Targeting of STAT3 Signaling Enhances Efficacy of Breast Cancer Immunotherapy

描述（由申请人提供）：本提案将检验以下假设：信号转导子和转录激活子3（STAT 3）的下调将优化乳腺癌免疫疗法的疗效，该疗法使用靶向肿瘤细胞和肿瘤微环境（TME）中免疫细胞的小基因疫苗。这种方法的基本原理是基于大量的证据，表明STAT 3转录因子抑制针对肿瘤细胞的免疫活化所必需的介质的表达，并在TME中传播肿瘤细胞和免疫细胞之间的串扰，导致显著的肿瘤诱导的免疫抑制。这三个具体目标旨在验证STAT 3作为有效靶标，以提供TME和靶向Legumain的小基因疫苗之间的必要协同作用，从而实现转移性乳腺癌的最佳免疫治疗。首先，将建立小探针STAT 3抑制剂（NSC 295558）的概念证明，一旦该抑制剂被掺入脂质体纳米颗粒（NP）中并特异性靶向并递送至这些细胞类型，该抑制剂就能有效下调肿瘤相关巨噬细胞（TAM）和鼠4 T1乳腺癌细胞中的STAT 3信号传导。通过将NP的氨基磷脂与Legumain的氮杂肽环氧化物抑制剂缀合来实现特异性靶向，Legumain是一种由TME中的TAM和4 T1肿瘤细胞特异性过表达的天冬酰胺酰内肽酶。然后测试靶向和负载的NP在体外选择性抑制STAT 3转录活性和诱导4 T1细胞和携带组成型激活的STAT 3的TAM的细胞凋亡的能力。第二，将建立原理证明，该策略在我们的BALB/c小鼠中的同基因、自发4 T1乳腺癌转移模型中在体内下调IL-6/JAK/STAT 3信号传导是有效的。重点将是评估STAT 3下调是否与T调节（TReg）细胞的下调以及编码IL-6和TGF-β的基因相关，这些基因是肿瘤诱导的免疫抑制的指标。还将评估STAT 3下调与TAM中促炎细胞因子和趋化因子表达增加以及与4 T1肿瘤细胞中免疫抑制因子消除的相关性。第三，建立概念证明，STAT 3的下调产生具有与我们的基于Legumain的小基因疫苗的抗肿瘤作用有效协同所需的最小免疫抑制活性的肿瘤微环境类型，以诱导长寿命的CD 8+记忆T细胞应答，其预防或延迟已建立的转移性乳腺癌的复发。重点将是对我们的联合免疫疗法的抗肿瘤作用负责的先天性和适应性免疫的细胞和分子免疫机制的阐明。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，即STAT 3转录因子的特异性下调减少免疫抑制因子的产生，从而产生有利的免疫肿瘤微环境，其与基于Legumain的小基因疫苗有效协同，以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.