Regulation of Snail1 in cancer metastasis

描述（由申请人提供）：癌症转移占绝大多数癌症相关死亡，仍然是临床医生和科学家面临的挑战。上皮间充质转化（EMT）通过激活细胞迁移和侵袭所需的基因来促进癌症转移。Snail1是EMT的关键调控因子，在上皮细胞中Snail1的蛋白水平、亚细胞定位和功能受到严格调控。我们利用人类kinome RNAi文库开发了一种高通量筛选，以鉴定Snail1的新的翻译后调节因子。通过该筛选鉴定了盘状蛋白结构域受体2 (DDR2)，我们发现DDR2缺失导致Snail1水平降低，而DDR2过表达与Snail1水平升高相关。我们已经证明DDR2在翻译后稳定了Snail1蛋白，Snail1 mRNA水平几乎没有变化，这种稳定依赖于通过ERK2的MAPK/ERK信号传导。此外，DDR2在人浸润性导管性乳腺癌中表达，而在正常乳腺组织和浸润性小叶性乳腺癌中不表达。这些初步研究结果表明，DDR2可能在乳腺癌转移过程中调控snail1介导的细胞侵袭中发挥重要作用。然而，DDR2信号稳定Snail1蛋白的分子机制以及DDR2在Snail1介导的乳腺癌细胞侵袭体内是否必需尚不清楚，本课题将通过三个具体目的进行研究。ERK2在Snail1稳定中的作用将通过确定ERK2和Snail1的物理相互作用，ERK2是否磷酸化Snail1以及在哪些位点磷酸化，以及这如何影响Snail1的细胞功能来研究。此外，DDR2在乳腺癌细胞侵袭中的体内作用将通过两种不同的模型系统来确定：鸡绒毛膜尿囊膜（CAM）实验和人类-小鼠导管内（MIND）异种移植模型。最后，我们将在人类乳腺癌的多个亚型中测量DDR2的表达，并确定这种表达是否与Snail1表达或其他乳腺癌预后因素相关。这项研究将揭示EMT在肿瘤侵袭和转移中的新分子机制，并将有助于未来开发更好的治疗乳腺癌患者的方法。

DESCRIPTION (provided by applicant): Cancer metastasis accounts for the vast majority of cancer-related fatalities and remains a challenge for both clinicians and scientists. The epithelia mesenchymal transition (EMT) contributes to cancer metastasis by activating genes required for cell migration and invasion. Snail1 is a key regulator of EMT, and Snail1's protein level, subcellular localization, and function are tightly regulated in epithelial cells. We developed a high throughput screen with a human kinome RNAi library to identify novel post-translational regulators of Snail1. The discoidin domain receptor 2 (DDR2) was identified through this screen and we found that DDR2 depletion resulted in decreased levels of Snail1, while DDR2 overexpression correlated with elevated levels of Snail1. We have shown that DDR2 stabilized Snail1 protein post-translationally with little change in Snail1 mRNA levels, and this stabilizatio was dependent on MAPK/ERK signaling through ERK2. Additionally, DDR2 was expressed in human invasive ductal breast cancer, but not normal breast tissue or invasive lobular breast cancer. These preliminary findings indicate that DDR2 likely plays an important role in regulating Snail1-mediated cell invasion during breast cancer metastasis. However, the molecular mechanism by which DDR2 signals to stabilize Snail1 protein and whether DDR2 is essential for Snail1- mediated breast cancer cell invasion in vivo is unknown and will be studied through three specific aims in this proposal. The role of ERK2 in Snail1 stabilization will be studied by determining how ERK2 and Snail1 physically interact, whether ERK2 phosphorylates Snail1 and at which sites, and how this affects the cellular function of Snail1. In addition, the in vivo roleof DDR2 in breast cancer cell invasion will be determined using two different model systems: the chicken chorioallantoic membrane (CAM) assay and a human-in-mouse intraductal (MIND) xenograft model. Finally, DDR2 expression will be measured in multiple subtypes of human breast cancer, and whether this expression correlates with Snail1 expression or other breast cancer prognostic factors will be determined. This research will unravel novel molecular mechanisms regulating EMT in cancer that contribute to invasion and metastasis, and will help to develop better therapies for treating breast cancer patients in the future.