Physical and functional interaction between MNT and CCDC6

ABSTRACT : CCDC6 gene was first isolated fused to RET, forming the PTC1 oncogene in papillary thyroid carcinomas. The gene product, termed H4(D10S170) or CCDC6, is a 65 kDa phosphoprotein located in both cytoplasm and nucleus, that contains a coiled coil domain for protein-protein interactions. Our preliminary results from proteomic screenings showed that CCDC6 interacts with MNT, a protein containing a basic-helix-loop-helix-leucine zipper (bHLH-LZ) protein from the MXD family. MNT is known by its role as an antagonist of the MYC oncogene in several model systems. In this project, we have confirmed the CCDC6-MNT interaction in the HEK293T cell line derived from human embryonic kidney. We aimed to delimitate the interaction domain transfecting HEK293T cells with CCDC6 and MNT mutants and performing co-immunoprecipitation assays. The results suggest that the C-terminal domain of CCDC6 and that both the LZ together and the N-terminal domains of MNT are necessary for the interaction. We have also studied the nuclear or cytoplasmatic localization of the MNT-CCDC6 complex in HeLa cells (derived from human cervical carcinoma), and we found that they interact mainly in the cytoplasm. In order to evaluate the effects of CCDC6-MNT on cell proliferation, we transfected HeLa and HeLa shCCDC6 cells (CCDC6 silenced) with an MNT overexpressing vector and performed clonogenic assays. Furthermore, to study the role of MNT-CCDC6 in DNA damage, a viability analysis was performed in HAP1 (derived from Chronic Myelogenous Leukemia) and HAP1 MNT KO cells treated with cisplatin and etoposide. Data suggest that MNT confers partial resistance against etoposide. Finally, we studied the effects of CCDC6 silencing on apoptosis in HAP1 MNT KO versus HAP1 control. For this purpose, we silenced CCDC6 with short-hairpin RNA (shRNA) lentiviral constructs, and the results showed that the silencing seems to play an antiapoptotic role, mostly in the absence of MNT. In summary, our work has shed light into different biochemical and biological functions of these two important oncoproteins.Máster en Biología Molecular y Biomedicin