The Role of JAK2 in ILEI-Induced Cancer Stem Cell Formation

 转移性疾病是导致大多数乳腺癌患者死亡的原因，但转移的分子机制尚不清楚。一种假设是，被称为癌症干细胞(CSCs)的原始乳腺癌细胞的一个特定子集有助于转移性突起(1)。研究表明，经历了转化生长因子-β诱导的上皮向间充质转化的细胞具有干细胞特性[2]。由于传统的化疗药物不能根除肿瘤干细胞，阐明转化生长因子-β诱导的肿瘤干细胞形成的分子机制可以针对肿瘤干细胞进行靶向治疗，并为癌症治疗提供一种范式转变。转化生长因子β诱导内胚层转化的途径之一是肿瘤抑制因子hnRNP-E1(3-5)。在正常情况下，hnRNP-E1结合并抑制与EMT相关的mRNAs的翻译，但转化生长因子-β处理使hnRNP-E1从mRNAs中释放出来，允许翻译，并诱导EMT。如前所述，经历了转化生长因子-β诱导的EMT的细胞具有血管干细胞特性，所以我们想知道转化生长因子-β/hnRNP-E1的下游效应子对血管干细胞特性的影响。我们从关注转化生长因子β和hnRNP-E1共同调控的基因开始我们的研究，并选择了细胞因子白介素样EMT诱导剂(ILEI)。初步结果表明，ILEI是激活STAT3信号和启动体外CSC形成所必需的，但不是充分的。我们以前的工作表明，JAK2作为STAT3信号的典型激活子，与ILEI一样受到转化生长因子-β/hnRNP-E1机制的调节。这些发现使我们推测，ILEI需要一个额外的转化生长因子-β/hnRNP-E1调节基因，如JAK2来磷酸化STAT3并启动CSC的形成。这一假设将通过以下具体目标加以阐述。目的1确定转化生长因子β和hnRNP-E1在JAK2表达中的作用。多聚体图谱和RNA免疫沉淀将确认JAK2是否受hnRNP-E1的翻译调控。目标2将确定JAK2在ILEI信号转导中的作用。JAK2将在几个细胞系中被调制，我们将测量它对STAT3激活以及乳房膜形成的影响。目的3验证ILEI信号在CSC形成中的体内意义。我们之所以对CSCs感兴趣，是因为它们在转移性生长中扮演着重要角色。因此，在我们的最终目标中，我们将使用小鼠乳腺癌转移模型，并应用JAK抑制剂ruxolitinib来阻断ILEI信号。这些目的将支持靶向ILEI信号作为一种治疗转移性乳腺癌的新方法。

 DESCRIPTION (provided by applicant) Metastatic disease causes the mortality of most breast cancer patients, but the molecular mechanism of metastasis remains unclear. One hypothesis is that a specific subset of primary breast cancer cells known as cancer stem cells (CSCs) contributes to metastatic outgrowths (1). It has been shown that cells that have undergone a TGF-β-induced epithelial-to-mesenchymal transition (EMT) possess CSC properties (2). Because traditional chemotherapeutics cannot eradicate CSCs, clarifying the molecular mechanism of TGF-β-induced CSC formation could allow for targeted therapies against CSCs and provide a paradigm shift for cancer care. One of the ways TGF-β can induce EMT is the tumor suppressor hnRNP-E1 (3-5). Under normal conditions hnRNP-E1 binds and inhibits the translation of EMT-related mRNAs, but TGF-β treatment releases hnRNP-E1 from the mRNAs, allows for translation, and induces EMT. As stated previously, cells that have undergone a TGF-β-induced EMT possess CSC properties so we wanted to know the downstream effectors of TGF- β/hnRNP-E1 that confer CSC properties. We began our search by focusing on genes that were regulated by both TGF-β and hnRNP-E1, and chose the cytokine Interleukin-like EMT Inducer (ILEI). Preliminary results have shown that ILEI is necessary but not sufficient to activate STAT3 signaling and initiate CSC formation in vitro. Our previous work suggested that JAK2, a canonical activator of STAT3 signaling, was regulated by the same TGF-β/hnRNP-E1 mechanism as ILEI. These findings lead us to hypothesize that ILEI requires an additional TGF-β/hnRNP-E1-regulated gene such as JAK2 to phosphorylate STAT3 and initiate CSC formation. This hypothesis will be addressed through the following Specific Aims. Aim 1 will establish the role of TGF-β and hnRNP-E1 in JAK2 expression. Polysome profiling and RNA-Immunoprecipitations will confirm whether JAK2 is translationally regulated by hnRNP-E1. Aim 2 will determine the role of JAK2 in ILEI signaling. JAK2 will be modulated in several cell lines, and we will measure its effect on STAT3 activation as well as mammosphere formation. Aim 3 will validate the in vivo significance of ILEI signaling in CSC formation. We are interested in CSCs because of their putative role in metastatic outgrowths. Thus, in our final aim we will use a murine breast cancer metastasis model and apply the JAK inhibitor ruxolitinib to block ILEI signaling. These Aims will support the targeting of ILEI signaling as a novel therapy against metastatic breast cancer.