Unbiased Piggybac Mutagenesis Screens Identify Tumorigenic and Metastatic Pathways in Breast Cancer

Breast cancer is the most frequent cancer-related death in female cancer patients and metastasis remains the primary cause of cancer-evoked mortality. Tumourigenesis and metastatic progression are complex processes, where cancer cells acquire specific mutations or genomic alterations. Despite some progress in understanding breast tumour biology, most of the molecular mechanisms leading to tumour progression and ultimately to metastasis remain undefined. Activating mutations in PIK3CA, which encodes for a catalytic subunit (p110a) of the phosphoinositide 3-kinase (PI3K), are among the most frequent driver alterations in human breast cancer leading to an hyperactivated PI3K pathway signaling. Oncogenic transformation often requires simultaneous and numerous driver oncogenic events. This results in tumour heterogeneity, which impacts the clinical outcome of the disease and make their discovery extremely necessary. In my PhD studies, we focused on the identification of genes involved in breast cancer tumourigenesis and metastasis using a transposon-induced mutagenesis screen. In particular, we performed an unbiased ex vivo piggyBac (PB) transposon insertional mutagenesis screen using cancer cells with an activating PIK3CAH1047R mutation to identify possible synergistic mechanisms of metastatic colonization. I identified and validated NFIB as an inducer of metastatic colonization in breast cancer. Mechanistically and functionally, I demonstrated that, NFIB increases the expression of the oxidoreductase ERO1A and the growth factor VEGFA, promotes metastasis and shortens overall survival of the animals. Finally, NFIB-ERO1A- VEGFA co-expression in basal-like breast cancers correlated with reduced patient survival. Furthermore, I generated a conditional PB PIK3CAH1047R mutant mammary cancer mouse model and performed an in vivo PB genome-wide unbiased mutagenesis screen to identify potential synergistic tumorigenic pathways. Serial transplantation of the mutant tumours enriched for tumour initiating cells and targeted sequencing of transposon integration sites revealed almost 7700 common insertion sites (CISs). The most frequently altered genes in the secondary transplanted tumours compared with the primary transplanted tumours may increase tumour initiating ability. My results not only describe potential new targetable pathways of breast cancer tumorigenesis and metastatic colonization, but also highlight the power of genetic screenings for identifying functionally relevant breast cancer driver pathways.