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

• 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
• 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

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 乳腺癌是美国女性第二大癌症相关死亡原因， 提供更好的治疗选择和增加筛查。一个特别有侵略性的 在这些死亡病例中，代表不成比例数量的亚型是基底样乳腺癌 癌症（BLBC）。BLBC的高死亡率源于缺乏有效、可持续的 化疗治疗选择与肿瘤往往发展耐药性治疗，尽管 鼓励治疗反应。BLBC患者中较高的复发率要求 开发针对不同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，而不是管腔乳腺癌细胞系，表现出 对前导链复制聚合酶（DNA）的部分抑制高度敏感 聚合酶链反应（POLE）。BLBC细胞中的POLE抑制导致复制叉停滞， DNA损伤，衰老样状态或细胞死亡。我们的初步数据， 我的申请的前提，揭示了BLBC细胞系中的POLE抑制，而不是其他细胞系中的POLE抑制。 乳腺癌细胞系，导致炎症细胞因子转录物的增加， 由NF-κ BB转录因子靶向，一种依赖于RELA表达的表型。 这种细胞因子的产生足以影响肿瘤免疫微环境， 在POLE后观察到BLBC异种移植物中F4/80阳性巨噬细胞的显著浸润 镇压因此，我的中心假设是，NF-κ BB激活后POLE抑制 选择性激活BLBC细胞中的细胞因子分泌程序。在目标1中，我将分析激活 通过免疫印迹法检测模式识别受体（PRR）和NF-κ B B的表达， NF-κ B B亚细胞定位和NF-κ BB荧光素酶报告基因。我将描述哪些PRR（CGAS- STING、MAVS-RIG-I）和NF-κ BB组分（IKK-g、p65、RELB）通过复制触发 应激，并使用CRISPR/Cas9产生缺乏关键信号传导组分的细胞系，以揭示 负责NF-κ BB活化的那些。在目标2中，我将确定POLE抑制的影响 对肿瘤内细胞因子表达和肿瘤细胞组成的影响。如果免疫浸润发生改变， 我将确定与免疫检查点抑制剂的合作程度。这项工作是 影响，因为它将区分是否复制应激诱导抑制领先 链复制触发免疫刺激或免疫抑制环境， 以增强未来的抗癌疗法。