Cell survival pathways and mechanisms of response in breast cancer
AuthorWhittle, James Richard
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2022-02-12.
© 2019 James Richard Whittle
Breast cancer is a heterogenous disease that can be stratified into at least six subtypes based on gene expression profiling. Each of these subtypes likely arise from a different cell of origin, through a repertoire of genetic aberrations that influences treatment decisions and subsequent resistance to therapy. Multiple mechanisms of resistance exist, including evasion of apoptotic cell death, which is a hallmark of cancer. The development of BH3 mimetics, which antagonise pro-survival proteins of the BCL2 family, is a new field of targeted cancer therapy. Initial studies observed that BCL2 is overexpressed in the majority of primary and metastatic estrogen receptor positive (ER+) breast cancer, and targeting BCL2 with ABT-199 (venetoclax), a potent BCL2 inhibitor, synergises with endocrine therapy in preclinical models of ER+ breast cancer. This thesis builds on these insights into the molecular events that are responsible for resistance to cell death. ER+ breast cancers also frequently exhibit deregulation of the retinoblastoma (Rb) pathway that includes cyclin-dependent kinase 4 and 6 (CDK4/6)/cyclin D1 (CCND1)/retinoblastoma (Rb), resulting in uncontrolled cellular proliferation. CDK4/6 inhibitors significantly augment tumor response when combined with endocrine therapy in ER+ breast cancer. Despite their potent antiproliferative activity, CDK4/6 inhibitors fail to induce apoptotic cell death. This thesis investigates the addition of the BH3 mimetic ABT-199 to endocrine therapy (fulvestrant) and the CDK4/6 inhibitor palbociclib in breast tumor organoids and patient-derived xenograft models of ER+ breast cancer. Triple therapy, which was well tolerated in vivo, produced a superior and more durable tumor response compared to single or doublet therapy. Increased apoptosis was observed in vitro and in vivo, with improved survival in PDX models. Notably, MHC class I and PDL1 expression increased in a syngeneic mouse model of ER+ breast cancer, accompanied by a reduction in regulatory T cells. Together, these findings provide a rationale for investigation of combination therapy in the clinic. The targeting of another pro-survival protein, MCL1, has been an intense area of interest as it is frequently dysregulated in cancer. In breast cancer, MCL1 is often amplified, particularly in triple negative and HER2-amplified subtypes, where high expression predicts resistance to treatment and poor outcomes. This thesis shows that a novel inhibitor of MCL1, S63845, demonstrated marked synergistic activity in preclinical models of breast cancer, supporting a rationale for clinical evaluation of MCL1 inhibitors in breast cancer. This work also explores how the accumulation of genetic hits to a key cell of origin influences the resulting breast cancer subtype. The precise initiating events that predispose the sequence from normal epithelium to neoplastic progression is poorly understood. Breast organoids were generated from human reduction mammoplasties, creating a tool to study the clonal evolution of breast cancer. CRISPR/Cas9 gene editing was used for targeted knockdown of four of the most commonly mutated tumour suppressor genes (P53, PTEN, RB1 and NF1) in cultured mammary progenitor cells. Mutated organoids gained long-term culturing capacity and formed tumours of luminal histology when transplanted into mice. Organoids were demonstrated to be responsive to endocrine therapy or cytotoxic chemotherapy, supporting the potential utility of this model to enhance our understanding of the cellular origins and genetic lesions underlying breast cancer.
KeywordsBreast cancer; Apoptosis; BCL2; MCL1; ABT-199; Venetoclax; S63845; Palbociclib; Organoids; CRISPR/Cas9; Gene editing; P53; PTEN; RB1; Patient derived xenograft
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- Medical Biology - Theses