Autocrine TGFBeta and Breast Cancer Cell Growth

说明应用程序的广泛长期目标和具体目标，并参考该项目与健康相关的内容。简明扼要地描述实现这些目标的研究设计和方法。避免总结过去的成就和使用第一人称。此描述的目的是在脱离应用程序时，作为对拟议工作的简洁而准确的描述。如果申请得到资助，在目前的资助周期中，我们发现在ER+乳腺癌和癌前乳腺上皮细胞中，维生素D3类似物诱导自分泌TGFbeta。在没有自分泌TGFbeta的情况下，维生素D3类似物的反应对抑制的抵抗力增加了100倍，初步证据表明，由TGFbeta信号激活的Smad3增强了维生素D3受体VDR的转录。然而，虽然将II型转化生长因子β受体(RII)导入RII缺失的MCF-7细胞可以完全恢复维生素D类似物的敏感性，但Smad3的转导并不能。这表明，除了SMAD3之外，还需要TGFbeta信号的第二个方面。因此，在特定的目标I中，我们将检验这样的假设，即Smad3与VDR的相互作用是TGFbeta/VDR串扰所必需的，但除了SMAD3途径外，TGFbeta介导的MAPK途径的激活也是必需的。我们和其他人已经观察到维生素D类似物会导致细胞凋亡。诱导细胞凋亡的机制在很大程度上还没有被探索，特别是关于转化生长因子β串扰的作用。转化生长因子β本身也可诱导细胞凋亡。因此，我们将验证这样的假设，即维生素D3类似物诱导的细胞凋亡是由药物治疗导致的自分泌转化生长因子β活性增强所介导的。在目前的资助期间，我们发现在ER+的乳腺癌细胞中，TGFbeta受体的表达受到转录抑制。用甲基酶抑制剂5-氮杂脱氧胞苷可以阻断这种抑制作用。令人惊讶的是，这种药物并没有逆转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的转录不是5氮杂脱氧胞苷的靶点。进一步的研究表明，抑制Sp1介导转录的SP3转录因子在ER+乳腺癌细胞系中升高。结果表明，SP3能抑制RII基因的表达，使其发生凝胶漂移，阻断RII启动子-报告基因活性。此外，5-氮杂胞苷处理后，SP3转录本减少。因此，似乎Sp1/SP3的动态平衡是抑制ER+乳腺癌细胞中RII转录的原因。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类似物与转化生长因子β的相互作用机制。确定内质网细胞中维生素D3的反应是否也与转化生长因子β串扰有关。2.确定维生素D3类似物在特定目的I中列出的细胞类型中诱导细胞凋亡的机制，并确定是否依赖自分泌转化生长因子β来抑制细胞凋亡。确定在ER+乳腺癌中Sp1和Sp3的动态平衡是如何调控RII转录的。

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.