Characterization and Regulation of High Risk and Maligna

高风险和恶性疾病的特征和监管

• 批准号: 6947445
• 负责人: DAVID DANFORTH
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6947445
• 关键词: BCL2 gene /protein; CD95 molecule; MCF7 cell; all trans retinol; apoptosis; breast neoplasms; cancer risk; cell growth regulation; cell line; drug interactions; female; human genetic material tag; human subject; mammary epithelium; microarray technology; neoplasm /cancer genetics; neoplastic cell; neoplastic growth; patient oriented research; preneoplastic state; tamoxifen; transforming growth factors; women's health

The antiestrogen tamoxifen and vitamin A-related compounds, the retinoids, each have strong antiproliferative effects on breast cancer cells. In combination these agents act synergistically to further inhibit growth, representing an important means for enhancing their antiproliferative actions. This has resulted in increased efficacy from both a therapeutic and chemopreventive standpoint. The present study was undertaken to define this synergism and determine the mechanism of action of this enhanced activity in combination. The effect of 4-hydroxytamoxifen (TAM) and all-trans retinoic acid (AT) in combination on proliferation of MCF-7 breast cancer cells was studied in vitro. It was found that TAM and AT, in combination, acted synergistically to cause a time-dependent and dose-dependent inhibition of MCF-7 cell growth. Both TAM and AT each blocked cell cycle progression throughout 7 days of treatment, but without any synergistic or additive effect on this process. TAM and AT acted synergistically, however, to stimulate apoptosis, with increased DNA fragmentation and downregulation of bcl-2 protein expression in a manner temporally equivalent to the synergistic inhibition of growth. The negative growth factor transforming growth factor beta (TGF) is secreted by these cells and was studied as a potential mediator of the synergistic effects of TAM + AT on apoptosis. While TAM but not AT stimulated TGF1 secretion, in combination TAM and AT acted synergistically to induce a 5 fold increase in TGF1 secretion over 72 hours. TGF1 alone had no apoptotic effects on these cells, but TGF1 in combination with AT acted synergistically to inhibit growth, to downregulate bcl-2 protein expression, and to stimulate apoptosis of these cells in a manner analogous to that noted for TAM + AT. Coincubation of TAM + AT with anti-TGF-beta antibody, however, did not block modulation of Bcl-2 protein expression or apoptosis. Together these findings indicate that the synergism of AT + TAM occurs independently of TGF-beta, and that TGF-beta may interact with AT in a novel manner to provide another important antiproliferative mechanism for breast cancer cells. Because AT is also a naturally occurring hormone, these findings may have broad implications for regulation of breast cancer cell growth. AT regulation of another important mediator of apoptosis, Fas antigen, is being studied. Fas antigen, a cell surface protein and member of the TNF receptor family, is a mediator of apoptosis in multiple cell types, but is not expressed in MCF-7 breast cancer cells. AT and interferon-gamma (IFN), in combination, act synergistically to inhibit growth of these cells, which encouraged us to examine their potential regulation of Fas-antigen expression. We found that AT and IFN in combination act synergistically to induce Fas protein expression in MCF-7 cells in a time-dependent and