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

描述（由申请人提供）：某些过度表达cyclin 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，与早期发生的转移性疾病相关。目前，这些患者的治疗方式与那些具有更有利的乳腺癌生物学的患者相似，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，开发靶向治疗策略对于提高患者生存率至关重要。TGF-¿调节的Smad3是细胞周期蛋白D/CDK4和ras激活的JNK的底物，在早期和晚期乳腺癌中分别表现出抑瘤/致癌的双重作用。癌细胞利用这种致癌转变的机制尚不清楚。我们假设Smad3的抑瘤和促转移作用是通过其位点特异性磷酸化介导的，在TGF-¿、cyclin D/CDK4、RAS/JNK与乳腺癌细胞的转移潜能之间提供了联系。本提案的具体目的将直接验证这一假设：目的1：确定CDK4和JNK介导的Smad3磷酸化是否调节TGF-¿诱导的侵袭性乳腺癌细胞中肿瘤抑制和促转移信号之间的转化。我们将引入Smad3磷酸化位点突变体或用CDK4或JNK抑制剂处理细胞，并通过mRNA和蛋白质分析评估Smad3依赖性细胞周期控制和EMT标记。目的2：阐明CDK4和JNK抑制在体外、3D培养和小鼠乳腺癌异种移植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转导或CDK4或JNK抑制剂处理的侵袭性乳腺癌细胞系进行迁移和侵袭试验，3D肿瘤和小鼠异种移植模型。目的3：研究CDK4和JNK介导的Smad3磷酸化对EMT聚焦信号网络中Smad3调控的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。我们将采用一种新的细胞阵列来确定CDK4和JNK抑制对Smad3相关emt促进转录因子活性的影响，这些转录因子与几种癌症相关的信号通路有关，并使用免疫组织化学来评估人类乳腺癌组织中研究蛋白的模式表达。总之，这项工作将有助于疾病的分子分期，并促进新的治疗策略的发现，这将扩大对侵袭性乳腺癌亚型患者的治疗选择。

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.