Intersection of autophagy and vesicle trafficking in Her2-positive breast cancer

Her2 阳性乳腺癌中自噬和囊泡运输的交叉点

基本信息

批准号：
10658423
负责人：
JUN-LIN GUAN
金额：
$ 40.76万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-13 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10658423
关键词：
Ablation Accounting Acetylation Autophagocytosis Bioinformatics Breast Cancer Cell Breast cancer metastasis CRISPR screen CRISPR/Cas technology Cell membrane Cell model Cell physiology Cells Complement Custom Cytoplasm Data Disease ERBB2 gene Endosomes Epidermal Growth Factor Receptor Event Family Functional disorder Funding Genes Goals Golgi Apparatus Health Hela Cells Human In Vitro Incidence Intervention Knock-in Knock-out Knowledge Libraries Lysosomes Malignant Neoplasms Mammary Neoplasms Mammary Tumorigenesis Maps Mediating Membrane Fusion Modeling Molecular Mouse Mammary Tumor Virus Mus Mutation Neoplasm Metastasis Oncogenic Patients Phosphorylation Proteins Receptor Protein-Tyrosine Kinases Recycling Regulation Regulator Genes Relapse Resistance Role Signal Transduction Therapeutic Trastuzumab Tyrosine Kinase Inhibitor Vesicle Woman combinatorial conditional knockout design driver mutation effective therapy efficacy evaluation extracellular vesicles genetic approach human disease improved outcome in vivo inhibition of autophagy insight knock-down loss of function malignant breast neoplasm member mouse model mutant neoplastic cell novel therapeutics patient responsibilities pharmacologic single-cell RNA sequencing small hairpin RNA small molecule inhibitor synergism targeted treatment therapy resistant trafficking virtual

Ablation Accounting Acetylation Autophagocytosis Bioinformatics Breast Cancer Cell Breast cancer metastasis CRISPR screen CRISPR/Cas technology Cell membrane Cell model Cell physiology Cells Complement Custom Cytoplasm Data Disease ERBB2 gene Endosomes Epidermal Growth Factor Receptor Event Family Functional disorder Funding Genes Goals Golgi Apparatus Health Hela Cells Human In Vitro Incidence Intervention Knock-in Knock-out Knowledge Libraries Lysosomes Malignant Neoplasms Mammary Neoplasms Mammary Tumorigenesis Maps Mediating Membrane Fusion Modeling Molecular Mouse Mammary Tumor Virus Mus Mutation Neoplasm Metastasis Oncogenic Patients Phosphorylation Proteins Receptor Protein-Tyrosine Kinases Recycling Regulation Regulator Genes Relapse Resistance Role Signal Transduction Therapeutic Trastuzumab Tyrosine Kinase Inhibitor Vesicle Woman combinatorial conditional knockout design driver mutation effective therapy efficacy evaluation extracellular vesicles genetic approach human disease improved outcome in vivo inhibition of autophagy insight knock-down loss of function malignant breast neoplasm member mouse model mutant neoplastic cell novel therapeutics patient responsibilities pharmacologic single-cell RNA sequencing small hairpin RNA small molecule inhibitor synergism targeted treatment therapy resistant trafficking virtual