dose-dependent manner over 48 hours. Induction of Fas mRNA was assessed by quantitative RT-PCR. AT + IFN synergistically induced Fas mRNA within 6 hours, indicating transcriptional regulation of Fas antigen expression. Following induction of Fas antigen, treatment with Fas antibody alone resulted in minimal cellular growth inhibition. Coincubation of Fas antibody with cycloheximide resulted in a significant decrease in cell viability and stimulation of apoptosis between 6 and 12 hours of treatment. This was associated with characteristic morphologic changes of apoptosis and DNA fragmentation, and indicated the presence of endogenous inhibitors to Fas signaling in these cells. Inhibition to FAS signaling was not associated with the expression of FLIPS or FLIPL. The lack of expression of caspase 3 protein and the strong expression of bcl-2 protein confirmed the presence of a type II FAS signaling pathway, and strongly suggested that bcl-2 expression was responsible for inhibition of FAS mediated apoptosis in these cells. Confirmatory studies to test the ability of Bcl-2 antisense oligonucleotides to enhance Fas-mediated apoptosis are in progress. The induction of Fas-mediated apoptosis by AT + IFN suggests important new treatment strategies for breast cancer, and further clarifies the anntiproliferative actions of these two naturally occurring agents. The cellular, metabolic and molecular changes which occur in a normal breast epithelial cell in the development of a breast cancer are poorly understood and need to be defined. An understanding of these changes are important for defining the carcinogenic pathway of breast cancer, for identification of prognostic biomarkers for risk assessment, identification of biomarkers for selection and monitoring of chemoprevention agents, and for the development and evaluation of new chemoprevention drugs and chemoprevention strategies. To further define these characteristics we have developed high risk breast epithelial cell lines from normal high risk tissue from women with invasive breast cancer. These cell lines have normal breast epithelial cell morphology and express cytokeratins 14 and 18, and thus contain both basal and luminal cell types. We examined growth and metabolic regulation by retinoids, vitamin-A related compounds which are naturally occuring and which have chemopreventive and therapeutic effects for breast cancer. Studies to date have shown that these cells secrete the negative growth factor transforming growth factor beta (TGF), predominantly in the isomeric form TGF2. All-trans retinoic acid (AT) stimulates secretion of TGF2, primarily in the latent form, and inhibits growth through blockade of cell cycle progression without stimulating apoptosis. Active TGF2 has strong antiproliferative effects on these breast epithelial cells, indicating secreted TGF may have an important paracrine or endocrine regulatory role in women at risk for breast cancer. Gene expression profiles and their regulation by AT and TGF are being studied by cDNA microarray. This will allow identification of distinctive patterns of global gene expression between these two prominent naturally occurring antiproliferative agents. Confirmation of expression differences will be confirmed with duplicate array and quantitative RT-PCR. To identify potential changes in the carcinogenic pathway of the high risk cells, gene expression profiles of high risk cells will also be compared with those of normal nonrisk breast