Abstract B58: Modeling oncogenic events in breast cancer precursor cells to study cancer evolution and genomic instability

Introduction: Over recent years, our knowledge of breast cancer biology has greatly improved; however, the mechanisms of tumor initiation and genomic drivers of this disease remain poorly understood. Hormone-negative breast cancers, such as triple-negative breast cancer (TNBC), are specifically challenging to treat, since they are genetically heterogeneous, often aggressive, and lack targetable driver mutation. Most TNBCs (80%) are basal-like (BLBC) and the majority of BLBCs harbor pathogenic mutations in TP53 tumor-suppressor gene (84%), indicating that TP53 plays a critical role in the pathogenesis of this cancer. Other less frequently observed alterations include mutation or epigenetic silencing of BRCA1/2 genes (36%), amplifications of oncogenes (MYC 40%, CCNE1 9%), and mutations or deletions of tumor suppressors (PTEN 35%, RB1 20%). Unfortunately, none of these genetic aberrations is currently druggable, mostly due to the lack of specific inhibitors. However, modeling these oncogenic events in precursor cells will enable us to study cancer evolution and acquisition of genomic instability. Experimental Procedures: We used basal-like mammary epithelial cells, MCF10A and MCF12A, to generate precursor models of TNBC. We overexpressed in these cell lines a hotspot p53 mutation (R175H), which demonstrates a dominant-negative phenotype. We confirmed that ectopic expression of R175H p53MUT impairs the ability of p53WT to properly respond to DNA damage induced by hydroxyurea or etoposide. As a result, cells fail to elevate the expression of p53 downstream targets, such as CDKN1A (cell cycle inhibitor), PUMA, BAX, and MDM2 (apoptosis regulators). Using CRISPR/Cas9-mediated genome editing we further performed depletion of RB1, PTEN, BRCA1, and BRCA2 genes in derived precursor models. Depletion of olfactory receptors OR10A4 and OR2W5 was performed as a control. In addition, we overexpressed S. pyogenes Cas9 endonuclease in these cell lines, in order to utilize them in CRISPR/Cas9-based gene knockout screens. Multiplexed functional analysis of CRISPR/Cas9-induced genomic alterations can be performed with lentiviral gRNA-based libraries in Cas9-expressing cells. All modified cell lines were clonally derived and gene depletion was validated with qRT-PCR and Western blot. Cas9 efficacy was confirmed with doxycycline-inducible RB1 knockout verified by T7 Endonuclease I genotyping assay. We further performed a range of phenotypic assays, such as proliferation, migration, anchorage-independent growth, and genomic instability assays, to measure the gain of tumorigenic properties upon acquisition of different genomic alterations. Summary: We established breast cancer precursor cell line models harboring genomic alterations in tumor suppressor genes commonly altered in TNBC, such as p53, RB1, PTEN, BRCA1, and BRCA2. Conclusions: Studying early transformation events leading to development of TNBC is necessary for understanding the etiology of this disease and identifying therapeutic vulnerabilities of precancerous lesions. To our knowledge, no comprehensive repertoire of breast cancer precursor models has been derived before. Also, ectopic expression of Cas9 in these cell lines makes them suitable for CRISPR/Cas9-based genomic screens to study disease evolution. CRISPR/Cas9 screens represent powerful tools to understand pathogenicity of different mutation events in disease initiation and progression. The use of genomically stable precursor cells in such studies makes the initial identification of putative drivers easier to define, in contrast to cancer cell lines, in which various compensatory pathways have evolved to cope with high genomic instability. Thus, established precursor models can be utilized in large-scale CRISPR/Cas9 screens focused on discovery of therapeutic targets, synthetic lethality, and genomic drivers of TNBC. Citation Format: Justyna Kanska, Kruttika Dabke, Simon A. Gayther. Modeling oncogenic events in breast cancer precursor cells to study cancer evolution and genomic instability [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B58.