In vitro validation of ubiquitin-related genes involved in the regulation of tumor-cell migration

JULIANA H. ENRIQUE´ STEINBERG; MARIO ROSSI; FABIANA ALEJANDRA ROSSI; EZEQUIEL CALVO-ROITBERG

Buenos Aires

Congreso; LVI SAIB / XV SAMIGE Meeting; 2020

The formation and progression of tumors is regulated by the abundance and activity of oncogenic proteins or tumor suppressors. Moreover, the development of metastasis foci in patients suffering from cancer represents a significant reduction in their survival and life quality. The Ubiquitin-Proteasome System (UPS) plays a fundamental role in the maintenance of protein homeostasis both in normal and stressed conditions, thus regulating almost every single cellular process. Since alterations in the ubiquitination cascade have been shown to be associated with malignant transformation, invasive potential of cells and metastasis, we sought to investigate the role of UPS in the regulation of tumor-cell migration. For this purpose, we performed a genetic screen using an shRNA library directed against UPS genes and Boyden chambers to analyze the migrating potential of breast cancer cells infected with this library. After the selection process, we characterized the non-migrating cell population and determined the relative abundance of each shRNA by Illumina Next Generation Sequencing. We obtained a list of 30 candidate genes, half of which had already been associated with the regulation of migration/invasion/tumorigenic processes or metastasis. In order to validate that silencing of the screen candidate genes reduces the migratory potential, we generated knockdown MDAMB231 cell lines by separately transducing three different shRNAs for each candidate gene, followed by multiple wound healing assays. We used an empty vector as a negative control and a shRNA well characterized in the past by our group as a positive control. We designed an innovative bioinformatic analysis to process the results. Using this approach, we were able to select three promising candidate genes for which at least two shRNAs independently impaired cell migratory ability. Our studies will not only be key to better understand the mechanisms that control migration but could also have therapeutic implications. Altogether, these findings demonstrate that shRNA screens using Boyden chambers are useful for identifying novel genes that regulate migration, which might represent novel therapeutic targets for the development or improvement of cancer treatments.