Role of RASSF9 in melanoma using the CRISPR/Cas9 system

Abstract

Melanoma is the most lethal type of skin cancer, caused by the transformation of melanocytes, cells that promote pigmentation of the skin. Melanoma can be divided into four distinct groups, with the two most prevalent presenting modified sequences of B-Raf (52%) or Ras proteins (28%). Ras is the most altered protein in solid tumors and its effectors may be good therapeutic targets. Members of the RASSF family (Ras-association Family) were characterized by their participation in the induction of Ras-mediated apoptosis. This family is subdivided into two subgroups. The first composed by the RASSF1-6 members that present the Ras interaction motif at the C-terminal portion. These are the most studied members of the family. In comparison, the RASSF7-10 members present the RalGDS/AF6 domain at the N-terminal portion and not much is known about the functions of these proteins. In cancer, these proteins have their expression often suppressed through hypermethylation of their promoters. RASSF9 was first described as P-CIP1, a vesicle transport related protein. This protein is capable of interacting with H-, N- and R-Ras and with ASPP1 / 2 during chromosomal segregation. In addition, RASSF9 is crucial for epidermal homeostasis and is induced by sun exposure. Importantly, there is no data about the function and importance of this protein in melanoma. In this project, we aim to understand the action of RASSF9 by modifying a non-metastatic melanoma line. To do so, we will perform knockout with the CRISPR/Cas9 system followed by characterization of changes in invasive and proliferative capacity in vitro and in vivo. In addition, we will evaluate the resistance to chemotherapy and possible alterations in the metabolic pathways due to the absence of RASSF9. With this work we intend to determine the participation of RASSF9 in different aspects necessary for the establishment and progression of melanoma. (AU)