Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.
Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA
J Immunother Cancer. 2021 Jul;9(7). doi: 10.1136/jitc-2020-002258.
There are highly effective treatment strategies for estrogen receptor (ER)+, progesterone receptor (PR)+, and HER2+ breast cancers; however, there are limited targeted therapeutic strategies for the 10%-15% of women who are diagnosed with triple-negative breast cancer. Here, we hypothesize that ER targeting drugs induce phenotypic changes to sensitize breast tumor cells to immune-mediated killing regardless of their ER status.
Real-time cell analysis, flow cytometry, qRT-PCR, western blotting, and multiplexed RNA profiling were performed to characterize ER+ and ER- breast cancer cells and to interrogate the phenotypic effects of ER targeting drugs. Sensitization of breast cancer cells to immune cell killing by the tamoxifen metabolite 4-hydroxytamoxifen (4-OHT) and fulvestrant was determined through in vitro health-donor natural killer cell IN-release killing assays. A syngeneic tumor study was performed to validate these findings in vivo.
Pretreatment with tamoxifen metabolite 4-OHT or fulvestrant resulted in increased natural killer (NK)-mediated cell lysis of both ER+ and ER- breast cancer cells. Through multiplexed RNA profiling analysis of 4-OHT-treated ER+ and ER- cells, we identified increased activation of apoptotic and death receptor signaling pathways and identified G protein-coupled receptor for estrogen (GPR30) engagement as a putative mechanism for immunogenic modulation. Using the specific GPR30 agonist G-1, we demonstrate that targeted activation of GPR30 signaling resulted in increased NK cell killing. Furthermore, we show that knockdown of GPR30 inhibited 4-OHT and fulvestrant mediated increases to NK cell killing, demonstrating this is dependent on GPR30 expression. Moreover, we demonstrate that this mechanism remains active in a 4-OHT-resistant MCF7 cell line, showing that even in patient populations with ER+ tumors that are resistant to the cytotoxic effects of tamoxifen, 4-OHT treatment sensitizes them to immune-mediated killing. Moreover, we find that fulvestrant pretreatment of tumor cells synergizes with the IL-15 superagonist N-803 treatment of NK cells and sensitizes tumor cells to killing by programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK) cells. Finally, we demonstrate that the combination of fulvestrant and N-803 is effective in triple-negative breast cancer in vivo.
Together, these findings demonstrate a novel effect of ER targeting drugs on the interaction of ER+ and, surprisingly, ER- tumors cells with the immune system. This study is the first to demonstrate the potential use of ER targeting drugs as immunomodulatory agents in an ER agnostic manner and may inform novel immunotherapy strategies in breast cancer.
对于雌激素受体 (ER)+、孕激素受体 (PR)+和 HER2+的乳腺癌，有非常有效的治疗策略；然而，对于 10%-15%被诊断为三阴性乳腺癌的女性，只有有限的靶向治疗策略。在这里，我们假设 ER 靶向药物诱导表型变化，使乳腺癌细胞对免疫介导的杀伤敏感，而不管其 ER 状态如何。
实时细胞分析、流式细胞术、qRT-PCR、Western blot 和多重 RNA 分析用于表征 ER+和 ER-乳腺癌细胞，并探究 ER 靶向药物的表型效应。通过体外健康供体自然杀伤细胞 IN 释放杀伤测定，确定他莫昔芬代谢物 4-羟基他莫昔芬 (4-OHT) 和氟维司群对乳腺癌细胞对免疫细胞杀伤的敏感性。进行了一项同种异体肿瘤研究，以验证这些发现的体内。
用他莫昔芬代谢物 4-OHT 或氟维司群预处理可增加 NK 介导的 ER+和 ER-乳腺癌细胞的裂解。通过对 4-OHT 处理的 ER+和 ER-细胞的多重 RNA 分析，我们发现凋亡和死亡受体信号通路的激活增加，并确定 G 蛋白偶联受体雌激素 (GPR30) 参与作为免疫调节的潜在机制。使用特定的 GPR30 激动剂 G-1，我们证明靶向激活 GPR30 信号会导致 NK 细胞杀伤增加。此外，我们表明，GPR30 敲低抑制了 4-OHT 和氟维司群介导的 NK 细胞杀伤增加，表明这依赖于 GPR30 表达。此外，我们证明该机制在 4-OHT 耐药 MCF7 细胞系中仍然活跃，表明即使在对他莫昔芬的细胞毒性作用耐药的 ER+肿瘤患者中，4-OHT 治疗也能使它们对免疫介导的杀伤敏感。此外，我们发现氟维司群预处理肿瘤细胞与 IL-15 超激动剂 N-803 联合作用可增强 NK 细胞对程序性死亡配体 1 (PD-L1) 靶向高亲和力自然杀伤 (t-haNK) 细胞杀伤的敏感性。最后，我们证明氟维司群和 N-803 的组合在体内对三阴性乳腺癌有效。
总之，这些发现表明 ER 靶向药物对 ER+和令人惊讶的 ER-肿瘤细胞与免疫系统相互作用的一种新作用。这项研究首次证明 ER 靶向药物作为免疫调节剂以 ER 不可知的方式使用的潜力，并可能为乳腺癌提供新的免疫治疗策略。
