ACCEPTED MANUSCRIPT Differential alterations of molecular signature of RBSP3, LIMD1 and CDC25A in normal oral epithelium during oral tumorigenesis

�The study aims at understanding the molecular profile of three candidate cell cycle regulatory genes RBSP3, LIMD1 and CDC25A in the basal/ parabasal layer of normal head and neck epithelium possessing stem cell like character, followed by alteration of signature during tumorigenesis. Immunohistochemical expression/ promoter methylation was used to determine the molecular signature of the genes in normal oral epithelium. Alteration of the expression profile during tumor development was studied by immunohistochemistry, promoter methylation, deletion and mutation in HPV/ tobacco etiological groups, followed by clinic-pathological correlation. In basal/parabasal layer, the molecular signature of the genes was low protein expression/ high promoter methylation of RBSP3, high expression/ low methylation of LIMD1 and high expression of CDC25A. Dysplastic epithelium maintained the signature of RBSP3 through high methylation/ additional deletion with loss of the signatures of LIMD1 and CDC25A via deletion/ additional methylation of LIMD1. Similarly, maintenance/ loss of signature in invasive tumors was due to recurrent deletion/ methylation. Thus, differential patterns of alteration of the genes might be pre-requisite for the development of dysplastic and invasive lesions. Expression of the genes at different tumor stages was similar due to comparable or increased alterations (deletion/ methylation). Tobacco negative HNSCC patients had significantly lower alterations of LIMD1 and CDC25A, along with better survival of HPV negative/ tobacco positive patients. Our data suggests the importance of the signature of the genes in normal basal oral epithelium and their role in the development and progression of head and neck lesions.