Activation of Inflammatory Responses Upon Replication Stress in Basal-Like Breast Cancer

基底样乳腺癌复制应激时炎症反应的激活

基本信息

批准号：
10619444
负责人：
Elizabeth Frances Gorodetsky
金额：
$ 5.27万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-04 至 2026-03-03
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10619444
关键词：
Affect Animals Basal Cell Cancer Biological Models Breast Cancer Cell Breast Cancer Model Breast Cancer Patient Breast Cancer cell line CRISPR/Cas technology CSF3 gene Cancer Etiology Cell Death Cell Line Cells Cessation of life Clinical DNA Damage DNA Polymerase II DNA replication fork Data Development Doxycycline Environment Exhibits Future Gene Expression Genotoxic Stress Goals Immune Immune checkpoint inhibitor Immune signaling Immune system Immunofluorescence Immunologic Immunological Models Immunooncology Immunotherapeutic agent Immunotherapy Infiltration Inflammatory Inflammatory Response Innate Immune System Intercalating Agents Interleukin-6 Intervention Ionizing radiation Knock-out Luciferases Macrophage Malignant Neoplasms Measures Modality Molecular Mus Mutagens NF-kappa B Neoplasm Metastasis Nude Mice Pathway interactions Patient-Focused Outcomes Pattern recognition receptor Phenotype Polymerase Post-Translational Protein Processing Production Prognosis Proteomics RELA gene Regimen Relapse Reporter Resistance development Signal Transduction Stimulator of Interferon Genes Therapeutic Titrations Topoisomerase Inhibitors Transcript Treatment Protocols Tumor-infiltrating immune cells Western Blotting Woman Work Xenograft procedure cancer cell cancer subtypes cancer therapy cell type clinically relevant cytokine experience immune cell infiltrate improved malignant breast neoplasm mammary molecular subtypes mortality neoplastic cell novel p65 programs replication stress response screening senescence small hairpin RNA stem targeted treatment therapeutically effective transcription factor transcriptomics treatment response tumor tumor microenvironment tumor-immune system interactions

项目摘要

Project Summary Breast cancer accounts for the second most cancer-related deaths in U.S. women despite the availability of improved treatment options and increased screening. A particularly aggressive subtype that represents a disproportionate number of these mortality cases is basal-like breast cancer (BLBC). The high mortality of BLBC stems from a lack of effective, sustainable chemotherapeutic treatment options with tumors often developing resistance to treatment despite encouraging treatment responses. This higher relapse rate among BLBC patients calls for the development of more effective therapeutic regimens targeting distinct molecular alterations of basal-like tumors. Strikingly, we find that BLBC, but not luminal breast cancer cell lines, exhibit a severe sensitivity to partial suppression of leading strand replicative polymerase, DNA polymerase epsilon (POLE). POLE suppression in BLBC cells leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. Our preliminary data, which form the premise of my application, reveals that suppression of POLE in a BLBC cell line, but not other breast cancer cell lines, results in an increase in inflammatory cytokine transcripts known to be targeted by the NF-𝜅B transcription factor, a phenotype that is dependent upon RELA expression. This cytokine production is sufficient to impact the tumor immune microenvironment, as I observed significant infiltration of F4/80 positive macrophages in BLBC xenografts upon POLE suppression. Therefore, my central hypothesis is that NF-𝜅B activation upon POLE inhibition activates a cytokine secretion program selectively in BLBC cells. In aim 1, I will analyze activation of pattern recognition receptors (PRRs) and NF-𝜅B via immunoblot, immunofluorescence for NF- 𝜅B subcellular localization, and an NF-𝜅B luciferase reporter. I will delineate which PRRs (cGAS- STING, MAVS-RIG-I) and NF-𝜅B components (IKK-g, p65, RELB) are triggered by replication stress, and use CRISPR/Cas9 to generate cell lines lacking key signaling components to uncover those responsible for NF-𝜅B activation. In aim 2, I will determine the impact of POLE suppression on intra-tumoral cytokine expression and tumor cell composition. If immune infiltration is altered, I will determine the degree of cooperation with immune checkpoint inhibitors. This work is impactful because it will discriminate whether replication stress induced by inhibiting leading strand replication triggers an immunostimulatory or immunosuppressive environment that could be targeted to enhance future anti-cancer therapies.

项目摘要乳腺癌占美国妇女第二大与癌症有关的死亡，要求改善治疗选择和筛查的可用性。一个特别激进的代表这些死亡率案例不成比例的亚型是基本的乳房癌症（BLBC）。 BLBC植物的高死亡率由于缺乏有效，可持续性肿瘤的化学治疗方法通常会产生对治疗目的地的抗性鼓励治疗反应。 BLBC患者中这种较高的继电器率要求开发针对明显分子改变的更有效的治疗方案基底样肿瘤。令人惊讶的是，我们发现BLBC，而不是腔内乳腺癌细胞系，暴露了对部分抑制铅链复制聚合酶DNA的严重敏感性聚合酶Epsilon（POL）。 BLBC细胞中的极极导致复制叉的失速， DNA损伤以及感应状态或细胞死亡。我们的初步数据，构成我的应用程序的前提表明，在BLBC细胞系中抑制极了，但没有其他乳腺癌细胞系，导致炎性细胞因子转录本已知为由NF-𝜅B转录因子靶向，该表型取决于Rela表达。这种细胞因子的产生足以影响肿瘤免疫微环境，因为观察到BLBC Xenographictics在极点上的F4/80阳性巨噬细胞显着浸润抑制。因此，我的中心假设是抑制极后NF-𝜅B激活在BLBC细胞中有选择地激活细胞因子分泌程序。在AIM 1中，我将分析激活通过免疫印迹，NF-免疫荧光的模式识别受体（PRRS）和NF-𝜅B 𝜅B亚细胞定位和NF-𝜅B荧光素酶报告基因。我将描述哪个prrs（cgas-- sting，mavs-rig-i）和nf-𝜅b组件（IKK-G，p65，relb）是通过复制而触发的压力，并使用CRISPR/CAS9产生缺乏关键信号组件的细胞系来发现负责NF-𝜅B激活的人。在AIM 2中，我将确定极力抑制的影响在肿瘤内细胞因子表达和肿瘤细胞组成上。如果免疫浸润发生了改变，我将确定与免疫切除点抑制剂的合作程度。这项工作是有影响力，因为它会区分是否通过抑制领先的重复应力引起的复制应力链复制会触发可能的免疫刺激器或免疫抑制环境目标是增强未来的抗癌疗法。