More than meets the EMT: Uncovering non-canonical ZEB1 proteoforms and their roles in cancer cell plasticity

The ability of cancer cells to undergo dynamic, non-genetic transitions between cell states is a major driver of tumour heterogeneity, metastasis, and therapy resistance. As a result, cancer cell plasticity remains one of the biggest obstacles to effective treatment. To achieve this, this thesis provides evidence that highlights the need to shift our understanding of cellular plasticity from focusing exclusively on well-characterised models such as the epithelial-to-mesenchymal transition (EMT), to considering a broader landscape of phenotypic states that cancer cells adopt, and the distinct molecular mechanisms that are regulated by them. ZEB1 is a key regulator of cancer cell plasticity, yet its role has largely been defined through its function as an EMT transcription . Here, I show that ZEB1 is a complex protein with functions extending well beyond EMT. Using a multi-omic approach, I explore ZEB1’s roles in triple-negative breast cancer, a subtype reliant on ZEB1-mediated plasticity. I am the first to characterise four major ZEB1 proteoforms, expressed as 125, 140, 200, and 260 kDa, that have distinct subcellular localisations. I confirm that larger proteoforms arise from extensive post-translational modifications, not splicing. Only the 140 and 200 kDa forms localise to the nucleus. Importantly, the 200 kDa nuclear proteoform is exclusive to cancer stem cell (CSC) populations, suggesting distinct proteoform-specific functions. To investigate ZEB1’s broader activity, I defined its interactome in CSCs and non-CSCs, revealing distinct protein networks involved in RNA processing, cell cycle, and cytokinesis. These findings, along with public interactome and imaging data, suggest a novel role for ZEB1 in mitotic processes. ZEB1 chromatin profiling revealed a shared core cistrome between CSCs and non-CSCs, regulating MYC, E2F, and metabolic pathways, likely driven by 140 kDa ZEB1. In contrast, EMT-related programs and AP-1 co-occupancy at distal enhancers are CSC-specific and linked to 200 kDa ZEB1. Contrary to its traditional classification as a repressor, ZEB1 frequently activates transcription in HCC38 cells. Transcriptomic analysis of CRISPR activation and inhibition ZEB1 TNBC lines supports a highly context-dependent regulatory role. Collectively, these findings redefine ZEB1 as a pleiotropic and context-responsive regulator of cancer cell plasticity, with distinct proteoforms enabling diverse transcriptional and non-transcriptional functions.