Roberto Stabile

This thesis investigates the complex role of epigenetics in driving phenotypic plasticity within colon and oral cancer. More specifically, it examines how chromatin remodelers and microRNAs affect cancer progression and metastasis by bridging the exploration of general mechanisms underpinning phenotypic plasticity in colon cancer with the detailed analysis of the role of the CDK2APN gene in modulating the competitive interplay between SWI/SNF and NuRD chromatin remodeling complexes in oral squamous cell carcinoma (OSCC).

Chapter 2 explores the elusive nature of phenotypic plasticity, a key feature of carcinoma cells that enables them to alter their morphological and functional characteristics during local invasion and metastasis. This chapter uncovers a quasi-mesenchymal subpopulation of colon cancer cells, termed EpCAMlo, characterized by increased motility, invasiveness, chemotherapy resistance, and high metastatic potential. Through comprehensive bulk and single-cell RNA sequencing, the study reveals enhanced Wnt/β-catenin signaling and various degrees of epithelial-to-mesenchymal transition (EMT), including hybrid E/M states. These findings are particularly significant as they highlight a correlation with the CMS4 subtype, notorious for poor prognosis and pronounced stromal components.

In Chapter 3, we extend the analysis to an additional subpopulation of epithelial cells within the SW480 colon cancer cell line. The identification of distinct subpopulations, i.e. resident cancer stem cells (rCSCs), migratory CSCs (mCSCs), and high-relapse cells (HRCs) from the same colon cancer cell line, highlights the relevance of intra-tumor heterogeneity and the distinct roles of the different cellular components in tumor dynamics. Spatial transcriptomic data from patient-derived cancers confirm the presence and distinct spatial distributions of these subpopulations, enhancing our understanding of their contributions to tumor behavior.

Transitioning from colon to oral cancer, Chapter 4 focusses on the suppression of the CDK2APN tumor suppressor gene in OSCC, encoding for a critical subunit of the NuRD complex. Here, the main research question was why, despite the low frequency of mutations in its coding sequence, the CDK2APN gene is frequently downregulated in oral cancer. To this aim, we identified several microRNAs that inhibit CDK2APN translation, and developed a novel combined ISH/IF approach to analyze their expression in the context of oral squamous tumor architecture. Furthermore, the epigenetic regulation exerted by the newly identified microRNAs has significant implications for patient survival, emphasizing the clinical relevance of epigenetic control in OSCC.

Following up on these issues, Chapter 5 adds another layer of complexity by exploring the dual roles of phenotypic plasticity and inflammation in cancer progression, and focus on their regulation through the competitive dynamics between the NuRD and SWI/SNF chromatin remodeling complexes in OSCC. Here, we reveal how perturbations in these complexes influence inflammatory pathways and epithelial-mesenchymal plasticity. Specifically, I demonstrate that the loss of CDK2APN enhances a pro-inflammatory secretome, promoting monocyte recruitment and differentiation into M2-like macrophages within the tumor microenvironment. These findings not only link chromatin remodeling to inflammation and EMP but also suggest the potential of CDK2APN and related chromatin remodelers as prognostic markers and therapeutic targets.

Finally, Chapter 6 synthesizes the insights gained from both cancer types and discusses innovative strategies to decode the complexities given by the different chromatin remodelers in the context of phenotypic plasticity in cancer. This chapter addresses the challenges and potential of targeting the epigenetic mechanisms that underlie this plasticity, proposing novel approaches for cancer therapy that leverage our understanding of chromatin dynamics and microRNA regulation. By integrating these findings, the thesis proposes a new framework for considering cancer treatment, emphasizing the potential to disrupt the epigenetic interactions that contribute to cancer cell adaptability and resilience.