The Dichotomous Role of TGF-beta/Smad3 Signaling in Breast Cancer

描述(申请人提供)：某些过度表达细胞周期蛋白D的侵袭性乳腺癌也可以表达过度活跃的RAS。患有相应肿瘤的患者预后较差，与早期发生转移性疾病有关。目前，这些患者的治疗方式与那些乳腺癌生物学更有利的患者相似，因此在预后方面没有显著改善。鉴于侵袭性乳腺癌亚型的异质性，开发有针对性的治疗策略对于提高患者存活率至关重要。转化生长因子β调控的Smad3是细胞周期蛋白D/CDK4和RAS激活的JNK的底物，在早期乳腺癌和晚期乳腺癌中分别显示出双重的肿瘤抑制和致癌作用。癌细胞利用这种致癌转移的机制尚不清楚。我们推测Smad3的抑瘤和促转移作用是通过其位点特异性的磷酸化来实现的，从而在转化生长因子β、细胞周期蛋白D/CDK4、RAS/JNK和乳腺癌细胞的转移潜能之间建立了联系。该方案的具体目的如下：目的1：确定CDK4和JNK介导的SMAD3的磷酸化是否调节侵袭性乳腺癌细胞中转化生长因子β诱导的抑癌和促转移信号之间的转换。我们将引入Smad3磷酸化位点突变或用CDK4或JNK抑制剂处理细胞，并通过mRNA和蛋白质分析评估Smad3依赖的细胞周期控制和EMT标记物。目的：探讨CDK4和JNK抑制在体外、3D培养和小鼠乳腺癌移植瘤模型中对肿瘤形成和转移的影响。我们将使用CDK4/JNK磷酸化位点突变的SMAD3转导的侵袭性乳腺癌细胞株，或者用CDK4或JNK抑制剂处理的侵袭性乳腺癌细胞株，进行迁移和侵袭实验，3D肿瘤和小鼠异种移植模型。目的：研究CDK4和JNK介导的Smad3磷酸化对EMT信号网络中Smad3调节转录因子活性的影响，并确定Smad3、细胞周期蛋白D和RAS相关蛋白在侵袭性乳腺癌亚型中的关键特征表达模式。我们将使用一种新的细胞阵列来确定CDK4和JNK抑制对SMAD3相关的EMT促进转录因子活性的影响，这些转录因子参与了几个与癌症相关的信号通路，并使用免疫组织化学来评估研究蛋白在人类乳腺癌组织中的模式表达。综上所述，这项工作将有助于疾病的分子分期，并有助于发现新的治疗策略，从而扩大侵袭性乳腺癌亚型患者的治疗选择。 公共卫生相关性：虽然转化生长因子-β/Smad3信号在早期癌症中具有肿瘤抑制作用，但其功能与晚期肿瘤的不良预后和转移有关。我们推测CDK4和Smad3的JNK磷酸化是侵袭性乳腺癌癌变的机制，药物靶向CDK4/JNK介导的Smad3磷酸化可能延缓疾病的进展。通过结合全球癌细胞信号网络研究SMAD3的作用和抑制，我们希望更有效地确定哪些患者将受益于CDK4和JNK抑制剂治疗。

DESCRIPTION (provided by applicant): Certain aggressive breast cancers that overexpress cyclin D can also express hyperactive Ras. Patients with corresponding tumors have poor outcomes associated with early onset of metastatic disease. Currently, these patients are treated in a similar manner to those with more favorable breast cancer biology and, hence, are not experiencing significant improvements in prognosis. Given the heterogeneity of aggressive breast cancer subtypes, the development of targeted treatment strategies is essential to improve patient survival rates. TGF- ¿-regulated Smad3, a substrate of both cyclin D/CDK4 and Ras-activated JNK, exhibits a dichotomous tumor suppressant/oncogenic role in early and late stage breast cancer, respectively. The mechanism by which this oncogenic shift is exploited by cancer cells remains unclear. We hypothesize that the tumor suppressant and pro-metastatic effects of Smad3 are mediated by its site-specific phosphorylation, providing a link between TGF-¿, cyclin D/CDK4, RAS/JNK and the metastatic potential of breast cancer cells. The specific aims of this proposal will directly test this hypothesis as follows: Aim 1: Determine if CDK4 and JNK mediated phosphorylation of Smad3 regulates the transformation between TGF-¿ induced tumor suppressive and pro-metastatic signaling in aggressive breast cancer cells. We will introduce Smad3 phosphorylation site mutants or treat cells with CDK4 or JNK inhibitors, and assess for Smad3-dependent markers of cell cycle control and EMT by mRNA and protein analysis. Aim 2: Elucidate the effect of CDK4 and JNK inhibition on tumor formation and metastasis in vitro, in 3D culture, and in a murine xenograft model of breast cancer. We will employ aggressive breast cancer cell lines transduced with CDK4/JNK phosphorylation site mutated Smad3 or treated with CDK4 or JNK inhibitor in migration and invasion assays, 3D tumor and murine xenograft models. Aim 3: Investigate the impact of CDK4- and JNK- mediated Smad3 phosphorylation on the activity of Smad3-regulated transcription factors in the context of an EMT focused signaling network, and determine key signature expression patterns of Smad3, cyclin D and Ras related proteins in aggressive breast cancer subtypes. We will employ a novel cell array to determine the effects of CDK4 and JNK inhibition on the activity of Smad3 associated EMT-promoting transcription factors that are implicated in several cancer-related signaling pathways, as well as use immunohistochemistry to assess the patterned expression of study proteins in human breast cancer tissues. Taken together, this work will contribute to the molecular staging of disease and facilitate discovery of novel therapeutic strategies that will expand treatment options for patients with aggressive breast cancer subtypes. PUBLIC HEALTH RELEVANCE: While TGF-¿/Smad3 signaling is tumor suppressive in early-stage cancer, its function correlates with poor prognosis and metastasis in later stage tumors. We hypothesize that CDK4 and JNK phosphorylation of Smad3 is the mechanism underlying this oncogenic transformation in aggressive breast cancer, and pharmacological targeting of CDK4/JNK mediated Smad3 phosphorylation may slow disease progression. By examining Smad3 action and inhibition in association with the global cancer cell signaling network, we hope to more effectively identify patients who will benefit from CDK4 and JNK inhibitor therapy.