Role of Eya3 in regulating the immune microenvironment to promote breast tumor progression

摘要 Eya蛋白（Eya 1-4）最初被发现是六个家族的必需共激活剂。 通过控制增殖、存活、上皮细胞与 间充质命运和许多组织中的总体细胞命运规范。Six/Eya综合体 在发育完成后下调，但在许多癌症中重新表达，包括乳腺癌。 癌除了作为Six家族蛋白质的转录辅因子外，Eyas还含有Tyr 在它们的C-末端结构域中的磷酸酶活性不仅与转录激活有关， 而且在运动性、DNA修复和对损伤的反应中的存活、血管生成和抗- 雌激素受体-b的肿瘤活性。目前，正在开发针对转录和转录的抑制剂， Eyas的酪氨酸磷酸酶活性，以剖析其不同的功能，并潜在地开发抗癌 剂. Eyas在其N-末端结构域中也含有Ser/Thr磷酸酶活性，这是完全可分离的 Tyr磷酸酶活性。这种活性在癌症中的功能是完全未知的， 任何正在开发的Eya抑制剂的靶向。有趣的是，Eya的N末端结构域并不 含有任何可识别的磷酸酶基序，我们的数据表明， Eya不是内在的，而是由于与蛋白磷酸酶2A（PP 2A）的相互作用。此外，本发明还提供了一种方法， 使用两种不同的免疫活性小鼠模型，我们首次表明，Eya 3，这是 与其他亚型相比，其在三阴性乳腺癌（TNBC）中过表达，并且含有 最高的Ser/Thr磷酸酶活性通过其对适应性免疫的作用影响肿瘤进展。我们 进一步证明Eya 3通过其PP 2A相关的Ser/Thr磷酸酶活性调节关键的 免疫检查点蛋白程序性死亡配体1（PD-L1）。因此，我们确定了一种新的肿瘤促进因子， Eya 3的功能仅在功能性适应性免疫系统的背景下揭示。本提案中 我们检验了Eya 3通过其募集PP 2A的能力调节乳腺肿瘤生长和进展的假设， 导致PD-L1水平升高和肿瘤特异性T细胞应答降低。 本项目的研究结果将极大地促进我们对Eya 3在乳腺肿瘤中作用的理解 进展，也可能揭示Eya 3通过其先前未知的与PP 2A相互作用的能力，是一种 适应性肿瘤免疫中重要的新角色。此外，我们可能会发现一种新的调节机制， 关键的免疫检查点蛋白PD-L1，其调控在很大程度上仍然难以捉摸。因此，我们的研究可以 产生新的生物标志物或治疗策略，以增强PD-1/PD-L1介导的治疗的功效。

Abstract The Eya proteins (Eya 1-4) were initially discovered as essential co-activators of the Six family of homeoproteins that regulate embryonic development via controlling proliferation, survival, epithelial versus mesenchymal fates, and overall cell fate specification in numerous tissues. The Six/Eya complex is downregulated after development is complete, but is re-expressed in numerous cancers, including breast cancer. In addition to acting as transcriptional cofactors for the Six family of proteins, Eyas also contain a Tyr phosphatase activity in their C-terminal domains that has been implicated not only in transcriptional activation, but also in motility, DNA repair and survival in response to damage, angiogenesis, and inhibition of the anti- tumor activity of estrogen receptor-b. Currently, inhibitors are being developed against the transcriptional and Tyr phosphatase activities of Eyas to dissect its different functions, and to potentially develop anti-cancer agents. The Eyas also contain a Ser/Thr phosphatase activity in their N-terminal domain, that is completely separable from the Tyr phosphatase activity. The function of this activity in cancer is completely unknown and it is not targeted by any of the Eya inhibitors under development. Intriguingly, the N-terminal domain of Eya does not contain any recognizable phosphatase motifs and our data suggest that the Ser/Thr phosphatase activity of Eya is not intrinsic, but is instead due to an interaction with the protein phosphatase 2A (PP2A). In addition, using two different immune competent mouse models, we show for the first time that Eya3, which is overexpressed in triple negative breast cancers (TNBC) as compared to other subtypes, and contains the highest Ser/Thr phosphatase activity, influences tumor progression via its effects on adaptive immunity. We further demonstrate that Eya3, through its PP2A-associated Ser/Thr phosphatase activity, regulates the key immune checkpoint protein programmed death-ligand 1 (PD-L1). Thus, we identified a novel tumor promoting function for Eya3 that is only revealed in the context of a functional adaptive immune system. In this proposal we test the hypothesis that Eya3 regulates breast tumor growth and progression via its ability to recruit PP2A, leading to increased levels of PD-L1 and a diminished tumor specific T-cell response. Results obtained in this project will significantly advance our understanding of the role of Eya3 in breast tumor progression, and may also reveal that Eya3, through its previously unknown ability to interact with PP2A, is an important new player in adaptive tumor immunity. Further, we may uncover a novel regulatory mechanism of a key immune checkpoint protein, PD-L1, whose regulation has remained largely elusive. Thus our studies may result in novel biomarker or therapeutic strategies to enhance efficacy of PD-1/PD-L1 mediated therapies.