Role of MSK1, ERa and Brf1 in alcohol-associated breast cancer

描述(由申请人提供)：酒精是饮食因素，与乳腺癌风险最一致。这种关联涉及雌激素受体(ER)，它在大约80%的乳腺癌病例中过度表达(ER+)。酒精在ER(+)乳腺癌患者中比在ER(-)乳腺癌患者中更明显，然而，其分子机制仍有待确定。癌细胞具有一致的核仁肥大的细胞学特征，其中rRNAs是由RNA聚合酶(Pol)I和Pol III合成的。病理学家一直将增大的核仁作为细胞转化和肿瘤发生的诊断指标。这表明原位转化与RNA PolI和III基因转录的解除密切相关，因为核仁的大小反映了rRNA合成的水平。RNA Pol III负责合成各种未翻译的RNA，包括5S rRNAs和tRNAs。解除对RNA POLIII依赖基因(POLIII基因)的管制将有助于提高细胞的翻译能力，这是促进细胞转化和肿瘤发展所必需的。酒精诱导的POLIII基因失控可能是乳腺癌发生的基础。我们以前的研究表明，MAP激酶调节Brf1和TBP的表达和Pol III基因的转录，并介导组蛋白H3(H3ph)的磷酸化。我们最近的研究表明，乙醇通过细胞培养和动物模型增强TBP和c-Jun的表达，激活MAP激酶，诱导POLIII基因转录。初步结果显示，酒精可诱导正常乳腺和乳腺癌细胞系中POL III基因转录。然而，对乳腺癌细胞的诱导率(5-6倍)高于正常乳腺细胞(2.5倍)。进一步的分析表明，诱导是内质网依赖的。雌激素受体配体E2(17b-雌二醇)诱导这些基因的表达(2倍)，而乙醇与E2共同作用，使POLIII基因转录增加12倍，导致I细胞增殖和转化。酒精激活了MAP激酶下游的MSK1(丝裂原和应激激活蛋白激酶1)，它介导组蛋白H3(H3ph)在丝氨酸10(H3S10ph)和丝氨酸28(H3S28ph)处的磷酸化，并调节基因表达和细胞转化。因此，我们假设酒精激活了MSK1，而MSK1调节H3ph。H3ph反过来上调Brf1的表达和Pol III基因的转录，以增强细胞的蛋白质合成能力，最终可能导致Era依赖型乳腺癌。这意味着酒精的诱导可能是一个早期事件，有助于ER(+)乳腺癌的发展。通过细胞培养和动物模型，我们将确定：1)酒精激活的MSK1是否介导Brf1和Pol III基因的转录，进而导致表型变化，以及是否通过其化学抑制剂和shRNA抑制MSK1，或使用MSK KO小鼠阻止酒精诱导的细胞转化和Pol III基因转录；2)酒精诱导的H3ph是否调节Brf1和Pol III基因的表达和细胞转化；3)在给予酒精或酒精加E2后，era和Brf1的表达是否影响Pol III基因的转录，以及是否通过其shRNA阻断Brf1的表达抑制裸鼠体内的肿瘤形成。这些研究旨在确定酒精诱导的Pol III基因失控在ER+乳腺癌发生中的分子机制。研究MSK1和Brf1 shRNAs在肿瘤形成中的作用可能为抑制肿瘤生长提供新的途径。我们的总体目标是调查 MSK1和Brf1在酒精诱导的反应中可能在ER+乳腺癌的发生中起关键作用。

DESCRIPTION (provided by applicant): 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.