Systematisches genomweites RNAi-Screen zur Identifizierung neuer synthetisch letaler Interaktionen in humanen Brustkrebs-Zelllinien: Auswirkungen auf die Entwicklung von therapeutischen molekularen Targets.
Zusammenfassung der Projektergebnisse
Targeted therapies are emerging as specific less toxic alternatives to classical cancer treatments. Unfortunately, a subgroup of 15-20% of breast tumors characterized for the absence of hormonal estrogen and progesterone (ER/PG) receptors and the growth factor receptor ErbB2, also called HER2/neu, do not respond to any of these tailored approaches, leaving chemotherapy as the only therapeutic option. This latter group of breast cancers is known as Triple Negative breast Tumors (TNT). It was performed an unbiased genome wide RNAi screens to identify genes that when silenced selectively affect viability of TNT cells in vitro. The possibility to interrogate the complete genome by loss-of-function studies provided us with the unique opportunity to validate master regulators (MRs) from the computational predictions for the identification of upstream modulators and downstream interacting molecules that recapitulate the essential functions of the MRs. This goal was pursued by the systematic completion of four specific aims; 1. RNAi screen of breast cancer cell lines using a 70,000 shRNA-mir library that targets the whole human genome, 2. Identification of selective depleted genes in TNT cell lines using next generation sequencing, 3. Selection of the exclusively depleted shRNAs in TNT cell lines, 4. Evaluation of the accumulated evidences for a filtered selection of molecular targets. Taken together, we could demonstrate that the shRNA screening can recapitulate the Basal and Luminal clustering as primary tumors subtypes do based on depletion/overrepresentation profile of the shRNAs. Furthermore, when we crossed our data from the shRNA screening with the ARACNe prediction to identify MRs for Basal vs. Luminal breast cancer tumors, from all candidate genes, E2F2 emerged as promising synthetic lethal for Basal/TNT breast cancer tumors. Moreover, this gene was tested using a cell proliferation/viability assay and it showed a limited but specific effect in the majority of the Basal/TNT cell lines tested. Other candidate genes emerged also form the ARACNe prediction, EN1 (Engrailed-1) gene, a transcription factor supposed to have a role in the control of development and associated with poor prognosis in patients with salivary adenoid cystic carcinoma. Our results showed that EN1 knockdown affected cell proliferation/viability of all Basal B cell lines in a range between 30% and 50%, pointing to EN1 as a promising candidate gene for the selectively killing of Basal B cell lines. Our experience with the shRNA screening and the validation of the data will serve to identifiy genes in Basal/TNT cell lines that are potential targets for specific therapies and will also serve to develop algorithms to identify the candidates genes emerging from shRNAs screening based on the RNAi technology.
