Involvement of EZH2-MYC loop and SALL4 in Epithelial-Mesenchymal Transition (EMT) and trastuzumab resistance process in HER2+ breast cancer

Breast cancer (BC) is the most common type of cancer in females worldwide. It is also the second leading cause of death in women. BC is covered with heterogeneity properties, that leads to poor prognosis and therapeutic resistance. It has always been essential to unveil the different molecular mechanisms involved in BC cancer progression, finding a suitable treatment for the patients. This thesis focuses on unwrapping the various molecular mechanisms involved in HER2+ BC subtypes, as this denotes an aggressive phenotype among other subtypes of BC. Downregulation of miR-33b has been documented in many types of cancers and involves proliferation, migration, and epithelial-mesenchymal transition (EMT). Furthermore, enhancer zeste homolog 2-gene (EZH2) is a master regulator of controlling the stem cell differentiation and cell proliferation processes. The implication of miR-33b in the EMT pathway and analyze the role of EZH2 in this process and interaction between them is one of the main spotlights of the thesis. miR-33b is downregulated in HER2+ BC cells vs healthy controls, where EZH2 has an opposite expression in vitro and patients’ samples. The upregulation of miR-33b suppressed proliferation, induced apoptosis, reduced invasion, migration and regulated EMT by an increase of E-cadherin and a decrease of ß-catenin and vimentin. The silencing of EZH2 mimicked the impact of miR-33b overexpression. Furthermore, the inhibition of miR-33b induces cell proliferation, invasion, migration, EMT, and EZH2 expression in non-tumorigenic cells. Notably, the Kaplan–Meier analysis showed a significant association between high miR-33b expression and better overall survival. These results suggest miR-33b as a suppressive miRNA that could inhibit tumour metastasis and invasion in HER2+ BC partly by impeding EMT through the MYC–EZH2 loop's repression. On the other hand, treatment for the HER2+ BC subtype is minimal. Trastuzumab is a monoclonal antibody, regularly used for the treatment of this specific subtype of BC. Although trastuzumab is currently considered one of the most effective oncology treatments, a significant number of patients with HER2-overexpressing breast cancer do not benefit from it. The other part of the thesis focuses on finding a novel molecular mechanism of one transcription factor (TF), Sal-like protein 4 (SALL4), a critical regulator of cancer aggressiveness and resistance treatment. HER2+ BC cells with acquired resistance to trastuzumab express a higher level of SALL4 as compared to the wild type cells. Gain and loss function experiments showed that less SALL4 expression conducted the restoration of the trastuzumab 21 | P a g e sensitivity significantly; however, the transient overexpression of SALL4 in parental cell lines induced high proliferation of the cells, resulting of the reduction of trastuzumab efficacy. Furthermore, SALL4 expression regulates the PI3K/AKT pathway, through controlling of PTEN expression. Moreover, AKT phosphorylation activated many downstream targets, such as BCL2, resulting in increased cell survival and proliferation. It has been observed that SALL4 expression regulates EMT pathway via controlling the MYC expression. SALL4 showed a physical interaction with RBBP4, a NuRD complex member, and regulates the downstream proteins such as PTEN and BCL2. This interaction also helps cells to be escaped from the trastuzumab treatment and therefore, targeting the SALL4–NuRD pathway in HER2+ BC, mostly in acquired resistance cell lines would be a promising therapeutic approach and better treatment for this specific type of cancer in future. SALL4 also predicted as a prognostic factor in all subtypes of BC through KM plotter. This study provides a viable molecular mechanism-drive therapeutic strategy for the significant subset of patients with HER2+ BC whose malignancies are driven by SALL4 expression

MicroRNA-33b Suppresses Epithelial-Mesenchymal Transition Repressing the MYC-EZH2 Pathway in HER2+ Breast Carcinoma

Downregulation of miR-33b has been documented in many types of cancers and is being involved in proliferation, migration, and epithelial-mesenchymal transition (EMT). Furthermore, the enhancer of zeste homolog 2-gene (EZH2) is a master regulator of controlling the stem cell differentiation and the cell proliferation processes. We aim to evaluate the implication of miR-33b in the EMT pathway in HER2+ breast cancer (BC) and to analyze the role of EZH2 in this process as well as the interaction between them. miR-33b is downregulated in HER2+ BC cells vs healthy controls, where EZH2 has an opposite expression in vitro and in patients' samples. The upregulation of miR-33b suppressed proliferation, induced apoptosis, reduced invasion, migration and regulated EMT by an increase of E-cadherin and a decrease of ß-catenin and vimentin. The silencing of EZH2 mimicked the impact of miR-33b overexpression. Furthermore, the inhibition of miR-33b induces cell proliferation, invasion, migration, EMT, and EZH2 expression in non-tumorigenic cells. Importantly, the Kaplan-Meier analysis showed a significant association between high miR-33b expression and better overall survival. These results suggest miR-33b as a suppressive miRNA that could inhibit tumor metastasis and invasion in HER2+ BC partly by impeding EMT through the repression of the MYC-EZH2 loop