Defining the Role of Pin1 and CDK-mediated Smad3 Phosphorylation in Triple Negative Breast Cancer Migration and Metastasis

定义 Pin1 和 CDK 介导的 Smad3 磷酸化在三阴性乳腺癌迁移和转移中的作用

• 批准号: 9258312
• 负责人: Alexandra L Thomas
• 金额: $ 2.18万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9258312
• 关键词: Affect; Area; Binding; Biological Assay; Breast; Breast Cancer Model; CCNE1 gene; CDK2 gene; CDK4 gene; Cancer Patient; Cancer cell line; Cell Cycle Regulation; Cell Differentiation process; Cell Proliferation; Cells; Cessation of life; Consequentialism; Cyclin D1; Cyclins; Disease; Disease Progression; Event; Fatty acid glycerol esters; Genetic Transcription; Harvest; Heterogeneity; Histologic; Hormonal; Hormone Receptor; In Vitro; Injectable; Knowledge; Label; Link; MDA MB 231; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Mediating; Monitor; Mus; Neoplasm Metastasis; Oncogenic; Operative Surgical Procedures; Organ; Patient-Focused Outcomes; Patients; Peptidylprolyl Isomerase; Pharmacologic Substance; Pharmacology; Phosphorylation; Phosphorylation Site; Primary Neoplasm; Process; Proline; Proteins; Radiation; Relapse; Reporter; Resistance; Role; Serine; Signal Pathway; Signal Transduction; Site; Testing; Therapeutic; Threonine; Transforming Growth Factor beta; Tumor Suppression; Ubiquitination; Work; Xenograft Model; cancer cell; cancer subtypes; cell motility; chemotherapy; cis-trans-Isomerases; fluorescence imaging; improved; in vivo; inhibitor/antagonist; insight; knock-down; malignant breast neoplasm; migration; mutant; new therapeutic target; novel; novel therapeutics; overexpression; public health relevance; receptor expression; restoration; success; targeted treatment; therapeutic target; transcription factor; triple-negative invasive breast carcinoma; tumor; tumor growth; tumor xenograft; tumorigenesis

 DESCRIPTION (provided by applicant): Triple negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes. Due to the heterogeneity of this subtype, there are currently no tailored therapies available for triple negative disease. Consequently, TNBC patients are treated with limited success and are prone to relapse and death because of the tendency of TNBCs to metastasize. There is an urgent need for improved therapeutic options for TNBC patients, and it is critical to investigate potential therapeutic targets to help improve patient outcomes. Cyclin D and E overexpression and the consequential impact on CDK4/2 activity correlate with aggressive breast cancer subtypes. Smad3, a key TGFβ signaling intermediate, is a substrate of CDK4/2. CDK4/2-mediated noncanonical Smad3 phosphorylation contributes to the oncogenic shift in TGFβ signaling in breast oncogenesis from promoting tumor suppression to supporting tumor growth. The impact of CDK-mediated Smad3 phosphorylation on cancer cell migration, invasion, and metastasis is an area of active study. CDK4/2 phosphorylation at the noncanonical T179 site (pT179) of Smad3 is associated with Pin1- Smad3 binding and promotion of migration and invasion. Pin1 is a peptydylprolyl cis/trans isomerase and is overexpressed in breast and other cancers. Pin1 expression levels correlate with increased breast tumor grade and poor patient outcomes. Our group has demonstrated an association with Pin1, Smad3 pT179, and CDK2 activity that impacts TNBC oncogenesis. We hypothesize that the interaction of Smad3 and Pin1, facilitated by CDK-mediated Smad3 phosphorylation, promotes cancer cell proliferation, migration and metastasis in cyclin- expressing TNBC. The specific aims of this proposal will directly test this hypothesis as follows: Aim 1: Examine the effect of Pin1 on Smad3-regulated cell cycle control in TNBC cell lines. We will knock-down (KD) Pin1 in TNBC cell lines and assay for changes in Smad3 stability and Smad3-dependent cell cycle control. We will employ a novel transcription factor reporter array to determine the impact of changes in Smad3 activity on cell proliferation and EMT-associated signaling pathways. Aim 2: Investigate the extent to which CDK2-mediated Smad3 phosphorylation impacts Pin1-Smad3 interaction and cell migration and invasion in TNBC cell lines. We will express Smad3 phosphorylation mutants or treat cells with a CDK2 inhibitor and assay for interaction with Pin1 and migration/invasion. Aim 3: Determine the extent to which inhibiting Pin1 and CDK-mediated Smad3 phosphorylation affects cancer metastasis in vivo. We will utilize mCherry-labeled MDA-MB-231 cells with Pin1 KD, transduced with Smad3 phosphorylation site mutants, or treated with CDK2 inhibitors as a xenograft model and monitor metastatic events with fluorescent imaging. Overall, this work will contribute to the growing body of evidence for the use of CDK2 inhibitors as a treatment option for TNBC patients and expand our knowledge of key signaling pathways for novel therapeutic discovery.

