Exploring epigenetic mechanisms to target mammary cancer cells: a comparative approach

Author(s)

Pedro Luiz Porfirio Xavier

• Document type: Doctoral Thesis
• Press: Pirassununga.
• Institution: Universidade de São Paulo (USP). Faculdade de Zootecnica e Engenharia de Alimentos (FZE/BT)
• Defense date: 2020-12-18

Examining board members

Heidge Fukumasu; Maria Lucia Zaidan Dagli; Raquel Carvalho Montenegro; Tathiane Maistro Malta Pereira; Juliano Rodrigues Sangalli

• Advisor: Heidge Fukumasu

Abstract

Cancer is one of the main causes of deaths in dogs worldwide, and mammary cancer in female dogs corresponding to more than 50% of all types of tumors. In addition, mammary tumors in dogs share many clinical and molecular similarities in comparison to breast cancer in women. Therefore, dogs are great models for comparative studies in oncology. Epigenetic mechanisms are key regulators of gene expression and dysregulation of these mechanisms are associated to the development of several types of cancer both in humans and dogs. Thus, canine model may be important for the development of innovative and potential therapies for cancer, especially for breast cancer. Thus, the main purposes of this thesis were: 1) to review the epigenetic mechanisms behind the development of different types of cancer in dogs, in comparison with abnormalities well described in human cancer; 2) to determine possible molecular targets associated with tumorigenicity and invasiveness in canine mammary cancer cells (CMC cells); 3) to determine potential epigenetic targets associated with self-renewal and tumorigenicity in CMC cells; 4) to observe the effects of a new dual BET/HDAC inhibitor in human breast cancer cells using 2D and 3D in vitro models. In the first chapter, we gathered findings demonstrating that some epigenetic abnormalities are similar in both human and canine cancer, suggesting a potential approach to use the canine model to determine new epigenetic mechanisms regulating cancer and to develop new therapies. In chapter 2, using in vitro models, we demonstrate that epithelial-mesenchymal (EMT)-associated transcription factors, ZEB1 and ZEB2, are associated with tumorigenicity and invasiveness of CMC cells, exhibiting higher expression in mesenchymal-like cells in comparison with epithelial-like cells. In addition, we observed that molecular pathways associated with EMT, invasiveness, and tumorigenicity, such as ECM organization and degradation, focal adhesion, TGF-β / BMP signaling, PI3K-AKT signaling, and WNT signaling are enriched to upregulated genes overexpressed in mesenchymal-like cells. Furthermore, this work was important to determine cell lines presenting higher tumorigenic potential in our cell bank. In chapter 3, we demonstrated that inhibition of a family of epigenetic reader proteins, known as BET proteins, by (+)-JQ1, is a promising strategy to suppress in vitro tumorigenicity and self-renewal of CMC cells. BET inhibition resulted in a decrease of tumorspheres formation in low-adherence plates and colony formation in soft-agar assay, classical models used to assess in vitro self-renewal and tumorigenicity. Furthermore, inhibition of BET proteins decreased expression of important genes associated with self-renewal pathways including WNT, NOTCH, Hedgehog and PI3K/AKT/mTOR. Besides that, BET inhibition decreased ZEB2 expression, a transcription factor important for the maintenance of self-renewal in CMC cells. Finally, in chapter 4, we observed that the dual BET/HDAC inhibitor, TW09, reduced cell viability and increased cell death in human breast cancer cells in comparison with individual inhibition of BET and HDAC proteins by (+)-JQ1 and CI994, respectively. In addition, TW09 reduced number of primary and secondary BC tumorspheres, suggesting effects regarding tumorigenicity and self-renewal potential of breast cancer cells. Thus, TW09 demonstrated good effects in these cells. We still intend to deeply study the effects of dual BET/HDAC inhibition by TW09 on breast cancer both in human and canine cells in order to confirm this strategy as a potential and innovative therapy for mammary cancer in both species. Finally, the results demonstrated in this thesis collaborate to the validation and discovery of important mechanisms behind mammary cancer and its phenotypes in dogs, besides highlight the canine model as a great model to perform comparative studies in oncology and search for new targets and therapies for cancer, since several of the mechanisms and effects observed in this model, corroborate those observed in human cancer cells. (AU)

• FAPESP's process: 17/11966-4 - Epigenetic modulations and self-renewal potential of canine mammary cancer cells: the search for potential therapeutic targets
• Grantee: Pedro Luiz Porfirio Xavier
• Support Opportunities: Scholarships in Brazil - Doctorate