Functional consequences of rearrangements to the FBXL17 gene detected in patient samples and cancer cell lines, on the E3 ubiquitin-ligase SCF(Fbxl17) activity

Abstract

Fbxo7 is one of the 69 human F-box proteins, which interacts with SKP, Cullin and RBX to form the largest family of ubiquitin-ligases known as SCF-type E3 ubiquitin ligases. In addition to the F-box domain of interaction with SKP1, the Fbxl17 contains the LRR domain (Leucine Rich Repeat) which interacts with its substrates. The FBXL17 gene is recurrently disrupted by rearrangements in the region that encodes its LRRs. From 1992 patient samples from the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) project, FBXL17 was mutated in 135. And in 746 cancer cell lines, array-CGH data showed breaks in FBXL17 in three breast lines, BT-474, HCC38, and HCC1395, and the oesophageal/gastric cardia adenocarcinoma line OE-19. Here, we propose to identify the SCF(Fbxl17) substrates with impaired ubiquitination caused by the LRR depletion in order to understand the mechanisms of the tumorigenicity induced by this mutation. To do this, we will develop in vitro ubiquitination reaction using SCF(Fbxl17) or SCF(Fbxll17-”3LRR) and the protein microarray (protoarrays) as a source of substrates. The differentially ubiquitinated candidates by the wild type complex will be validated in cellulo and in vitro and submitted to the Ubiquitin Chain REstriction Analysis (UbiCRest) in order to identify the ubiquitin chain introduced by the SCF(Fbxl17). Cells expressing the Fbxl17-”3LRR (HCC1395, OE-19) and Fbxl17 (MCF7, HB4a) will be used in the functional assays. We have published our expertise in this subject and I will collaborate with the Dr. Marcelo Gomes from the Dep. of Biochemistry and Immunology Faculty of Medicine of Ribeirao Preto/USP and Dra Heike Laman and Dr Paul Edwards from the Dep of Pathology from University of Cambridge/UK. The main goal of this research plan is to understand the FBXL17 function in the development of breast cancer. We hope to contribuite with the definition of the FBXL17 as a potential cancer biomarker and therapeutic target. (AU)