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: 9121294
• 负责人: Alexandra L Thomas
• 金额: $ 3.73万
• 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 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9121294
• 关键词: Affect; Area; Binding; Biological Assay; Breast; Breast Cancer Cell; Breast Cancer Model; Breast Cancer Patient; Breast Cancer cell line; CCNE1 gene; CDK2 gene; CDK4 gene; Cell Cycle Regulation; Cell Proliferation; Cells; Cessation of life; Cyclin D1; Cyclins; Disease; Disease Progression; Event; Fatty acid glycerol esters; Harvest; Heterogeneity; Hormonal; Hormone Receptor; In Vitro; 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; 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（由申请人提供）：三阴性乳腺癌（TNBC）是最具侵袭性的乳腺癌亚型之一。由于这种亚型的异质性，目前还没有针对三阴性疾病的定制疗法。因此，TNBC患者的治疗成功率有限，并且由于TNBC的转移倾向而易于复发和死亡。迫切需要改进TNBC患者的治疗选择，并且研究潜在的治疗靶点以帮助改善患者结局至关重要。细胞周期蛋白D和E过表达及其对CDK 4/2活性的影响与侵袭性乳腺癌亚型相关。Smad 3是一种关键的TGFβ信号传导中间体，是CDK 4/2的底物。CDK 4/2介导的非经典Smad 3磷酸化有助于乳腺癌发生中TGFβ信号从促进肿瘤抑制到支持肿瘤生长的致癌转变。CDK介导的Smad 3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。CDK 4/2在Smad 3的非经典T179位点（pT 179）的磷酸化与Pin 1-Smad 3结合以及促进迁移和侵袭相关。Pin 1是一种肽基脯氨酰顺/反异构酶，在乳腺癌和其他癌症中过表达。Pin 1表达水平与乳腺肿瘤分级增加和患者预后不良相关。我们的研究小组已经证明了Pin 1，Smad 3 pT 179和CDK 2活性与影响TNBC肿瘤发生的相关性。我们假设，Smad 3和Pin 1的相互作用，促进CDK介导的Smad 3磷酸化，促进癌细胞增殖，迁移和转移的细胞周期蛋白表达TNBC。本提案的具体目的将直接测试该假设如下：目的1：检查Pin 1对TNBC细胞系中Smad 3调节的细胞周期控制的影响。我们将在TNBC细胞系中敲低（KD）Pin 1，并测定Smad 3稳定性和Smad 3依赖性细胞周期控制的变化。我们将采用一种新的转录因子报告阵列，以确定Smad 3活性的变化对细胞增殖和EMT相关信号通路的影响。目标二：研究CDK 2介导的Smad 3磷酸化影响TNBC细胞系中Pin 1-Smad 3相互作用以及细胞迁移和侵袭的程度。我们将表达Smad 3磷酸化突变体或用CDK 2抑制剂处理细胞，并测定与Pin 1的相互作用和迁移/侵袭。目的3：确定抑制Pin 1和CDK介导的Smad 3磷酸化在体内影响癌症转移的程度。我们将利用mCherry标记的具有Pin 1 KD的MDA-MB-231细胞、用Smad 3磷酸化位点突变体转导的细胞或用CDK 2抑制剂处理的细胞作为异种移植模型，并用荧光成像监测转移事件。总的来说，这项工作将有助于越来越多的证据表明CDK 2抑制剂作为TNBC患者的治疗选择，并扩大我们对新治疗发现的关键信号通路的了解。