Study of synthetic lethality in HPV-transformed cells.

Author(s)

Walason da Silva Abjaude

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2016-12-02

Examining board members

Enrique Mario Boccardo Pierulivo; Roger Chammas; Erico Tosoni Costa; Rodrigo da Silva Galhardo; Bryan Eric Strauss

• Advisor: Enrique Mario Boccardo Pierulivo

Abstract

Human Papillomaviruses (HPV) are non-enveloped DNA viruses that infect epithelial cells. Persistent infection with some HPV types is the main risk factor for the development of cervical cancer. DNA repair machinery plays an essential role in several stages of the HPV life cycle and is crucial for tumor cells survival. During malignant transformation, HPV E6 and E7 oncoproteins induce structural and numerical chromosome alterations and modulate DNA damage response. These observations suggest that cellular DNA repair machinery may play a dual role in both HPV biology and pathogenesis. In the present study, we sought to investigate the role of DNA repair proteins in cervical cancer derived cells biology. In order to achieve this goal, the expression of 189 genes was silenced in HeLa (HPV18) and SiHa (HPV16) cells as well as in primary human keratinocytes (PHK) using lentiviral vectors expressing specific shRNA. The effect of gene silencing was determined by cell viability assay, cell growth analysis, clonogenic and soft agar colony formation test. We observed that ATM, BRCA1, CHEK2 and HMGB1 down-regulation decreased growth rate, clonogenic potential and cellular anchorage-independent growth of HPV-transformed cervical cancer-derived cell lines with no effect in normal keratinocytes. Treatment of cells with drugs that inhibit ATM and CHEK2 activity showed that tumor cells are more sensitive to the inhibition of these proteins than PHK. Besides, we show that PHK expressing HPV16 E6 alone or along with HPV16 E7 were more sensitive to these inhibitors than control PHK or PHK expressing only E7. Moreover, PHK expressing E6 mutants defective for p53 degradation were less sensitive than PHK expressing E6wt. Moreover, to potentiate the effect observed by the ATM and CHEK2 inhibition, we treated cells lines with Doxorubicin and Cisplantin. We observed that tumor cells lines and PHK expressing HPV16 E6 or HPV16 E6/E7 were more sensitive to DNA damage induction. Altogether, these results indicated that these genes are required for HPV-transformed cells survival. Besides, our results suggest that this effect is related to HPV16 E6 oncoprotein expression and its capacity to degrade p53. (AU)

• FAPESP's process: 12/16512-8 - Study of synthetic lethality in HPV-transformed cells
• Grantee: Walason da Silva Abjaude
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)