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，仍然是临床医生和科学家面临的挑战。上皮间质转化（EMT）通过激活细胞迁移和侵袭所需的基因而促进癌症转移。Snail 1是EMT的关键调节因子，Snail 1的蛋白水平、亚细胞定位和功能在上皮细胞中受到严格调节。我们开发了一个高通量筛选与人类kinome RNAi文库，以确定新的翻译后调节Snail 1。通过该筛选鉴定了盘状结构域受体2（DDR2），我们发现DDR2缺失导致Snail 1水平降低，而DDR2过表达与Snail 1水平升高相关。我们已经证明DDR2在转录后稳定了Snail 1蛋白，而Snail 1 mRNA水平几乎没有变化，并且这种稳定依赖于通过ERK 2的MAPK/ERK信号转导。此外，DDR2在人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Snail 1介导的细胞侵袭中起重要作用。然而，DDR2信号稳定Snail 1蛋白的分子机制以及DDR2是否是Snail 1介导的乳腺癌细胞体内侵袭所必需的尚不清楚，本提案将通过三个具体目标进行研究。ERK 2在Snail 1稳定中的作用将通过确定ERK 2和Snail 1如何物理相互作用，ERK 2是否磷酸化Snail 1以及在哪些位点，以及这如何影响Snail 1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，将在多种亚型的人乳腺癌中测量DDR2表达，并确定该表达是否与Snail 1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的新分子机制，这些机制有助于侵袭和转移，并将有助于开发未来治疗乳腺癌患者的更好疗法。

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.