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

项目摘要 乳腺癌是美国女性癌症相关死亡人数第二多的原因，尽管 改进的治疗选择和增加的筛查的可用性。一个特别咄咄逼人的 在这些死亡病例中，代表不成比例数量的亚型是基底样乳房 癌症(BLBC)。BLBC的高死亡率源于缺乏有效的、可持续的 化疗方案--尽管肿瘤经常对化疗产生抗药性 令人鼓舞的治疗反应。BLBC患者较高的复发率要求 针对不同的分子改变开发更有效的治疗方案 基底样瘤。令人惊讶的是，我们发现BLBC，而不是腔内乳腺癌细胞系，表现出 对部分抑制先导链复制聚合酶DNA的严重敏感性 聚合酶epsilon(极)。BLBC单元中的极点抑制导致复制分叉停滞， DNA损伤，以及类衰老状态或细胞死亡。我们的初步数据构成了 我应用前提，揭示了BLBC细胞系中的极点抑制，而不是其他 乳腺癌细胞株，导致已知的炎性细胞因子转录本增加 以核因子-𝜅B转录因子为靶点，这是一种依赖于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，因为我 BLBC异种移植瘤极上可见大量F4/80阳性巨噬细胞 压制。因此，我的中心假设是，在电极抑制时，NF-𝜅B被激活 选择性地激活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、免疫荧光检测模式识别受体(PRRs)和核因子-𝜅B 𝜅B亚细胞定位，以及一个NF-𝜅B荧光素酶报告。我将描述哪些PRR(cGAS- STIN、MAVS-RIG-I)和NF-𝜅B组件(IKK-g、P65、RELb)由复制触发 应激，并使用CRISPR/Cas9来产生缺乏关键信号成分的细胞系来揭示 那些负责核因子-𝜅B激活的人。在目标2中，我将确定极点压制的影响 肿瘤内细胞因子表达与肿瘤细胞成分的关系。如果免疫渗透被改变， 我将确定与免疫检查点抑制剂的合作程度。这项工作是 有影响力，因为它会区分是否通过抑制引导诱导复制应激 链复制触发免疫刺激或免疫抑制环境，可能 有针对性地加强未来的抗癌治疗。

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.