Novel Roles of LY6K in Glioblastoma Tumorigenicity

项目总结 胶质母细胞瘤(GBM)是恶性程度最高的脑癌，患者预后极差。截然不同 分子亚型，以原神经型(PN)、经典型和间叶型(MES)为特征，以及遗传性 异质性使基底膜肿瘤对目前的治疗方法产生抗药性。除了遗传异质性引起的 在分化的细胞中，GBM肿瘤还含有一小部分起始于胶质瘤或干细胞样癌。 细胞(GSC)。我们实验室的基因表达谱研究表明，患者来源的GSCs也可以 分类为表型与GBM相似的亚型。我们发现了三千多个差异基因 在PN和类似MES的GSCs之间表达。其中，淋巴细胞抗原复合体6，K基因座(LY6K) 是差异表达最多的基因之一。LY6K是一种来自LY6家族的GPI锚定蛋白。五花八门 LY6家族的成员与人类癌症有关，包括乳腺癌、食道癌和肺癌 癌症。在我们的初步研究中，我们测试了LY6K上调在GBM肿瘤发生中的作用，并开始 研究LY6K作用的潜在机制。我们发现1)LY6K的高水平表达与 2)Mes样GSCs中LY6K的表达明显受抑 降低体外和体内的致瘤行为；3)LY6K和EGFR-ERK信号在 受辐射的PN样GSCs，否则两者的水平都检测不到；4)抑制LY6K的表达 减少ERK1/2的激活，这可能是EGFR信号的下游效应。因此，我们假设 GSCs中高水平的LY6K促进了GBM肿瘤的发生。此外，根据我们的辐射数据，我们假设 LY6K可能通过增强EGFR信号和随后的ERK激活发挥作用，从而促进 抗辐射能力。为了检验这些假设，我们制定了两个具体目标。第一个目标是检查 LY6K作为癌基因在GSCs中的作用我们将在体外和体内进行致瘤性测试，以确定 GSCs中LY6K表达的调节是否调节肿瘤的发生。我们也会研究这个机制。 通过检测LY6K启动子甲基化检测LY6K在GSCs和GBM临床标本中的表达。其次，我们 我将研究LY6K和EGFR信号之间的关系，因为它与辐射抗性有关。至 确定LY6K是否调节EGFR信号，我们建议修改LY6K在GSCs中的表达 并衡量由此带来的福利变化。随后，我们将确定哪些特定的域在 LY6K负责通过结构域缺失实验增强EGFR信号。从以下方面获得的洞察 这些目的将进一步加深我们对LY6K在EGFR信号转导中的作用以及对GBM致瘤性的理解。

PROJECT SUMMARY Glioblastoma (GBM) is the most malignant brain cancer, with extremely poor prognosis in patients. Distinct molecular subtypes, characterized as proneural (PN), classical, and mesenchymal (MES), as well as inherited heterogeneity render GBM tumors resistant to current therapies. In addition to the genetic heterogeneity arising from the differentiated cells, GBM tumors also contain a small population of glioma-initiating or stem-like cancer cells (GSCs). Gene expression profiling studies from our lab showed that patient-derived GSCs can also be classified into subtypes phenotypically similar to GBM. We found over three thousand genes that are differentially expressed between PN and MES-like GSCs. Among these, Lymphocyte Antigen Complex 6, Locus K (LY6K) was one of the top differentially expressed genes. LY6K is a GPI-anchored protein from the LY6 family. Various members of the LY6 family have been implicated in human cancers, including breast, esophageal, and lung cancers. In our preliminary studies, we tested the roles of LY6K upregulation in GBM tumorigenesis and started to examine the underlying mechanism of LY6K action. We found that 1) high levels of LY6K expression correlates with poor prognosis of patients with GBM; 2) suppression of LY6K expression in MES-like GSCs significantly reduced tumorigenic behaviors in vitro and in vivo; 3) LY6K and EGFR-ERK signaling both become activated in irradiated PN-like GSCs which otherwise have undetectable levels of both; 4) suppression of LY6K expression decreases ERK1/2 activation, which is likely a downstream effect of EGFR signaling. Thus, we hypothesize that high levels of LY6K in GSCs promotes GBM tumorigenesis. Moreover, based our radiation data, we hypothesize that LY6K may function by enhancing EGFR signaling and subsequent ERK activation, thus promoting radioresistance. To test these hypotheses, we have developed two specific aims. The first aim examines the function of LY6K as an oncogene in GSCs. We will perform in vitro and in vivo tumorigenicity assays to determine whether modulation of LY6K expression in GSCs regulates tumorigenicity. We will also examine the mechanism of LY6K expression in GSCs and GBM clinical samples by examining LY6K promoter methylation. Secondly, we will investigate the relationship between LY6K and EGFR signaling, as it pertains to radioresistance. To determine whether LY6K modulates EGFR signaling, we propose to modify the expression of LY6K in GSCs and measure the resulting changes in pERK. Subsequently, we will determine which specific domains within LY6K are responsible for EGFR signal enhancement, via domain-deletion experiments. The insight gained from these aims will further our understanding the function LY6K in EGFR signaling as well as GBM tumorigenicity.