Targeting of STAT3 Signaling Enhances Efficacy of Breast Cancer Immunotherapy

描述（由申请人提供）：该提案将验证信号换能器和转录激活因子3 （STAT3）的下调将优化靶向肿瘤细胞和肿瘤微环境（TME）免疫细胞的minigene疫苗的乳腺癌免疫治疗效果的假设。这种方法的基本原理是基于大量证据表明，STAT3转录因子抑制肿瘤细胞免疫激活所需介质的表达，并在TME中传播肿瘤细胞和免疫细胞之间的串扰，导致明显的肿瘤诱导的免疫抑制。这三个特定目标旨在验证STAT3作为一个有效的靶点，在TME和靶向Legumain的迷你基因疫苗之间提供必要的协同作用，以实现转移性乳腺癌的最佳免疫治疗。首先，将建立一个小探针STAT3抑制剂（NSC295558）的概念证明，一旦将该抑制剂纳入脂质体纳米颗粒（NPs）并特异性靶向并递送到这些细胞类型中，该抑制剂就可以有效下调肿瘤相关巨噬细胞（tam）和小鼠4T1乳腺癌细胞中的STAT3信号。特异性靶向是通过将NPs的氨基磷脂与Legumain的环氧化氮肽抑制剂偶联来实现的，Legumain是一种天冬酰胺内肽酶，在TME中被TAMs和4T1肿瘤细胞特异性过表达。然后将测试靶向和负载的NPs在体外选择性抑制STAT3转录活性的能力，以及诱导4T1细胞和含有组成性激活STAT3的tam细胞凋亡的能力。其次，在我们的BALB/c小鼠同基因自发性4T1乳腺癌转移模型中，将建立原理证明该策略在体内下调IL-6/JAK/STAT3信号是有效的。重点将是评估STAT3下调是否与t -调节性（TReg）细胞下调以及编码IL-6和TGF-¿的基因相关，这些基因是肿瘤诱导免疫抑制的指标。STAT3下调的相关性也将通过tam中促炎细胞因子和趋化因子的表达增加以及4T1肿瘤细胞中免疫抑制因子的消融来评估。第三，建立概念证明，STAT3的下调创造了一种肿瘤微环境，这种肿瘤微环境具有最小的免疫抑制活性，需要有效地与我们的legumainminigene疫苗的抗肿瘤作用协同作用，以诱导长寿命的CD8+记忆T细胞反应，从而防止或延迟已确定的转移性乳腺癌的复发。重点将阐明先天免疫和适应性免疫的细胞和分子免疫机制，这些机制负责我们联合免疫治疗的抗肿瘤作用。公共卫生相关性：本研究与公共卫生的相关性是在小鼠模型系统中建立概念证明，STAT3转录因子的特异性下调可减少免疫抑制因子的产生，从而创造有利的免疫肿瘤微环境，该微环境可与基于Legumain的minigene疫苗有效协同，显著改善已建立的转移性乳腺癌的免疫治疗。

DESCRIPTION (provided by applicant): This proposal will test the hypothesis that downregulation of the signal transducer and activator of transcription 3 (STAT3) will optimize the efficacy of breast cancer immunotherapy with a minigene vaccine targeting tumor cells and immune cells in the tumor microenvironment (TME). The rationale for this approach is based on a large body of evidence indicating that the STAT3 transcription factor inhibits the expression of mediators necessary for immune activation against tumor cells and propagates crosstalk between tumor cells and immune cells in the TME leading to marked tumor-induced immunosuppression. The three specific aims are designed to validate STAT3 as an effective target to provide the necessary synergy between the TME and a minigene vaccine targeting Legumain to achieve optimal immunotherapy of metastatic breast cancer. First, proof of concept will be established for a small probe STAT3 inhibitor (NSC295558) to effectively downregulate STAT3 signaling in tumor-associated macrophages (TAMs) and murine 4T1 breast cancer cells once this inhibitor is incorporated into liposomal nanoparticles (NPs) and specifically targeted and delivered to these cell types. Specific targeting is achieved by conjugating the aminophospholipid of NPs with an azapeptide epoxide inhibitor of Legumain, an asparaginyl endopeptidase specifically overexpressed by TAMs and 4T1 tumor cells in the TME. Targeted and loaded NPs will then be tested for their ability to selectively inhibit STAT3 transcriptional activity in vitro and to induce apoptosis of 4T1 cells and TAMs harboring constitutively activated STAT3. Second, proof of principle will be established that this strategy is valid in downregulating IL-6/JAK/STAT3 signaling in vivo in our syngeneic, spontaneous 4T1 breast cancer metastasis model in BALB/c mice. Emphasis will be on assessing whether STAT3 downregulation correlates with downregulation of T-regulatory (TReg) cells and with genes encoding IL-6 and TGF-¿ that are indicators of tumor induced immunosuppression. Correlation of STAT3 downregulation will also be assessed with increased expression of proinflammatory cytokines and chemokines in TAMs as well as with the ablation of immunosuppressive factors in 4T1 tumor cells. Third, to establish proof of concept that downregulation of STAT3 creates the type of tumor microenvironment with the minimal immunosuppressive activity required to effectively synergize with the anti-tumor effects of our Legumain-based minigene vaccine to induce a long-lived CD8+ memory T cell response which prevents or delays the recurrence of established metastatic breast cancer. Emphasis will be on elucidation of cellular and molecular immunological mechanisms of innate and adaptive immunity responsible for anti-tumor effects of our combination immunotherapy. PUBLIC HEALTH RELEVANCE: The relevance of this research to public health is to establish proof of concept in a mouse model system that specific downregulation of the STAT3 transcription factor decreases the production of immunosuppressive factors and thereby creates a favorable immunological tumor microenvironment which effectively synergizes with a Legumain- based minigene vaccine to significantly improve immunotherapy of established metastatic breast cancer.