Role of HPV-E7-c-Abl-p63 axis in DNA damage repair: implications for radio/chemoresistance in cervical cancer

Abstract

Cervical cancer, caused by HR-HPV infection, is the fourth most common cancer and leading cause of cancer death in women worldwide. Treatment failure and metastasis are common due to intrinsic or acquired chemo/radio resistance, posing a life-threatening problem. The E7 oncoprotein of HR-HPV induces Delta Np63 alpha protein expression to repair DNA damage in cervical cancer cells after ionizing radiation. c-Abl, non-receptor tyrosine kinase, regulates Delta Np63 alpha protein stability in cervical cancer cells under resting conditions and after DNA damage. The HPV-E7/c-Abl/p63 signalling axis has been hypothesised to play a role in chemo/radiotherapy resistance in cervical cancer. To test this, expression of Delta Np63 alpha protein was silenced by siRNA to investigate its role as a DDR modulator in cervical cancer cells. Then, the effect of Delta Np63 alpha silencing on cell cycle progression and DDR activation was assessed after DNA damage using western blotting and flowcytometry. Depletion of Delta Np63 alpha protein in cervical cancer cells increased the expression level of cleaved PARP (an apoptosis marker) and phosphorylation of H2AX (a DNA damage marker) after induction of DNA damage by cisplatin, mitomycin C, bleomycin and ionising radiation. The analysis of the cell cycle indicates that in the absence of the Delta Np63 alpha protein, the cells experienced a significant delay and prolonged response to DNA damage, with a higher proportion of arrested cells at the G1, S, and G2 phases of the cell cycle. Modulation of Delta Np63 alpha expression in cervical cancer cells through pharmacological inhibition of c-Abl activity was also investigated, in order to increase sensitivity of cervical cancer cells to the therapy. CaSki cells were incubated with imatinib to inhibit c-Abl activity and, as a consequence, reduce the expression of Delta Np63 alpha protein. Then, DNA damage was induced by a 24-hour bleomycin treatment. Cell cycle progression and DDR activation were analysed using flow cytometry and western blotting. The results show that modulation of the Delta Np63 alpha protein by imatinib led to a delayed repair response to the bleomycin-induced DNA damage, which was associated with a significant increase in Gamma H2AX levels. This study concludes that the HPV-E7/c-Abl/p63 signalling axis plays a crucial role in the development of chemo/radiotherapy resistance in cervical cancer cells. Targeting this signalling axis could lead to the development of new therapeutic drugs that can overcome chemo/radiotherapy resistance in cervical cancer, ultimately improving patient survival rates.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Stephen Taylor (Supervisor) & Richard Edmondson (Supervisor)