) of SH3GL2 (63–77%) and CDC25A (37–64%) were seen in the dysplastic and HNSCC samples. Two novel single nucleotide polymorphism (SNPs) were found in the promoter region of SH3GL2. Reduced expression of these genes showed concordance with their alterations. Overexpression of EGFR and p-EGFR were significantly associated with reduced expression and alterations of SH3GL2 and CDC25A respectively. In-vitro demethylation experiment by 5-aza-2′-deoxycytidine (5-aza-dC) showed upregulation of SH3GL2 and CDC25A and downregulation of EGFR expression in Hep2 cell line. Poor patient outcome was predicted in the cases with alterations of SH3GL2 and CDC25A in presence of human papilloma virus (HPV) infection. Also, low SH3GL2 and high EGFR expression was a predictor of poor patient survival. Thus, our data suggests that overexpression of EGFR due to its reduced degradation and dephosphorylation is needed for development of HNSCC.

Highlights

Head and neck squamous cell carcinoma is the sixth most common cancer worldwide and it accounts for 30–40% of all cancer types in the Indian subcontinent [1]
It was evident that binding of EGF to epidermal growth factor receptor (EGFR) triggered a series of biochemical events including autophosphorylation of specific tyrosine residues in its kinase domain [4] i.e activated EGFR protein level will be elevated without changing total EGFR protein level [7,8]
High cytoplasmic as well as membrane bound expression of EGFR was seen in basal layer followed by gradual decrease in parabasal and spinous layers (Figure 2a)

Summary

Introduction

Head and neck squamous cell carcinoma is the sixth most common cancer worldwide and it accounts for 30–40% of all cancer types in the Indian subcontinent [1]. In the progression model of HNSCC, number of alterations like overexpression of epidermal growth factor receptor (EGFR) protein, deletion in chromosome 9p21, p16/p14 inactivation, trisomy of chromosome 7 and telomerase activation were suggested to be associated with the development of hyperplastic lesions of head and neck [2]. Among these alterations, overexpression of EGFR is quite important due to its regulation of multiple cell signaling cascades. Frequent deletion (29–37%), promoter hypermethylation (42– 46%) and reduced expression (68%) of SH3GL2 (located at chromosome 9p22.2) have been reported in head and lesions [1]

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