Epigenetic alterations in rostate cancer : elucidating the role of DNA methylation and histone modifications as well as the methylation patterns of prostate cancer associated genes in the development and evolution of the disease

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Λογοθέτη, Σουζάνα
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Prostate cancer (PCa) is an exemplar of heterogeneous disease that remains the most frequent and second most lethal solid tumor in men. The most important barrier in PCa therapy is the lack of markers that would allow the selection of the most appropriate therapeutic strategy for each patient. Aggressive Variant of Prostate Cancer (AVPC) is a subset of PCa patients with particularly virulent clinical characteristics and limited therapeutic options. PCa heterogeneity, therapy resistance, stemness, and lethal progression have been attributed to lineage plasticity, which refers to the ability of neoplastic cells to confront ominous microenvironmental pressures by altering their epigenetic profiles and as a result their phenotypic characteristics. Our overall hypothesis is that PCa heterogeneity can be described by epigenetic alterations and that epigenetic markers along with the presence of highly plastic cell state (HPCS) clusters that enable tumor adaptability in ominous microenvironmental pressures can be used as measurable metrics of aggressive PCa disease. In the present thesis, we aimed to identify epigenetic biomarkers that can better describe the intratumoral heterogeneity as well as epigenetic and transcriptional biomarkers that can be linked to lineage plasticity in an effort to identify candidate measures of PCa aggressiveness. In addition, we aimed to identify markers that can be linked to the effects of epigenetic modulator (Valemetostat) and chemotherapeutic (cabazitaxel, carboplatin) drug administration in lineage plasticity properties. For that purpose, we used patient-derived xenograft murine models of androgen indifferent PCa to perform genomic (T200.1 targeted DNA-sequencing, single cell DNA sequencing), epigenetic (Methylated CpG Island Amplification and Microarray-MCAM, Reduced representation bisulfite sequencing- RRBS), transcriptomic (Clariom S Affymetrix microarray, single cell and bulk-RNA sequencing) and proteomic (Reverse-Phase Protein Array -RPPA) studies. We used two AVPC PDX models (MDA PCA 144-4 and 144-13) that came from the same patient at the same time point but have diverse morphology to analyze intratumoral heterogeneity and identified differentially methylated genes, such as OTX2, SIX3 and DRD4 with potential interactions to the RB1, TP53 and PTEN networks that play pivotal roles in aggressive PCa. Next, we sought to identify a molecular signature that can be linked to lineage plasticity and aggressive potential. We showed that two AVPC PDX models (MDA PCa 177-B and MDA PCa 189-1), that come from the same patient at different time points in the course of his disease, share a common ancestor while exhibiting distinct phenotypic profiles, thus serving as experimental models for studying lineage plasticity in PCa. We identified a list of hyper- and hypo- methylated genes as well as active promoters, enhancers and transcriptional factors (TFs) that are uniquely present in the AVPC models compared to publicly available data of non-neoplastic specimens. In addition, scRNAseq analysis revealed the presence of a highly plastic cell state (HPCS) cluster in these AVPC models as determined by CytoTRACE score (≥), high Lineages’ Diversity score and trajectory inference, that was not present in normal prostate, primary low grade PCa and castrate-resistant prostate cancer specimens. We then, performed in vitro and in vivo studies of the effect of the chemotherapeutic drugs carboplatin and cabazitaxel, in cell proliferation and tumor growth and correlated it to protein expression aberrations. Carboplatin administration led to a slower increase of tumor volume compared to the control group, whereas cabazitaxel ± carboplatin led to total tumor eradication. These observations reflected dysregulation of multiple protein levels (such as LDHA, PTEN, Cadherin, GR, etc.). A pilot study of the effect of the epigenetic modulator Valemetostat (EZH1/2 inhibitor) was also performed in vitro. In conclusion, the epigenetic landscape and the emergence of HPCS play a pivotal role in PCa evolution and aggressiveness and enable the identification of patients whose tumor is destined to follow more aggressive disease.
Prostate cancer, AVPC, Lineage plasticity, Aggressive variant prostate cancer, Patient-derived xenografts, Next-Generation Sequencing (NGS), Bioinformatics, Epigenetics, DNAmethylation sequencing, ChIPseq, RPPA, RRBS, scRNAseq, Chemotherapy, MCAM, Pathway enrichment analysis