展开

项目摘要

Breast cancer is the most common malignancy among US women and remains a major health threat with high incidence and lethality due to therapeutic resistance and metastasis. Breast cancer is also a heterogenous disease with different subtypes, including HER2+ subtype accounting for about 25% of patients. Despite the remarkable progress in recent years including anti-HER2 targeted therapy, our understanding of the mechanistic basis for breast cancer, particularly metastasis is still very limited. The long-term goal of the proposed studies is to understand the molecular and cellular mechanisms of metastasis and therapeutic resistance of HER2+ and other breast cancers that are ultimately responsible for patient lethality. In the prior funding period, we generated and analyzed mouse models for HER2+ breast cancer with deletion of an essential autophagy gene FIP200 or Fip200-4A mutation specifically blocking its autophagy activity and discovered that blocking FIP200-mediated autophagy abolished mammary tumorigenesis and metastasis through a new mechanism directly regulating the oncogenic driver HER2 itself. We showed that autophagy blockade abolishes mammary tumorigenesis and metastasis by decreasing levels of HER2 on the plasma membrane of mouse and human tumor cells due to aberrant HER2 trafficking from the Golgi to endosomes and multiple vesicular bodies (MVBs) and eventually released from tumor cells in small extracellular vesicles (sEVs). Additionally, employing single-cell RNA sequencing (scRNA-seq) and bioinformatics analysis, we developed a workflow to determine pharmacological interventions that would yield similar effects as FIP200 ablation. We also found FIP200 acetylation at K276 by CBP that regulates its stability. Lastly, we performed an in vivo CRISPR-Cas9 screen of a custom designed library of autophagy regulatory genes using our unique HER2+ mammary tumor cells and identified p47 as a putative suppressor for HER2+ breast cancer metastasis. Previous studies showed a role for p47 in regulating membrane fusion events, autophagy, and NFκB signaling, suggesting potential crosstalk between autophagy with vesicle trafficking in breast cancer metastasis. Based on these strong preliminary data and using our unique mouse and cell models, we propose to 1) determine the mechanism of autophagy regulation of HER2 trafficking in mouse and human breast cancer cells, 2) identify pharmacological agents that can disrupt the functions of FIP200 in mouse and human breast cancer cells as well as patient-derived models, and 3) explore the role and mechanisms of regulation of HER2+ breast cancer metastasis by p47. Together, these studies will provide significant insights into the molecular and cellular mechanisms of breast cancer metastasis that may contribute to novel therapies for this devastating disease.

乳腺癌是美国妇女中最常见的恶性肿瘤，仍然是主要的健康威胁由于热抗性和转移而引起的高入射和致死性。乳腺癌也是异质的具有不同亚型的疾病，包括HER2+亚型约25％的患者。尽管有近年来，包括抗HER2靶向疗法在内的显着进步，我们对机械的理解乳腺癌，特别是转移的基础仍然非常有限。拟议的研究的长期目标是了解转移的分子和细胞机制以及HER2+和最终导致患者致死性的其他乳腺癌。在以前的资金期间，我们产生了并分析了HER2+乳腺癌的小鼠模型，并删除了必需的自噬基因FIP200或 FIP200-4A突变专门阻止其自噬活动，并发现阻断FIP200介导的自噬通过一种直接调节的新机制消除了乳腺肿瘤发生和转移致癌驱动器Her2本身。我们表明自噬封锁废除了乳腺肿瘤的发生和通过降低小鼠和人肿瘤细胞质膜上HER2的水平的转移从高尔基人到内体和多个囊泡体（MVB）的异常贩运HER2贩运从小细胞外蔬菜（SEV）中从肿瘤细胞中释放出来。额外使用单细胞RNA 测序（SCRNA-SEQ）和生物信息学分析，我们开发了一个工作流程来确定药物干预措施将产生与FIP200消融相似的影响。我们还发现K276的FIP200乙酰化 CBP调节其稳定性。最后，我们执行了一个自定义设计的体内CRISPR-CAS9屏幕使用我们独特的HER2+乳腺肿瘤细胞的自噬调节基因库，并将p47鉴定为一个 HER2+乳腺癌转移的推定抑制剂。先前的研究表明P47在调节中的作用膜融合事件，自噬和NFκB信号传导，表明自噬之间潜在的串扰囊泡在乳腺癌转移中的运输。基于这些强大的初步数据，并使用我们的独特小鼠和细胞模型，我们建议1）确定HER2贩运自噬调节的机制在小鼠和人类乳腺癌细胞中，2）确定可以破坏功能的药物小鼠和人类乳腺癌细胞中的FIP200以及患者衍生的模型，3）探索角色和 P47调节HER2+乳腺癌转移的机制。这些研究将共同提供对乳腺癌转移的分子和细胞机制的重要见解，可能有助于为这种毁灭性疾病的新疗法。