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。具有相应肿瘤的患者具有与转移性疾病的早期发作相关的不良结局。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，预后没有显著改善。鉴于侵袭性乳腺癌亚型的异质性，制定有针对性的治疗策略对提高患者生存率至关重要。TGF-β- 调节Smad 3，细胞周期蛋白D/CDK 4和Ras激活的JNK的底物，分别在早期和晚期乳腺癌中表现出二分的肿瘤抑制/致癌作用。这种致癌转变被癌细胞利用的机制仍不清楚。我们推测Smad 3的肿瘤抑制和促转移作用是由其位点特异性磷酸化介导的，提供了TGF-β，细胞周期蛋白D/CDK 4，RAS/JNK和乳腺癌细胞转移潜力之间的联系。本提案的具体目的将直接测试该假设如下：目的1：确定CDK 4和JNK介导的Smad 3磷酸化是否调节侵袭性乳腺癌细胞中TGF-β诱导的肿瘤抑制和促转移信号传导之间的转化。我们将引入Smad 3磷酸化位点突变体或用CDK 4或JNK抑制剂处理细胞，并通过mRNA和蛋白质分析评估细胞周期控制和EMT的Smad 3依赖性标志物。目标二：阐明CDK 4和JNK抑制对体外、3D培养和小鼠乳腺癌异种移植模型中肿瘤形成和转移的影响。我们将采用CDK 4/JNK磷酸化位点突变的Smad 3转导或CDK 4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。目标3：研究CDK 4和JNK介导的Smad 3磷酸化对EMT聚焦信号网络背景下Smad 3调节的转录因子活性的影响，并确定侵袭性乳腺癌亚型中Smad 3、细胞周期蛋白D和Ras相关蛋白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CDK 4和JNK抑制对Smad 3相关EMT促进转录因子活性的影响，这些转录因子参与了几种癌症相关的信号传导途径，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。总之，这项工作将有助于疾病的分子分期，并促进发现新的治疗策略，这将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TGF-β/Smad 3信号在早期癌症中具有肿瘤抑制作用，其功能与晚期肿瘤的不良预后和转移相关。我们假设Smad 3的CDK 4和JNK磷酸化是侵袭性乳腺癌致癌转化的潜在机制，并且CDK 4/JNK介导的Smad 3磷酸化的药理学靶向作用可能会减缓疾病进展。通过研究Smad 3的作用和抑制与全球癌细胞信号网络的关系，我们希望更有效地识别将从CDK 4和JNK抑制剂治疗中受益的患者。

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.