Quantitative proteomic identification of novel, bioactive substrates of matrix metalloproteinase (MMP)-11 and system-wide identification of new proteases involved in breast cancer progression and metastasis

蛋白质水解酶如基质金属蛋白酶（MMPs）并不像它的名字所暗示的那样仅仅是切割细胞外基质，而是通过对生物活性蛋白和信号分子的不可逆加工来调节肿瘤细胞的血管生成、生长、侵袭、转移和表型进化。MMP家族的一名成员MMP-11首次在浸润性乳腺癌的反应性基质中被发现，此后在200多篇论文中发现，MMP-11与乳腺癌患者预后不良密切相关。但是，尽管有广泛的表征，MMP-11没有已知的天然底物来解释其在乳腺癌中的关键活性。因此，我将采用Christopher Overall教授实验室开发的新型全系统定量蛋白质组学技术来鉴定和表征乳腺癌发生过程中MMP-11的生物活性底物。为了了解蛋白酶在乳腺癌发展和转移中的作用，不仅要确定单个蛋白酶的底物谱，而且要确定和表征参与这些过程的新蛋白酶。因此，在第二个项目中，我将确定蛋白酶降解，即所有蛋白酶在小鼠乳腺癌模型中的表达水平。为此，将使用在宿主实验室设计和制造的称为CLIP-CHIPTM的寡核苷酸微阵列，该芯片包含所有已知的人和小鼠蛋白酶、无活性同源物和抑制剂。在癌症或反应性基质组织中高度表达的蛋白酶将被分析其被称为底物降解物的底物光谱。这些实验将进一步深入了解包括基质金属蛋白酶在内的蛋白酶在癌症进展中的作用，特别是它们如何修饰药物靶底物的活性，从而有助于乳腺癌药物靶标验证。

Rather than just cleaving extracellular matrix as their name suggests, proteolytic enzymes such as matrix metalloproteinases (MMPs) modulate angiogenesis, growth, invasion, metastasis, and phenotypic evolution of cancer cells by the irreversible processing of bioactive proteins and signaling molecules. One member of the MMP family, MMP-11, was first discovered in the reactive stroma of invasive breast carcinoma, and since then in over 200 publications is very strongly correlated with poor patient outcome in breast carcinoma. But despite extensive characterization, MMP-11 has no known natural substrates to account for its pivotal activities in breast cancer. Therefore, I will employ novel, systemwide quantitative proteomic technologies developed in the laboratory of Prof. Christopher Overall to identify and characterize bioactive substrates of MMP-11 in breast carcinogenesis. To understand the role of proteases in breast cancer development and metastasis it is not only important to define the substrate spectrum of an individual protease but also to identify and characterize new proteases involved in these processes. Therefore, in a second project I will determine the protease degradome, that is the expression levels of all proteases in a murine breast cancer model. For this purpose, an oligonucleotide microarray termed the CLIP-CHIPTM comprising all known human and murine proteases, inactive homologues and inhibitors that was designed and made in the host laboratory will be used. Interesting proteases that are highly expressed in cancer or reactive stromal tissues will be analyzed for their spectrum of substrates termed the substrate degradome. These experiments will provide further insight into the role of proteases including matrix metalloproteinases in cancer progression, especially how they modify the activity of drug target substrates and so contribute to drug target validation in breast cancer.