RNDI- Mediated Breast Cancer Suppression

RNDI-介导的乳腺癌抑制

基本信息

批准号：
9099744
负责人：
FILIPPO G GIANCOTTI
金额：
$ 50.9万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9099744
关键词：
4T1 Ablation Adhesions Alleles Anchorage-Independent Growth BRAF gene Back Binding Biochemical Breast Breast Cancer Cell Breast Cancer Model Breast Carcinoma Breast Epithelial Cells CD100 antigen Cancer cell line Cell Aging Cell Cycle Cells Clinical Collaborations Coupling Cultured Cells DNA Microarray Chip Doctor of Medicine Doctor of Philosophy Drug Combinations ERBB2 gene Epigenetic Process Epithelial Family GTPase-Activating Proteins Gene Deletion Genetic Guanosine Triphosphate Phosphohydrolases Human Resources Injection of therapeutic agent Laboratories Lesion Lung MCF10A cells MEK inhibition MEKs Maintenance Malignant Epithelial Cell Malignant Neoplasms Mammary Neoplasms Mammary Tumorigenesis Mediating Mesenchymal Microarray Analysis Molecular Mouse Mammary Tumor Virus Mus Mutation Neoplasm Metastasis Neoplastic Cell Transformation Oncogenic Pathway interactions Play Point Mutation Property RNA Interference Research Role Sampling Signal Transduction Signaling Protein Structure TP53 gene Tail Testing Tetanus Helper Peptide Therapeutic Tumor Suppressor Proteins Veins Xenograft Model base c-myc Genes cancer cell cell motility cell transformation design embryonic stem cell feeding improved in vivo inhibitor/antagonist malignant breast neoplasm matrigel mouse model mutant new technology novel novel therapeutics overexpression plexin preclinical efficacy preclinical study programs ras GTPase-Activating Proteins receptor reconstitution resistance mechanism rho senescence stem treatment strategy triple-negative invasive breast carcinoma tumor initiation tumorigenesis tumorigenic

项目摘要

Project 4 Title : RND1-mediated Breast Cancer Suppression Leader: Filippo G. Giancotti, M.D., Ph.D. Key Personnel: Young-Mi Kim, Ph.D, Postdoctoral Research Scholar Surajit Sinha, Ph.D., Postdoctoral Research Scholar PROJECT SUMMARY: We have identified RND1, encoding a Rho family GTPase, as a tumor suppressor inactivated in aggressive breast cancers. Upon silencing of Rnd1, mammary epithelial cells underwent an Epithelial-to-Mesenchymal Transformation (EMT) and became senescent, implying that they had suffered an oncogenic insult. Expression of c-Myc rescued Rnd1-depleted cells from senescence and enabled them to form invasive, multi-acinar structures in 3D Matrigel. Moreover, silencing of Rnd1 conferred tumorigenic and invasive properties upon the stem-like Comma-1D cells. Conversely, expression of Rnd1 suppressed the ability of the basal-like 4T1 cells to colonize the lung upon tail-vein injection. Intriguingly, depletion of Rnd1 potently activated oncogenic Ras signaling and mechanistic studies indicated that Rnd1 suppresses Ras by binding to and activating the atypical Ras-GAP domain of Plexin B1. Analysis of clinical samples revealed that genetic and epigenetic mechanisms contribute to inactivate RND1 and thereby activate Ras signaling in a substantial fraction of basal-like and Triple Negative (TN) breast cancers. Based on these new findings, we propose to further examine the tumor suppressor function of Rnd1 in cultured cells and mouse models and to develop strategies for the treatment of Rnd1-deficient breast cancers. In Specific Aim 1, we will examine the molecular mechanisms through which Rnd1 suppresses Ras signaling, EMT, and oncogenesis. Our Preliminary Studies indicate that Rnd1 suppresses Ras signaling and EMT by binding to the insert segment of the split Ras GAP domain of Plexin B1, which acts selectively on Rap1. To examine the mechanism by which Rnd1 suppresses Ras signaling, EMT, and oncogenesis, we will use genetic reconstitution with wild type and mutant forms of various signaling proteins followed by biochemical, phenotypic and functional analysis. Based on the known functions of Rap1, we will examine if Rap1 promotes Ras signaling by inactivating p120 Ras-GAP as well as by activating BRAF. In addition, we will explore why loss of Rnd1 produces a dominant effect in mammary epithelial cells expressing additional Ras-GAP proteins. These studies will be conducted in collaboration with Neal Rosen (Project 3). In Specific Aim 2, we will examine if genetic inactivation of Rnd1 contributes to initiate and maintain mammary tumorigenesis and promotes metastasis in mice. Our Preliminary Studies indicate that inactivation of Rnd1 confers tumorigenic and invasive properties upon Comma-1D cells. To examine if inactivation of Rnd1 is sufficient to initiate and is necessary to maintain mammary tumorigenesis in vivo or it cooperates with other oncogenic lesions to mediate these effects, we will make use of a new technology developed by the Lowe laboratory (Core B: Speedy Mice). Tet-inducible shRNAs will be introduced in ES cells at the Col1 locus by using a cassette exchange strategy and in then in mice by using tetraploid complementation. Mice carrying these alleles will be crossed to MMTV-rtTTA mice and the compound mice crossed to MMTV-Myc mice as well as Tp53+/- mice. In vivo and ex vivo studies will be conducted to study the effect of ablation of Rnd1, alone or in combination with overexpression of Myc or inactivation of p53 on mammary tumor initiation, maintenance, and metastasis. In this Aim, we will also examine the role of Id1 as an additional cooperating oncogenic alteration. These studies will be performed in collaboration with Robert Benezra (Project 2). In Specific Aim 3, we will develop pharmacological strategies for the treatment of Rnd1-deficient breast cancers. Our Preliminary Studies indicate that inhibition of MEK suppresses the proliferation, invasion, and anchorage-independent growth of MCF-10A cells transformed by loss of Rnd1. To examine if inhibition of MEK is a potentially effective therapeutic strategy for Rnd1-deficient breast cancers, we will conduct preclinical studies on multiple Rnd1- deficient breast cancer cells. In collaboration with Neil Rosen and Jos� Baselga (Project 3), we will examine the preclinical efficacy of two novel allosteric MEK inhibitors. Since the effect of pharmacological inhibition of the Ras-ERK pathway is often limited by release of negative feed back loops, which operate to restrain its activation, or other resistance mechanisms, we will identify the specific mechanism of resistance operating in Rnd1-deficient cancer cells and develop pharmacological combinations designed to improve the effect of MEK inhibitors. Potentially effective drug combinations will undergo testing in xenograft models.

