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

TGF-β/Smad3 信号转导在乳腺癌中的二分作用

• 批准号: 8315358
• 负责人: Elizabeth Tarasewicz
• 金额: $ 4.22万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8315358
• 关键词: Binding; Biological; Biological Assay; Breast Cancer Cell; C-terminal; CDK2 gene; CDK4 gene; Cancer Biology; Cancer cell line; Cell Cycle Regulation; Cell Line; Cells; Cyclin D1; Development; Diagnostic Neoplasm Staging; Disease; Disease Progression; E-Cadherin; ERBB2 gene; Exhibits; Heterogeneity; Human; Image; Immigration; Immunohistochemistry; In Vitro; Link; MAPK14 gene; MAPK8 gene; MMP2 gene; Malignant Neoplasms; Mediating; Messenger RNA; Modeling; Molecular; Molecular Profiling; Mus; Mutate; Neoplasm Metastasis; Nuclear; Oncogenic; Outcome; Patients; Pattern; Phenotype; Phosphorylation; Phosphorylation Site; Plasminogen Activator Inhibitor 1; Play; Protein Analysis; Proteins; RAS Superfamily Proteins; Role; Signal Pathway; Signal Transduction; Signaling Protein; Site; Staging; Subgroup; Survival Rate; Testing; Tissues; Transfection; Transforming Growth Factor beta; Tumor stage; Variant; Vimentin; Work; Xenograft Model; Xenograft procedure; c-myc Genes; cancer cell; cancer therapy; chemotherapy; early onset; effective therapy; experience; improved; inhibitor/antagonist; malignant breast neoplasm; mutant; novel; novel therapeutics; occludin; outcome forecast; overexpression; patient population; protein expression; receptor; transcription factor; translational approach; treatment strategy; tumor; tumor progression

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.

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。患有相应肿瘤的患者预后较差，且早期发生转移性疾病。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特征的患者相似，因此，预后并没有显着改善。鉴于侵袭性乳腺癌亚型的异质性，制定靶向治疗策略对于提高患者生存率至关重要。 TGF-β调节的Smad3是细胞周期蛋白D/CDK4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。癌细胞利用这种致癌转变的机制仍不清楚。我们假设 Smad3 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，从而在 TGF-¿、细胞周期蛋白 D/CDK4、RAS/JNK 和乳腺癌细胞的转移潜力之间提供了联系。该提案的具体目标将直接检验该假设如下： 目标 1：确定 CDK4 和 JNK 介导的 Smad3 磷酸化是否调节侵袭性乳腺癌细胞中 TGF-¿ 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 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 和蛋白质分析评估细胞周期控制和 EMT 的 Smad3 依赖性标记。目标 2：阐明 CDK4 和 JNK 抑制对体外、3D 培养以及乳腺癌小鼠异种移植模型中肿瘤形成和转移的影响。我们将在迁移和侵袭测定、3D 肿瘤和小鼠异种移植模型中使用用 CDK4/JNK 磷酸化位点突变的 Smad3 转导或用 CDK4 或 JNK 抑制剂处理的侵袭性乳腺癌细胞系。目标 3：在 EMT 信号网络背景下研究 CDK4 和 JNK 介导的 Smad3 磷酸化对 Smad3 调节的转录因子活性的影响，并确定侵袭性乳腺癌亚型中 Smad3、细胞周期蛋白 D 和 Ras 相关蛋白的关键特征表达模式。我们将采用一种新型细胞阵列来确定 CDK4 和 JNK 抑制对 Smad3 相关 EMT 促进转录因子（这些转录因子参与多种癌症相关信号通路）活性的影响，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。总而言之，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，从而扩大侵袭性乳腺癌亚型患者的治疗选择。 公共健康相关性：虽然 TGF-¿/Smad3 信号传导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相关。我们假设 Smad3 的 CDK4 和 JNK 磷酸化是侵袭性乳腺癌中这种致癌转化的机制，并且 CDK4/JNK 介导的 Smad3 磷酸化的药理学靶向可能会减缓疾病进展。通过检查 Smad3 的作用和抑制与全球癌细胞信号网络的关联，我们希望更有效地识别将从 CDK4 和 JNK 抑制剂治疗中受益的患者。