The evolutionary trajectory of hormone-dependent breast cancer (HDBC) is shaped by endocrine therapy and interlaced with resistance-arising due to selective pressure acting at the cellular level. In vitro evidence showed that HDBC progresses through a multi-faceted route. It is initially characterized by exponential cellular growth, an in-between resting period in response to therapy called “dormancy’’, and a relapse, where cells resume proliferation after acquiring resistance (“awakening”). While extensive large-scale genomics studies have revealed an insufficient array of putative coding drivers explaining the progression of the malignancy, investigation on the non-coding regulatory genome (CREs) mutations’ role in its multi-step evolution is still in its infancy.
Of all the CREs, insulators are a frequent mutational hotspot in cancer. For their role in fostering enhancer-promoter contacts and maintaining physical boundaries between adjacent genomic regions when bound by CTCF, these elements were the first to being investigated in my work. Starting from a mutation that emerged from a luminal A breast cancer patient, I subsequently knocked out a cluster of insulators at the affected TAD (Topologically Associated Domain) to uncover global transcriptional rewiring and understanding clustered CTCF dynamics. This small-scale pilot functional study was followed by the validation of findings that emerged from a large-scale pooled CRISPR perturbation screen. The study, called SID (Systematic Identification of Epigenetically Defined Cis Regulatory Elements), systematically perturbed with a dcas9KRAB interference system a window of 12 Mbp regulatory elements comprising HDBC relevant enhancer, promoters, and CTCF. This unveiled time and context regulatory dependencies in the guise of significantly enriched or depleted sgRNA that involved CREs mapping at MYD88, TLR5, UNC93B1, and USP8 genes. As a conclusive work, I characterized the topology of awakenings through live imaging and started to hypothesize the role of cell-to-cell communication in HDBC evolution by exploring the role of PARD3.
In brief, my work added some pieces of understanding to untangle the role of CREs in hormone-dependent breast cancer evolution. By exploring their significance from different angles and experimental procedures, I could also unveil the importance of understanding and modelling all the stages of the disease, uncovering the complexity underlying it.