项目4 标题：RND1 介导的乳腺癌抑制领导者：Filippo G. Giancotti，医学博士、哲学博士主要人员：Young-Mi Kim，博士，博士后研究学者 Surajit Sinha，博士，博士后研究学者项目概要：我们已经鉴定出编码 Rho 家族 GTPase 的 RND1 作为一种在侵袭性肿瘤中失活的肿瘤抑制因子。 Rnd1 沉默后，乳腺上皮细胞经历了上皮间质转化。转化（EMT）并变得衰老，这意味着它们遭受了致癌性的侮辱。 c-Myc 将 Rnd1 耗尽的细胞从衰老中拯救出来，并使它们能够形成侵入性的多腺泡此外，Rnd1 的沉默赋予了肿瘤发生和侵袭的特性。干细胞样 Comma-1D 细胞离线时，Rnd1 的表达抑制了基底样 4T1 细胞的能力。尾静脉注射后在肺部定植，有趣的是，Rnd1 的消耗有效激活了致癌 Ras。信号传导和机制研究表明，Rnd1 通过结合并激活非典型蛋白来抑制 Ras Plexin B1 的 Ras-GAP 结构域分析揭示了遗传和表观遗传机制。有助于使 RND1 失活，从而激活大部分 basal-like 和 Ras 信号传导基于这些新发现，我们建议进一步检查肿瘤。 Rnd1 在培养细胞和小鼠模型中的抑制功能，并制定治疗策略 Rnd1 缺陷型乳腺癌在具体目标 1 中，我们将研究其分子机制。 Rnd1 抑制 Ras 信号传导、EMT 和肿瘤发生。通过与 Plexin B1 的分裂 Ras GAP 结构域的插入片段结合来抑制 Ras 信号传导和 EMT，选择性作用于 Rap1 来检查 Rnd1 抑制 Ras 信号传导、EMT 的机制。和肿瘤发生，我们将利用各种信号传导的野生型和突变型进行基因重组基于 Rap1 的已知功能，对蛋白质进行生化、表型和功能分析。我们将检查 Rap1 是否通过灭活 p120 Ras-GAP 以及激活 BRAF 来促进 Ras 信号传导。此外，我们将探讨为什么 Rnd1 的缺失会对乳腺上皮细胞产生显着影响这些研究将与 Neal Rosen 合作进行。（项目 3）在具体目标 2 中，我们将检查 Rnd1 的基因失活是否有助于启动和维持。我们的初步研究表明，其失活可促进小鼠乳腺肿瘤的发生并促进转移。 Rnd1 赋予 Comma-1D 细胞致瘤性和侵袭性特性检测 Rnd1 失活是否会产生影响。足以启动并且对于维持体内乳腺肿瘤发生是必要的，或者它与其他为了介导这些影响，我们将利用 Lowe 开发的新技术实验室（核心 B：Speedy 小鼠）将通过 Col1 位点将 Tet 诱导的 shRNA 引入 ES 细胞中。使用盒交换策略，然后在小鼠中使用四倍体互补。这些等位基因将与 MMTV-rtTTA 小鼠杂交，复合小鼠也会与 MMTV-Myc 小鼠杂交作为Tp53+/-小鼠，将进行体内和离体研究以研究单独或单独消融Rnd1的效果。与 Myc 过表达或 p53 失活相结合对乳腺肿瘤的发生、维持和在这个目标中，我们还将研究 Id1 作为额外的协同致癌改变的作用。这些研究将与 Robert Benezra 合作进行（项目 2），我们将在具体目标 3 中进行。制定治疗 Rnd1 缺陷型乳腺癌的药理学策略。表明 MEK 的抑制可抑制细胞的增殖、侵袭和不依赖贴壁的生长通过 Rnd1 缺失转化 MCF-10A 细胞以检查 MEK 的抑制是否具有潜在的效果。 Rnd1缺陷型乳腺癌的治疗策略，我们将对多种Rnd1-进行临床前研究我们将与 Neil Rosen 和 José Baselga（项目 3）合作检查缺陷的乳腺癌细胞。两种新型变构MEK抑制剂的临床前疗效自药理抑制作用。 Ras-ERK 通路通常受到负反馈环路释放的限制，负反馈环路的作用是抑制其激活，或其他抵抗机制，我们将确定抵抗作用的具体机制 Rnd1 缺陷的癌细胞并开发旨在提高 MEK 效果的药物组合潜在有效的药物组合将在异种移植模型中进行测试。