epithelial cells using the reference cell line MCF10A as an internal standard. In this manner important differences in the progression to the high risk state can be identified, as well as characterization of their regulation by important chemopreventive modulators. Two clinical trials which compliment these studies have been initiated. The first trial is Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and cDNA Gene Expression Profile. This is a collaborative study in which we will characterize the molecular, cytologic, and architectural changes in breast ductal epithelium associated with high risk for sporadic breast cancer. Breast ductal epithelial cells will be collected by breast duct lavage from the contralateral breast in postmenopausal women with ipsilateral breast cancer. These cells will be compared to ductal epithelial cells from the breast of female postmenopausal normal volunteers who are not at increased risk for breast cancer. Ductal epithelial cell specimens will be analyzed cytologically for the presence of hyperplasia, atypia, or in situ changes, and correlated with ductal architecture determined by duct endoscopy.

抗雌激素他莫昔芬和维生素 A 相关化合物（类维生素A）均对乳腺癌细胞具有很强的抗增殖作用。这些药物联合起来协同作用，进一步抑制生长，是增强其抗增殖作用的重要手段。从治疗和化学预防的角度来看，这都提高了疗效。本研究旨在定义这种协同作用并确定这种增强活性的联合作用机制。体外研究了4-羟基他莫昔芬（TAM）和全反式维甲酸（AT）联合对MCF-7乳腺癌细胞增殖的影响。结果发现，TAM 和 AT 联合作用，可协同作用，对 MCF-7 细胞生长产生时间依赖性和剂量依赖性抑制。 TAM 和 AT 均在 7 天的治疗过程中阻断细胞周期进程，但对此过程没有任何协同或附加作用。然而，TAM 和 AT 协同作用刺激细胞凋亡，增加 DNA 碎片并下调 bcl-2 蛋白表达，其方式在时间上相当于协同抑制生长。负生长因子转化生长因子β (TGF) 由这些细胞分泌，并被研究作为 TAM + AT 对细胞凋亡的协同作用的潜在介质。虽然 TAM 而不是 AT 刺激 TGF1 分泌，但 TAM 和 AT 组合可协同作用，在 72 小时内诱导 TGF1 分泌增加 5 倍。单独的TGF1对这些细胞没有凋亡作用，但TGF1与AT组合可协同抑制生长，下调bcl-2蛋白表达，并以类似于TAM + AT的方式刺激这些细胞的凋亡。然而，TAM + AT 与抗 TGF-β 抗体共孵育不会阻断 Bcl-2 蛋白表达或细胞凋亡的调节。这些发现共同表明，AT + TAM 的协同作用独立于 TGF-β 发生，并且 TGF-β 可能以一种新的方式与 AT 相互作用，为乳腺癌细胞提供另一种重要的抗增殖机制。由于 AT 也是一种天然存在的激素，因此这些发现可能对乳腺癌细胞生长的调节具有广泛的影响。 另一种重要的细胞凋亡介质 Fas 抗原的 AT 调节正在进行研究。 Fas 抗原是一种细胞表面蛋白，属于 TNF 受体家族成员，是多种细胞类型中细胞凋亡的介质，但在 MCF-7 乳腺癌细胞中不表达。 AT 和干扰素-γ (IFN) 联合使用可协同抑制这些细胞的生长，这促使我们研究它们对 Fas 抗原表达的潜在调节作用。我们发现 AT 和 IFN 组合可协同作用，在 48 小时内以时间依赖性和剂量依赖性方式诱导 MCF-7 细胞中 Fas 蛋白的表达。通过定量 RT-PCR 评估 Fas mRNA 的诱导。 AT + IFN 在 6 小时内协同诱导 Fas mRNA，表明 Fas 抗原表达的转录调节。 Fas 抗原诱导后，单独用 Fas 抗体处理导致最小的细胞生长抑制。 Fas 抗体与放线菌酮共孵育导致细胞活力显着降低，并在治疗 6 至 12 小时期间刺激细胞凋亡。这与细胞凋亡和 DNA 片段化的特征性形态变化有关，并表明这些细胞中存在 Fas 信号传导的内源性抑制剂。 FAS 信号传导的抑制与 FLIPS 或 FLIPL 的表达无关。 Caspase 3 蛋白表达的缺乏和 bcl-2 蛋白的强表达证实了 II 型 FAS 信号通路的存在，并强烈表明 bcl-2 表达负责抑制这些细胞中 FAS 介导的凋亡。测试 Bcl-2 反义寡核苷酸增强 Fas 介导的细胞凋亡能力的验证性研究正在进行中。 AT + IFN 诱导 Fas 介导的细胞凋亡为乳腺癌提供了重要的新治疗策略，并进一步阐明了这两种天然药物的抗增殖作用。 人们对乳腺癌发展过程中正常乳腺上皮细胞中发生的细胞、代谢和分子变化知之甚少，需要对其进行定义。了解这些变化对于确定乳腺癌的致癌途径、识别用于风险评估的预后生物标志物、识别用于选择和监测化学预防药物的生物标志物以及开发和评估新的化学预防药物和化学预防策略非常重要。为了进一步定义这些特征，我们从患有浸润性乳腺癌的女性的正常高风险组织中开发了高风险乳腺上皮细胞系。这些细胞系具有正常的乳腺上皮细胞形态并表达细胞角蛋白 14 和 18，因此包含基底细胞和腔细胞类型。我们研究了类维生素A、维生素A相关化合物对生长和代谢的调节，这些化合物是天然存在的，对乳腺癌具有化学预防和治疗作用。迄今为止的研究表明，这些细胞分泌负生长因子转化生长因子β（TGF），主要是TGF2的异构形式。全反式视黄酸 (AT) 主要以潜在形式刺激 TGF2 的分泌，并通过阻断细胞周期进程来抑制生长，而不刺激细胞凋亡。活性 TGF2 对这些乳腺上皮细胞具有很强的抗增殖作用，表明分泌的 TGF 可能对有乳腺癌风险的女性具有重要的旁分泌或内分泌调节作用。基因表达谱及其 AT 和 TGF 的调节正在通过 cDNA 微阵列进行研究。这将允许鉴定这两种重要的天然存在的抗增殖剂之间的全局基因表达的独特模式。表达差异的确认将通过重复阵列和定量 RT-PCR 来确认。为了确定高风险细胞致癌途径的潜在变化，还将使用参考细胞系 MCF10A 作为内标，将高风险细胞的基因表达谱与正常非风险乳腺上皮细胞的基因表达谱进行比较。通过这种方式，可以识别向高风险状态进展的重要差异，以及重要化学预防调节剂对其调节的特征。 两项补充这些研究的临床试验已经启动。第一项试验是方案 02-C-0077，通过细胞学、乳腺导管内窥镜检查和 cDNA 基因表达谱表征高风险乳腺导管上皮。这是一项合作研究，我们将描述与散发性乳腺癌高风险相关的乳腺导管上皮的分子、细胞学和结构变化。乳腺导管上皮细胞将通过乳腺导管灌洗从患有同侧乳腺癌的绝经后妇女的对侧乳房中收集。这些细胞将与来自绝经后正常女性志愿者乳房的导管上皮细胞进行比较，这些志愿者患乳腺癌的风险并不增加。对导管上皮细胞样本进行细胞学分析，以确定是否存在增生、异型性或原位变化，并与导管内窥镜检查确定的导管结构相关联。