Autocrine TGFBeta and Breast Cancer Cell Growth

自分泌 TGFBeta 与乳腺癌细胞生长

• 批准号: 6619638
• 负责人: MICHAEL G BRATTAIN
• 金额: $ 27.69万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6619638
• 关键词: JUN kinase; apoptosis; autocrine; breast neoplasms; cholecalciferol; estrogen receptors; gel mobility shift assay; mammary gland; neoplasm /cancer pharmacology; neoplastic growth; nutrition aspect of cancer; nutrition related tag; transcription factor; transforming growth factors; vitamin analog

State the application's broad long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This description is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this During the present cycle of funding we found that autocrine TGFbeta was induced by vitamin D3 analogs in ER+ breast cancer and pre-malignant mammary epithelial cells. Vitamin D3 analog response was 100 fold more resistant to inhibition in the absence of autocrine TGFbeta and preliminary evidence suggests that Smad3 activated by TGFbeta signaling enhances vitamin D3 receptor VDR directed transcription. However, while transfection of the type II TGFbeta receptor (RII) into RII null MCF-7 cells completely restores vitamin D analog sensitivity, transfection of Smad3 does not. This indicates that a second aspect of TGFbeta signaling is required in addition to Smad3. Thus, in Specific Aim I we will test the hypothesis that Smad3 interactions with the VDR are required for TGFbeta/VDR crosstalk, but that in addition to this Smad3 pathway, TGFbeta mediated activation of the MAPK pathway is also required. We and others have observed that vitamin D analogs cause apoptosis. The mechanism by which apoptosis is induced is largely unexplored, particularly with respect to the role of TGFbeta crosstalk. TGFbeta itself also induces apoptosis. Consequently, we will test the hypothesis that vitamin D3 analog induced apoptosis is mediated by the enhanced autocrine TGFbeta activity resulting from treatment with the drug. During the present period of funding we found that TGFbeta receptor expression in ER+ breast cancer cells is transcriptionally repressed. The repression can be blocked by treatment with the methylase inhibitor, 5 aza deoxycytidine. Surprisingly, this agent did not reverse methylation of the RII promoter, but instead increased the cellular level of Sp1 which is underexpressed in ER+ breast cancer cells. Sp1 is required for RII transcription. Interestingly, Sp1 transcription was not targeted by 5 aza deoxycytidine. Further, investigation showed that Sp3 transcription factor which represses Sp1 mediated transcription was elevated in ER+ breast cancer cell lines. Sp3 was shown to be a repressor of RII by southwestern, gel shift and blockade of RII promoter-reporter activity after Sp3 transfection. In addition Sp3 transcripts were decreased as a result of 5 aza cytidine treatment. Consequently, it appears as though Sp1/Sp3 homeostasis is responsible for repression of RII transcription in ER+ breast cancer cells. This hypothesis will be tested in Specific Aim III. The Specific Aims for the renewal project are: 1. Determine the mechanism of crosstalk between vitamin D3 analogs and TGFbeta in ER+ breast cancer cells, immortalized mammary epithelial cells and normal mammary epithelial cells. Determine whether vitamin D3 response in ER- cells is also associated with TGFbeta crosstalk. II. Determine the mechanism of induction of apoptosis by vitamin D3 analogs in the cell types listed in Specific Aim I and determine whether apoptosis inhibition is dependent upon autocrine TGFbeta. III. Determine how Sp1 and Sp3 homeostasis is controlled in ER+ breast cancer to regulate RII transcription.

说明申请的广泛的长期目标和具体目标，并提及项目的健康相关性。 简要描述研究设计和实现这些目标的方法。 避免总结过去的成就和使用第一人称。 本说明书旨在作为与申请分开时所提出的工作的简洁和准确的描述。 在目前的资助周期中，我们发现自分泌TGF β在ER+乳腺癌和癌前乳腺上皮细胞中由维生素D3类似物诱导。 维生素D3类似物响应在不存在自分泌TGF β的情况下对抑制的抗性高100倍，并且初步证据表明由TGF β信号传导激活的Smad 3增强维生素D3受体VDR定向转录。 然而，虽然将II型TGF β受体（RII）转染到RII缺失的MCF-7细胞中完全恢复了维生素D类似物的敏感性，但Smad 3的转染不能。 这表明除了Smad 3之外，还需要TGF β信号传导的第二个方面。 因此，在特定目的I中，我们将检验以下假设：Smad 3与VDR的相互作用是TGF β/VDR串扰所必需的，但是除了该Smad 3途径之外，还需要TGF β介导的MAPK途径的激活。我们和其他人已经观察到维生素D类似物引起细胞凋亡。 诱导细胞凋亡的机制在很大程度上未被探索，特别是关于TGF β串扰的作用。 TGF β本身也诱导细胞凋亡。因此，我们将测试维生素D3类似物诱导的细胞凋亡是由药物治疗引起的自分泌TGF β活性增强介导的假设。在目前的资助期间，我们发现ER+乳腺癌细胞中TGF β受体的表达受到转录抑制。 用甲基化酶抑制剂5氮脱氧胞苷处理可阻断阻遏作用。令人惊讶的是，这种药物没有逆转RII启动子的甲基化，而是增加了在ER+乳腺癌细胞中低表达的Sp1的细胞水平。 Sp1是RII转录所必需的。 有趣的是，Sp1转录不被5氮脱氧胞苷靶向。 此外，研究表明，抑制Sp1介导的转录的Sp3转录因子在ER+乳腺癌细胞系中升高。 Sp3被证明是一个阻遏物的RII的西南，凝胶移位和封锁的RII启动子-报告活性后，Sp3转染。 此外，由于5氮杂胞苷处理，Sp3转录物减少。 因此，似乎Sp1/Sp3稳态负责ER+乳腺癌细胞中RII转录的抑制。 这一假设将在《具体目标III》中得到检验。 更新项目的具体目标是：1。确定ER+乳腺癌细胞、永生化乳腺上皮细胞和正常乳腺上皮细胞中维生素D3类似物和TGF β之间的串扰机制。 确定ER-细胞中维生素D3的反应是否也与TGF β的相互作用有关。二.确定维生素D3类似物在特定目标I中列出的细胞类型中诱导细胞凋亡的机制，并确定细胞凋亡抑制是否依赖于自分泌TGF β。三.确定如何在ER+乳腺癌中控制Sp1和Sp3稳态以调节RII转录。