Autocrine TGFBeta and Breast Cancer Cell Growth

自分泌 TGFBeta 与乳腺癌细胞生长

• 批准号: 6794648
• 负责人: MICHAEL G BRATTAIN
• 金额: $ 28.02万
• 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/6794648
• 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.

陈述该应用程序的广泛长期目标和具体目标，以参考项目的健康相关性。 简单地描述实现这些目标的研究设计和方法。 避免摘要过去的成就和使用第一人称。 此描述旨在作为与应用程序分离时对拟议工作的简洁而准确的描述。 如果使用该应用，则在目前的资金周期中，我们发现Autocrine TGFBETA是由ER+乳腺癌和乳腺癌前乳腺上皮细胞中的维生素D3类似物诱导的。 在没有自分泌TGFBETA的情况下，维生素D3模拟反应对抑制的耐药性更高，而初步证据表明，TGFBETA信号激活的SMAD3增强了维生素D3受体D3受体VDR的定向转录。 但是，尽管将II型TGFBETA受体（RII）转染到RII NULL MCF-7细胞中完全恢复了维生素D模拟敏感性，但SMAD3的转染却没有。 这表明除SMAD3外，还需要TGFBETA信号的第二个方面。 因此，在特定的目的中，我们将测试以下假设：TGFBETA/VDR Crosstalk需要SMAD3与VDR的相互作用，但是除了此SMAD3途径外，TGFBETA介导的MAPK途径的激活也需要。我们和其他人观察到维生素D类似物会引起凋亡。 诱导凋亡的机制在很大程度上没有探索，尤其是在TGFBETA串扰的作用方面。 TGFBETA本身也诱导凋亡。因此，我们将检验以下假设：维生素D3类似物诱导的凋亡是由使用药物治疗的自分泌TGFBETA活性增强所介导的。在目前的资金中，我们发现ER+乳腺癌细胞中的TGFBETA受体表达受到转录抑制。 可以通过用甲基酶抑制剂为5 AZA脱氧胞苷的治疗来阻止抑制作用。令人惊讶的是，该试剂没有逆转RII启动子的甲基化，而是增加了在ER+乳腺癌细胞中不受欢迎的SP1的细胞水平。 SP1是RII转录所必需的。 有趣的是，SP1转录不是5个AZA脱氧胞苷的靶向。 此外，研究表明，在ER+乳腺癌细胞系中，抑制SP1介导的转录的SP3转录因子升高。 SP3被西南部，SP3转染后的RII启动子重复蛋白活性的凝胶移位显示为RII的阻遏物。 另外，由于5个AZA胞苷处理，SP3转录本减少了。 因此，似乎SP1/SP3稳态似乎负责抑制ER+乳腺癌细胞中RII转录。 该假设将在特定的目标III中进行检验。 更新项目的具体目的是：1。确定ER+乳腺癌细胞中维生素D3类似物和TGFBETA之间串扰的机理，永生的乳腺上皮细胞和正常的乳腺上皮细胞。 确定ER细胞中的维生素D3反应是否也与TGFBETA串扰有关。 ii。确定在特定目标I中列出的细胞类型中维生素D3类似物诱导凋亡的机理，并确定凋亡抑制是否取决于自分泌TGFBETA。 iii。确定在ER+乳腺癌中如何控制SP1和SP3稳态以调节RII转录。