Regulation of MT-MMPs by Trafficking in Cancer Cells

描述（由申请人提供）：我们的长期目标是确定膜型基质金属蛋白酶（MT-MMPs）如何调节肿瘤的侵袭和转移。尽管基质金属蛋白酶（MMPs）与恶性癌症的发展有关，但肿瘤细胞利用这些破坏性蛋白酶的机制仍未得到解决。MT-MMPs是具有跨膜结构域的MMPs的一个子集，能够将这些酶集中在质膜上对抗细胞外基质（ECM）屏障，代表了肿瘤细胞在肿瘤侵袭和转移过程中如何利用蛋白水解活性的新范式。我们最近证明了MT1-MMP在网格蛋白包被的囊泡中受内吞作用调节，以控制其在细胞表面的蛋白水解活性。我们确定它的细胞质结构域是内吞作用所必需的，去除它会导致该酶的功能获得突变体。这种内吞缺陷突变体在促进前列腺癌细胞的侵袭性方面比野生型MT1-MMP更有效，这表明肿瘤细胞的侵袭性可以通过mt - mmp的运输来调节。利用MT1-MMP和MT3-MMP之间的嵌合体，我们首次获得了内化的MT-MMPs可以循环回到细胞表面的证据。在本应用中，我们建议整合MT1-MMP和MT3- MMP的研究，以验证肿瘤细胞通过控制mt -MMP通过其细胞质结构域的运输来调节其侵袭性的假设。首先，我们将定义MT1-MMP和MT3-MMP从细胞表面运输到细胞内不同的区室，并表征生长因子和细胞外基质对其运输的影响。其次，我们将定义MT1- MMP和MT3-MMP的细胞质结构域在介导它们的运输/再循环中的作用，并表征它们与细胞成分的相互作用。第三，我们将定义运输对MT1-MMP和MT3-MMP在三维I型胶原基质内或通过其介导的细胞生长和侵袭的调节作用。这些方法的结果可能会对肿瘤细胞如何通过调节MT-MMPs的贩运获得生长和侵袭性优势产生见解，并且这些见解可能会导致开发用于治疗和化学预防癌症的新型药物。

DESCRIPTION (provided by applicant): Our long term objective is to determine how membrane-type matrix metalloproteinases (MT-MMPs) regulate tumor invasion and metastasis. Although matrix metalloproteinases (MMPs) have been implicated in the progression of malignant cancer, the mechanism by which tumor cells deploy these destructive proteinases remains unresolved. MT-MMPs are a subset of MMPs with transmembrane domains capable of focusing these enzymes on plasma membrane against extracellular matrix (ECM) barriers, representing a novel paradigm on how tumor cells deploy proteolytic activity during tumor invasion and metastasis. We have recently demonstrated that MT1-MMP is regulated by endocytosis in clathrin- coated vesicles to control its proteolytic activity on cell surface. We established that its cytoplasmic domain is required for endocytosis and its removal gave rise to a gain-of-function mutant of this enzyme. This endocytosis-deficient mutant is more potent than wild type MT1-MMP in promoting invasiveness in prostate cancer cells, suggesting that the invasiveness of tumor cells can be regulated through the trafficking of MT-MMPs. Employing a chimera between MT1-MMP and MT3-MMP, we obtained the first evidence that internalized MT-MMPs can be recycled back to cell surface. In this application, we propose to integrate the studies of MT1-MMP and MT3- MMP to test the hypothesis that tumor cells modulate their invasiveness by controlling the trafficking of MT-MMPs through their cytoplasmic domains. First, we will define the trafficking of MT1-MMP and MT3-MMP from cell surface to various intracellular compartmetns and characterize the impact of growth factors and extracellular matrix on their trafficking. Secondly, we will define the role of cytoplasmic domains of MT1- MMP and MT3-MMP in mediating their trafficking/recycling and characterize their interactions with cellular components. Thirdly, we will define the regulatory role of trafficking on MT1-MMP and MT3-MMP mediated cell growth and invasion within or through 3-dimensional type I collagen matrix. Results from these approaches may yield insights on how tumor cells gain a growth and invasive advantage by regulating the trafficking of MT-MMPs and such insights may lead to the development of novel drugs for therapy and chemoprevention against cancer.