RNDI- Mediated Breast Cancer Suppression

RNDI-介导的乳腺癌抑制

• 批准号: 9521927
• 负责人: FILIPPO G GIANCOTTI
• 金额: $ 50.56万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9521927
• 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; Cell model; Cells; Clinical; Collaborations; Complement; 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; Injections; Laboratories; Lesion; 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; Pharmacology; Phenotype; Play; Point Mutation; Property; RAS inhibition; RNA Interference; Research; Role; Sampling; Signal Transduction; Signaling Protein; Structure; TP53 gene; Tail; Testing; Tetanus Helper Peptide; Treatment Efficacy; 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; lung colonization; 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; small hairpin RNA; stem; treatment strategy; triple-negative invasive breast carcinoma; tumor initiation; tumorigenesis; tumorigenic

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 负责人：M.D.，Ph.D。Filippo G. Giancotti 关键人员：Young-Mi Kim博士，博士后研究学者 Surajit Sinha博士，博士后研究学者 项目摘要： 我们已经确定了RND1，编码RHO家族GTPase，是侵略性抑制肿瘤 乳腺癌。在沉默的RND1沉默后，乳腺上皮细胞接受上皮到间质细胞 转化（EMT）并变得感觉，这意味着它们遭受了致癌损伤。表达 C-Myc的of sectument拯救了RND1耗尽的细胞，并使它们形成了侵入性的多ACinar 3D Matrigel中的结构。此外，RND1的沉默赋予了肿瘤和侵入性特性 茎状的逗号1D细胞。相反，RND1的表达抑制了基本样4T1细胞的能力 在注射尾静脉时将肺定居。有趣的是，RND1的耗竭可能激活了致癌性RA 信号传导和机械研究表明，RND1通过结合并激活非典型 plexin b1的Ras-间隙结构域。临床样本的分析表明遗传和表观遗传机制 有助于灭活RND1，从而激活大量基本样本中的RAS信号传导 三重阴性（TN）乳腺癌。根据这些新发现，我们建议进一步检查肿瘤 RND1在培养的细胞和小鼠模型中的抑制功能，并制定治疗的策略 RND1缺乏乳腺癌。在特定目标1中，我们将检查分子机制 RND1抑制RAS信号，EMT和肿瘤发生。我们的初步研究表明RND1 通过结合plexin b1的分裂Ras间隙结构段的插入段来抑制RAS信号传导和EMT 在Rap1上有选择地起作用。检查RND1抑制RAS信号传导的机制，EMT， 和肿瘤发生，我们将使用各种信号的野生型和突变形式的遗传重构 蛋白质随后进行生化，表型和功能分析。基于Rap1的已知功能， 我们将检查RAP1是否通过灭活P120 RAS间隙以及激活BRAF来促进RAS信号传导。 此外，我们将探讨为什么RND1的损失在乳腺上皮细胞中产生主要作用 表达其他RAS间隙蛋白。这些研究将与Neal Rosen合作进行 （项目3）。在特定目标2中，我们将检查RND1的遗传失活是否有助于启动和维持 乳腺肿瘤发生并促进小鼠的转移。我们的初步研究表明 RND1承认逗号1D细胞上的致瘤性和侵入性特性。检查RND1的失活是否是 足以启动，并且有必要维持体内乳腺肿瘤发生或与其他合作 致癌病变以介导这些作用，我们将利用Lowe开发的新技术 实验室（核心B：快速小鼠）。 TET诱导的shRNA将在Col1基因座的ES细胞中引入 使用盒式交换策略，然后在小鼠中使用四倍体完成。携带的小鼠 这些等位基因将越过MMTV-RTTTA小鼠，并将复合小鼠交叉至MMTV-MYC小鼠 作为TP53 +/-小鼠。将进行体内和体内研究，以研究RND1的消融的影响 结合MYC过度表达或p53在乳腺肿瘤开始，维持和 转移。在此目标中，我们还将研究ID1作为额外的合作致癌改变的作用。 这些研究将与罗伯特·贝尼兹拉（Robert Benezra）合作进行（项目2）。在特定的目标3中，我们将 制定药物治疗RND1缺陷乳腺癌的策略。我们的初步研究 表明对MEK的抑制会抑制与无关的增殖，侵袭和无关的生长 MCF-10A细胞通过RND1的损失而变化。检查抑制MEK是否有效 RND1缺乏乳腺癌的治疗策略，我们将对多个RND1-进行临床前研究 乳腺癌细胞不足。与尼尔·罗森（Neil Rosen）和乔斯·巴塞尔加（Jos Baselga）合作（项目3），我们将研究 两种新型变构MEK抑制剂的临床前有效性。由于药物抑制的影响 RAS-ERK途径通常受到负馈回循环的释放的限制，该循环可限制其 激活或其他电阻机制，我们将确定在 RND1缺陷癌细胞并开发旨在改善MEK效果的药物组合 抑制剂。潜在的有效药物组合将在色谱模型中进行测试。