Browsing by Subject "EZH2"

Breast cancer is globally the leading cause of death in women. ER positive, HER2 negative breast cancer is the most common subgroup, covering two thirds of all breast cancer cases. The different isoforms of ERα, ERα66 and ERα36 are responsible of genomic and non-genomic ER signaling respectively. Tamoxifen is one of the most used drugs in ERα+ breast cancer. As a SERM tamoxifen blocks the activity of ERα66, but plays as an agonist for ERα36, which is associated with tamoxifen resistance. Tamoxifen resistance concerns more than 25% patients with ERα+ breast cancer but the molecular mechanisms that lead to development of resistant disease remain uncovered. Thus, the aim of this thesis was to reveal how two different ERα isoforms are used and regulated in tamoxifen resistance in two commonly used ERα+ breast cancer cell lines MCF7 and T47D. We studied the effect of hormones to tamoxifen sensitivity and to utilization of ERα isoforms. Additionally, we compared the transcriptomics of resistant and parental cells in both cell lines and tested how inhibition of key regulators affect the sensitivity against tamoxifen. In this thesis we report that MCF7 and T47D cell lines obtain different mechanisms of tamoxifen resistance, and that the development of tamoxifen resistance is a parallel process with the cell identity switch from luminal to basal. The EZH2 is involved in maintaining the luminal progenitor type of mammary cells, whereas c-Myc is highly expressed in the resistant cell lines. Hence, EZH2 and c-Myc are key players in development of tamoxifen resistance and could be considered as therapy targets in ERα+ breast cancers.

Lung cancer is one of the most common and deadliest cancers worldwide, but the mechanisms behind different types of lung cancer are still poorly understood. Non-small cell lung cancer makes up 80% of lung cancers, and some epigenetic mechanisms have been proposed for it. Epigenetic modifications are a way of influencing the expression of genes by inhibition or activation. PRC2 is an epigenetic modulator that catalyses the formation of methyl groups on histone 3 lysine 27, which is an epigenetic mark with repressive nature. PRC2 has been proposed to be downstream of AMPK, an energy sensor of the cell, which is phosphorylated by LKB1 under energy stress conditions. Inactivating mutations in LKB1 are known to cause and worsen non-small cell lung cancer, and the overexpression of EZH2, the catalytic subunit of PRC2, has similar effects. Therefore, establishing a novel downstream mechanism linking LKB1, AMPK, and PRC2 together could explain one mechanism for NSCLC tumorigenesis. Changes in metabolism are a feature of cancer cells, and this pathway could also link energy stress and cancer together. Mouse embryonic fibroblast and H358 cell lines overexpressing wild type EZH2, mutant EZH2 and GFP were generated and treated with the glycolysis inhibitor 2-deoxyglucose to study the effects of energy stress. Levels of histone methylation and phosphorylation statuses of AMPK and its downstream target ACC were assessed with Western blotting, and expression levels of potential PRC2 target genes with RT-qPCR. The study setting proved to be functional for the response of AMPK to energy stress conditions, as both AMPK and ACC were phosphorylated in the presence of 2-DG. In mouse embryonic fibroblasts, PIM1 showed different gene expression with wild type and mutant EZH2, suggesting that its activation would be regulated through the phosphorylation of the T311 site of EZH2 during energy stress. The results from histone methylation statuses did not follow the hypothesis, possibly because of the lack of specificity of detecting global H3K27me3. Other target genes besides PIM1 in MEFs did not show significant changes in expression level. Considering that the incorporation of the mutant EZH2 into PRC2 complexes was not validated, additional research would be needed to confirm or deny the explained mechanism between PRC2 and AMPK.