Validation analysis of genes potentially involved in the squamous differentiation response of oral carcinoma cells to cell cycle stress

Abstract

Head and neck cancer is rated as the sixth most common cancer worldwide among which oral squamous cell carcinoma (OSCC) is the most frequently diagnosed type. Human Papilloma Virus infection and the intake of tobacco and alcohol are the main risk factors in this pathology. The accumulation of genetic mutations and damage are thought to induce the development of cancer. Head and Neck epithelia possess different DNA-damage response mechanisms. The host laboratory has previously described a novel epidermal keratinocyte DNA-damage differentiation response (DDDR) to cell cycle stress. They also have found this squamous DDDR in non-lesional keratinocytes from head and neck. The loss of this mechanism would allow cells with genomic instability to proliferate resulting in carcinogenesis. Currently, the molecules controlling the DDDR are unknown. The lab recently run RNAseq experiments on oral carcinoma cells that respond or do not respond to the DDDR. I have studied five of the genes selected in the RNAseq study as potential regulators of the DDDR. We induced DDDR with Nocodazole (mitotic inhibitor), a drug used as a chemotherapy agent in four OSCCs. We observed that OSCC samples present an altered DDDR pattern. We therefore studied upon Nocodazole treatment, by RTqPCR, immunofluorescence or western blot, the expression of the candidate genes as well as two squamous differentiation markers (IVL and KRT13). The results show changes of four of the genes that suggest they might participate in the DDDR response. These genes have functions in cell cycle-dependent protein degradation, in cell differentiation, in suppression of proliferation, in DNA endoreplication or in programmed cell death. Although these assertions have to be validated with future experiments, the findings open a new path for an involvement of four of the genes analysed, in the squamous DDDR.