 描述(申请人提供)：三阴性乳腺癌(TNBC)是最具侵袭性的乳腺癌亚型之一。由于这一亚型的异质性，目前还没有针对三阴性疾病的量身定做的治疗方法。因此，TNBC患者的治疗效果有限，而且由于TNBCs有转移的趋势，容易复发和死亡。迫切需要改进TNBC患者的治疗方案，研究潜在的治疗靶点以帮助改善患者预后至关重要。细胞周期蛋白D和E的过度表达及其对CDK4/2活性的影响与侵袭性乳腺癌亚型有关。Smad3是一种关键的转化生长因子β信号中间体，是CDK4/2的底物。CDK4/2介导的非典型性Smad3磷酸化参与了转化生长因子β信号在乳腺癌发生中由促进肿瘤抑制向支持肿瘤生长的致癌转变。CDK介导的SMAD3磷酸化对癌细胞迁移、侵袭和转移的影响是一个活跃的研究领域。Smad3非典型T179位点(PT179)的CDK4/2磷酸化与Pin1-Smad3结合、促进迁移和侵袭有关。Pin1是一种酪氨酰基的顺式/反式异构酶，在乳腺癌和其他癌症中过表达。Pin1的表达水平与乳腺肿瘤分级增加和患者预后不良相关。我们的小组已经证明了Pin1、SMAD3、pT179和CDK2活性与影响TNBC肿瘤发生的关联。我们推测，在CDK介导的SMAD3磷酸化的促进下，Smad3和Pin1的相互作用促进了表达周期蛋白的TNBC中癌细胞的增殖、迁移和转移。这项建议的具体目的将直接检验这一假设如下：目的1：研究Pin1在SMAD3调控的TNBC细胞系细胞周期控制中的作用。我们将在TNBC细胞系中敲除(KD)Pin1，并检测SMAD3稳定性和SMAD3依赖的细胞周期控制的变化。我们将使用一个新的转录因子报告阵列来确定SMAD3活性的变化对细胞增殖和EMT相关信号通路的影响。目的：研究CDK2介导的Smad3磷酸化在多大程度上影响Pin1-Smad3相互作用和细胞在TNBC细胞中的迁移和侵袭。我们将表达Smad3磷酸化突变体或用CDK2抑制剂处理细胞，并检测与Pin1的相互作用和迁移/侵袭。目的：确定抑制Pin1和CDK介导的Smad3磷酸化对体内肿瘤转移的影响程度。我们将利用mCherry标记的带有Pin1 kD的MDA-MB-231细胞，用Smad3磷酸化位点突变体转导，或用CDK2抑制剂处理作为异种移植模型，并用荧光成像监测转移事件。总体而言，这项工作将有助于越来越多的证据表明使用CDK2抑制剂作为TNBC患者的治疗选择，并为新的治疗发现扩大我们对关键信号通路的知识。