Regulation of Snail1 in cancer metastasis

描述（由申请人提供）：癌症转移占癌症相关死亡的绝大多数，对于临床医生和科学家来说仍然是一个挑战。上皮间质转化（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的关键调节因子，Snail1的蛋白水平、亚细胞定位和功能在上皮细胞中受到严格调控。我们利用人类激酶组 RNAi 文库开发了一种高通量筛选，以鉴定 Snail1 的新型翻译后调节因子。通过该筛选鉴定了盘状蛋白结构域受体 2 (DDR2)，我们发现 DDR2 耗竭导致 Snail1 水平降低，而 DDR2 过表达与 Snail1 水平升高相关。我们已经表明，DDR2 在翻译后稳定了 Snail1 蛋白，而 Snail1 mRNA 水平几乎没有变化，并且这种稳定依赖于通过 ERK2 的 MAPK/ERK 信号传导。此外，DDR2 在人类浸润性导管乳腺癌中表达，但在正常乳腺组织或浸润性小叶乳腺癌中不表达。这些初步研究结果表明，DDR2 可能在乳腺癌转移过程中调节 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是否对于Snail1介导的乳腺癌细胞体内侵袭至关重要尚不清楚，将通过本提案中的三个具体目标进行研究。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，以及磷酸化如何影响 Snail1 的细胞功能，我们将研究 ERK2 在 Snail1 稳定中的作用。此外，DDR2在乳腺癌细胞侵袭中的体内作用将使用两种不同的模型系统来确定：鸡绒毛尿囊膜（CAM）测定和人鼠导管内（MIND）异种移植模型。最后，将在人类乳腺癌的多个亚型中测量 DDR2 的表达，并确定该表达是否与 Snail1 表达或其他乳腺癌预后因素相关。这项研究将揭示调节癌症中 EMT 促进侵袭和转移的新分子机制，并将有助于在未来开发更好的治疗乳腺癌患者的疗法。

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.