Abstract B29: ZNF516 a potential tumor suppressor gene candidate is implied in tumor progression in cervical cancer

Abstract

Abstract Background: In Cervical Cancer (CC) the role of HPV is fundamental; however, not all HPV infected women will develop this disease. Therefore, other mechanisms, such as silencing of tumor suppressor genes (TSG), could be implied in cervical carcinogenesis. In a previous study, we constructed methylation microarrays, where we found promoter aberrant hypermethylation of ZNF516 gen in CC, postulating it as TSG candidate. The aim of this study was to characterizer ZNF516 role in cervical carcinogenesis and cell cycle. Materials and methods: ECT1 E6/E7 (immortalized normal squamous epithelia cell line) and three CC cell lines: SiHa, C-4I and C-33A were cultivated for experiments. ZNF516 expression was determined by qRT-PCR and western blot. Treatment with 10 μM 5-aza-2′deoxycytidine (5- aza) was performed to evaluate a possible epigenetic regulation. Transfection with pCMV6-ZNF516-GFP (ZNF516) and pCMV6-GFP (Empty) was carried out in C-33A cell line through lipofection followed by G418 selection to obtain stable transfection. Viability and clonogenic assay was performed to evaluate transfected cell behaviour. Also, immnunohistochemical analysis was performed in 509 cervical biopsy tissues (55 normal; 188 low-squamous intraepithelial lesions; 205 high- squamous intraepithelial lesions and 57 squamous cervical cancers). Results: ZNF516 mRNA expression was significantly downregulated in SiHa (p<0.01) and C-33A (p<0.001) respect to ECT1 E6/E7. Western blot showed a deregulated protein expression of ZNF516 in all CC cell lines. After treatment with 5-aza, mRNA (p<0.05) and protein expression of ZNF516 of all CC cell lines were restored. ZNF516 transfected C-33A showed a significant colony formation (p<0.01) and viability (p<0.01) decrease compared with C-33A transfected with the empty vector. Nuclear expression of ZNF516 in cervical biopsies showed a diminished expression in SCC respect to normal epithelia (p<0.001) Conclusions: There is a clear ZNF516 dysregulation in CC cell lines, and the inactivation mechanism seem to be methylation. Restored expression of ZNF516 in C-33A cell line modifies the tumor phenotype, decreasing cellular viability and colony formation. These results suggest that ZNF516 could be a TSG, and its inactivation promotes CC developing. Citation Format: Carmen Gloria Ili, Tamara Viscarra, Juan Carlos Araya, Jaime Lopez, Barbara Mora, Javier Retamal, Enrique Bellolio, Susana Aedo, Juan Carlos Roa, Priscilla Brebi. ZNF516 a potential tumor suppressor gene candidate is implied in tumor progression in cervical cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B29.