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（由适用提供）：三重阴性乳腺癌（TNBC）是最具侵略性的乳腺癌亚型之一。由于该亚型的异质性，目前尚无针对三重阴性疾病的量身定制疗法。因此，由于TNBC倾向于转移，TNBC患者的成功率有限，并且很容易接力和死亡。迫切需要改善TNBC患者的治疗选择，至关重要的是研究潜在的治疗靶标以帮助改善患者的预后。细胞周期蛋白D和E的过表达以及对CDK4/2活性的结果影响与侵袭性的乳腺癌亚型相关。 SMAD3是一种键TGFβ信号中间体，是CDK4/2的底物。 CDK4/2介导的非规范SMAD3磷酸化有助于从促进肿瘤抑制到支持肿瘤生长的乳腺癌发生中TGFβ信号传导的致癌转移。 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积极研究的领域。 SMAD3的非规范T179位点（PT179）处的CDK4/2磷酸化与PIN1-SMAD3结合和迁移和入侵的促进有关。 PIN1是一种小蛋白酶/反式异构酶，在乳腺癌和其他癌症中过表达。 PIN1表达水平与乳腺肿瘤级别增加和患者结局差相关。我们的小组已证明与PIN1，SMAD3 PT179和CDK2活性有关联，从而影响TNBC肿瘤发生。我们假设Smad3和Pin1的相互作用在CDK介导的SMAD3磷酸化支持下促进了cyclin-表达TNBC的癌细胞增殖，迁移和转移。该提案的具体目的将直接检验该假设如下：目标1：检查PIN1对TNBC细胞系中SMAD3调节细胞周期控制的影响。我们将在TNBC细胞系中敲低（KD）PIN1，并测定SMAD3稳定性和SMAD3依赖性细胞周期控制的变化。我们将采用新型的转录因子报告基因阵列来确定SMAD3活性变化对细胞增殖和与EMT相关的信号通路的影响。目标2：研究CDK2介导的SMAD3磷酸化的程度对TNBC细胞系中的PIN1-SMAD3相互作用以及细胞迁移以及侵袭。我们将用CDK2抑制剂表达SMAD3磷酸化突变体或治疗细胞，并测定与PIN1和迁移/侵袭的相互作用。目标3：确定抑制PIN1和CDK介导的SMAD3磷酸化的程度会影响体内癌症转移。我们将使用PIN1 KD使用MCHERRY标记的MDA-MB-231细胞，用SMAD3磷酸化位点突变体翻译，或用CDK2抑制剂作为异种移植物模型进行处理，并用荧光成像监测转移性事件。总体而言，这项工作将有助于增加使用CDK2抑制剂作为TNBC患者的治疗选择的证据，并扩大我们对新型热发现的关键信号通路的了解。