Alcohol is the dietary factor, which is most consistently associated with breast cancer risk. This association involves the estrogen receptor (ER), which is over-expressed (ER+) in around 80% of breast cancer cases. Alcohol-association is more pronounced in ER(+) breast cancer cases than in ER(-) breast cancer cases, however, the molecular mechanism remains to be determined. Cancer cells have a consistent cytological feature of nucleolar hypertrophy, where rRNAs are synthesized by RNA polymerases (Pol) I and Pol III. Pathologists have been using enlarged nucleoli as a diagnostic indicator of cell transformation and neoplasia. It indicates that transformation in situ is tightly linked to the deregulation of RNA Pol I and III gene transcription, because the size of the nucleolus reflects the levels of rRNA synthesis. RNA Pol III is responsible for the synthesis of a variety of untranslated RNAs, including 5S rRNAs and tRNAs. Deregulation of RNA Pol III-dependent genes (Pol III genes) would serve to enhance the translational capacity of cells, which is required to promote cell transformation and tumor development. Alcohol-induced deregulation of Pol III genes may be fundamental to the development of breast cancer. Our previous studies demonstrated that MAP kinases modulated Brf1 and TBP expression and Pol III gene transcription and mediated phosphorylation of histone H3 (H3ph). Our recent studies have demonstrated that ethanol activates MAP kinase and induces Pol III gene transcription through enhanced TBP and c-Jun expression by using cell culture and an animal model. Preliminary results have revealed that alcohol induces Pol III gene transcription in both normal breast and breast cancer cell lines. However, the induction in breast cancer cells (5-6 fold) is higher than i normal breast cells (2.5 fold). Further analysis indicates that the induction is ER dependent. The ER ligand, E2 (17b-estradiol) causes an induction (<2 fold) of these genes, whereas ethanol works with E2 to create an additional increase (12 fold) in Pol III gene transcription, resulting i cell proliferation and transformation. Alcohol activated MSK1 (mitogen- and stress-activated protein kinase 1), a downstream component of MAP kinases, which mediates phosphorylation of histone H3 (H3ph) at serine 10 (H3S10ph) and serine 28 (H3S28ph) and modulates gene expression and cell transformation. Thus, we hypothesize that alcohol activates MSK1, which mediates H3ph. H3ph in turn upregulates Brf1 expression and Pol III gene transcription to enhance the protein synthetic capacity of cells, which can eventually lead to ERa-dependent breast cancer. This implies that the induction by alcohol may be an early event, contributing to the development of ER(+) breast cancer. By using cell culture and animal models, we will determine: 1) if alcohol-activated MSK1 mediates Brf1 expression and Pol III gene transcription, which in turn causes phenotypic changes, and if inhibition of MSK1 by its chemical inhibitor and shRNA or using a MSK KO mouse blocks alcohol-induced cell transformation and Pol III gene transcription;2) if alcohol-induced H3ph modulates Brf1 and Pol III gene expression and cell transformation;3) if alteration of ERa and Brf1 expression affects transcription of Pol III genes and if blocking Brf1 expression by its shRNA inhibits tumor formation in nude mouse upon administration of alcohol or alcohol plus E2. These studies are designed to determine the molecular mechanism of alcohol- induced deregulation of Pol III genes in the development of ER+ breast cancer. Investigating the effects of MSK1 and Brf1 shRNAs on tumor formation may provide a new approach to inhibit tumor growth. Our overall objective is to investigate the role of MSK1 and Brf1 in the alcohol-induced response that may be critically important in ER+ breast cancer development.
The project seeks to identify a novel class of targets, MSK1, ERa, and Brf1, triggered by alcohol response. By using cell culture and animal models, we will elucidate the molecular mechanism of alcohol-associated breast cancer and explore their roles of MSK1, ERa, and Brf1 in deregulation of RNA Pol III gene transcription, cell transformation and tumor formation. We will establish the specific pathway by determining whether MSK1, a downstream component of MAP kinases, mediats alcohol- induced histone H3 phosphorylation, Brf1 expression and Pol gene transcription. We will knock down MSK1 and Brf1 by its shRNAs to inhibit Pol III gene transcription and tumor formation. Our new discovers of alcohol-induced MSK1 activation, hisotne H3 phosphorylation, ERa and Brf1 expression and Pol III gene transcription will provide valuable information to develop new therapeutic strategy for alcohol-associate breast cancer.
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