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Doctoral thesis

Multi-omic analysis reveals disruption of cell lineage integrity after EHF loss in prostate cancer

  • 2024

PhD: Università della Svizzera italiana

English Prostate cancer is one of the most prevalent malignances among men, and the failure of first treatment lines uncovers the necessity of alternative therapeutic strategies. The occurrence of prostate cancer is led by alterations in the epithelial compartment, in which the transition to undifferentiation state of epithelial cells, as well as the evolution of stem-like tumor cells, are sustained by epigenetic, transcriptional, and metabolic reprogramming. Transformed cancerous cells may adapt to a wide range of microenvironments and metabolic conditions, and this phenotypic plasticity is a fundamental source of tumor heterogeneity, metastatic spread, and therapeutic resistance: blocking and reverting plasticity mechanisms is a promising strategy to treat prostate cancer. EHF, belonging to ETS family, is one of the most expressed transcription factors in the prostate epithelial compartment and preserves epithelial integrity by regulating differentiation, as well as stem properties. Another key ETS factor is ERG, which is overexpressed in half of prostate cancer patients and is associated with the loss of several tumor suppressors, promoting tumor growth, inflammatory and angiogenic response. Investigating the regulatory role of EHF in maintaining prostate epithelial integrity, as well as the role of ERG in this process, is of meaningful importance. Three mouse models were generated and characterized to investigate how the loss of Ehf supports malignant transformation and find potential weaknesses for therapeutic purposes: EHFko, with a conditional prostate-specific knockout of Ehf gene; ERG, with the over-expression of ERG in the epithelial compartment; ERG-EHFko, with both perturbations. The transcriptomic and proteomic characterization at bulk level points out that Ehf loss leads to the up regulation of multiple inflammatory and stemness-related pathways, including EMT and Wnt signaling, which are associated with the acquisition of invasiveness, cancer stem cell phenotype, resistance to apoptosis, and treatment resistance. Bulk transcriptome of ERG-EHFko genotype suggests that ERG synergistically cooperates with Ehf loss, enhancing the alterations promoted in EHFko condition. Interestingly, the unbalance recorded in murine models reflects the properties of the aggressive castration-resistant prostate cancer patients with low levels of EHF. After the bulk characterization, for a better understanding of the heterogeneity and complexity of the several cell types composing the transformed prostates, the single-cell RNA-sequencing was performed on the whole prostates of murine models, enabling the possibility to discover altered pathways, infer cell trajectories and study the cell-cell interaction network. This data confirms that Ehf deficiency induces an ambiguous transcriptional program in which epithelial cells acquire stem cell-like, highly undifferentiated, pan plastic properties, switching toward a mesenchymal phenotype. Il6/Jak/Stat3 signaling and AP-1 complex are key drivers of epithelial remodeling, and the pharmacological blocking of Il6/Jak/Stat3 pathway results effective in the re-establishment of the normal-like phenotype of organoids from Ehf depleted mouse models. Moreover, through single-cell data, the complex network of interaction among cell compartments was outlined; particularly, this reveals a profound tumor microenvironment reprogramming, which comprises the activation of type 3 immunity, the exhaustion of Cd8+ T cells, and the polarization toward M2 macrophages. The main findings were validated in silico in prostate cancer patient databases, in vitro through functional assays in tissue organoids and mouse allografts, and in vivo in murine models. The comprehension of a clinically aggressive castration-resistant prostate cancer subtype was improved through the complete characterization of Ehf-deficient mouse models, and a new potential therapeutic approach has been established.
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