Characterizing oncogenic function of ITCH in melanoma

摘要 瘙痒泛素E3连接酶被认为是免疫细胞中的关键分子。的角色 瘙痒在肿瘤发生中的作用是有争议的，因为它在肿瘤和免疫细胞中都有多种功能。 因此，考虑到肿瘤和免疫细胞，瘙痒在肿瘤发生中的深入特征是 填补了我们目前对瘙痒在癌症中的作用的了解的空白。我们最近报道了一起 瘙痒通过促进BRAF野生型黑色素瘤细胞BRAF活化而发挥致癌作用。我们的新品 获得的初步结果揭示了瘙痒通过调节CRAF和CRAF在黑色素瘤细胞中的多方面作用 其他致癌信号。我们的结果还表明，瘙痒在黑色素瘤中具有免疫抑制作用。 肿瘤免疫微环境。基于这些数据，我们假设在BRAF野生型黑色素瘤中 在黑色素瘤细胞和肿瘤免疫微环境中，瘙痒都是一种治疗漏洞。在……里面 黑色素瘤细胞，瘙痒维持致癌途径，包括RAF-MEK-ERK和其他维持其 健康；另一方面，免疫细胞中瘙痒的激活会滋生免疫抑制肿瘤 微环境。因此，以瘙痒为靶点可能是一种改变癌症免疫设定点的新策略。三 提出的目的是为了检验我们的假设。在目标1中，我们将定义K27如何连接RAF的泛素化 ITCH蛋白调节MEK-ERK信号转导。我们将表征瘙痒是否促进了非典型 CRAF的泛素化和随后激活的方式与BRAF相似，特别是在细胞环境中 其中CRAF在激活MEK-ERK信号中起核心作用。此外，我们将确定B55-PP2A是否 磷酸酶作为RAF蛋白上聚泛素链的阅读器蛋白而发挥作用 去除抑制的N-末端磷酸化。在目标2中，我们将描述RAF非依赖性致癌 瘙痒的功能。我们将检查瘙痒是否促进了其他致癌途径的激活，从而实现了 促进生存和促进转移的作用。在目标3中，我们将重点研究黑色素瘤细胞自主 以及体内瘙痒的非自主功能。我们将通过使用几个小鼠黑色素瘤来剖析这些角色 模特们。为了确定瘙痒是否会造成免疫抑制微环境，小鼠同基因黑色素瘤 将使用Itch/和Itch-/-小鼠生成模型，并将执行免疫分析以搜索 内在机制(S)。此外，Tich/和Tich-/-小鼠将与特征良好的小鼠杂交 黑色素瘤模型评估其在肿瘤细胞和免疫微环境中的作用。

Abstract The ITCH ubiquitin E3 ligase has been well characterized as a key molecule in immune cells. The roles of ITCH in tumorigenesis are rather controversial due to its versatile functions in both tumor and immune cells. Therefore, an in-depth characterization of ITCH in tumorigenesis considering both tumor and immune cells is warranted to fill the gap in our current knowledge of ITCH’s function in cancer. We recently reported an oncogenic function of ITCH through promoting BRAF activation in BRAF wild-type melanoma cells. Our newly obtained preliminary results unveil a multifaceted role of ITCH in melanoma cells by modulating CRAF and other oncogenic signals. Our results also suggest an immunosuppressive function of ITCH in the melanoma tumor immune microenvironment. Based on these data, we hypothesize that in BRAF wild-type melanoma cells, ITCH is a therapeutic vulnerability in both melanoma cells and the tumor immune microenvironment. In melanoma cells, ITCH sustains oncogenic pathways including RAF-MEK-ERK and others to maintain their fitness; activation of ITCH in immune cells, on the other hand, fosters an immunosuppressive tumor microenvironment. Hence targeting ITCH could be a novel strategy to alter the cancer-immune set point. Three aims are proposed to test our hypothesis. In Aim 1, we will define how K27-linked ubiquitination of RAF proteins by ITCH modulates MEK-ERK signaling. We will characterize if ITCH facilitates the atypical ubiquitination and subsequent activation of CRAF in a similar way to BRAF, especially in the cellular contexts where CRAF plays a central role in activating MEK-ERK signals. Moreover, we will determine if the B55-PP2A phosphatase functions as the reader protein for the poly-ubiquitin chains assembled on RAF proteins to remove the inhibitory N-terminal phosphorylations. In Aim 2, we will characterize RAF-independent oncogenic functions of ITCH. We will examine if ITCH facilitates the activation of other oncogenic pathways that fulfill its pro-survival and pro-metastatic roles. In Aim 3, we will focus on investigating both melanoma cell-autonomous and non-autonomous functions of ITCH in vivo. We will dissect these roles by using several mouse melanoma models. To define if ITCH creates an immunosuppressive microenvironment, mouse syngeneic melanoma models will be generated using Itch+/+ and Itch-/- mice, and immuno-profiling will be carried out to search for the underlying mechanism(s). Further, Itch+/+ and Itch-/- mice will be crossed with well-characterized murine melanoma models to assess its roles in both the tumor cell and immune microenvironment.