Validation of putative metastasis suppressor genes identified by an in vivo functional shRNA screen in a triple-negative breast cancer model


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Rebeka Tomasin1,Richard Redvers2,Marcia Cominetti1,Kaylene Simpson3,Robin Anderson2
1Federal University of São Carlos,2Olivia Newton John Cancer Research Institute,3Peter MacCallum Cancer Centre - Victorian Comprehensive Cancer Research Institute

Abstract

Background

When diagnosed early, breast cancer can be treated with a great rate of success. However, once the disease has progressed to metastatic stage, reaching organs such as lungs and bones, the treatments available are extremely limited and the prognosis is poor. This is particularly true for the triple-negative subtype. By identifying genes that regulate metastasis, it would be possible to predict the patients whose the disease is more likely to spread, and it would create an opportunity to develop more efficient therapies.

Method

Using a lentiviral shRNA library, we initiated an in vivo functional screen aiming to identify genes whose reduction in expression levels, in non/poorly metastatic triple-negative breast cancer cell lines (67NRch1/66cl4ch14, respectively), would lead to spontaneous metastasis in mice.

Results

Dozens of putative metastasis suppressors were identified by conventional and next generation sequencing. These candidates were further subjected to an in vitro RNAi screen involving viability, wound healing and adhesion assays in 66cl4ch14 cells. Integrative analysis of both screens (in vivo and in vitro), literature review, RNA-seq and bioinformatics analysis of gene expression and clinical outcome (Oncomine/Breastmark) indicated high confidence candidates to be taken for further analysis and additional validation in vivo.

Conclusion

Additional in vivo and in vitro experiments, aiming to validate the candidates genes as bona fide metastasis suppressor genes, as well as elucidate their possible mechanisms of action are ongoing.