Matrix remodeling in tumor growth and metastasis in a murine breast cancer model

描述（由申请人提供）：我的研究旨在了解成纤维细胞激活蛋白（FAP）的致瘤作用，FAP是一种具有二肽基肽酶（DPP）和内肽酶/胶原酶活性的膜结合丝氨酸蛋白酶。FAP是独特的，因为它在90%的人类上皮源性癌症的癌相关成纤维细胞（CAFs）中表达，但在正常或转化（恶性/肿瘤）上皮细胞中不表达。重要的是，我们实验室（J.Tchou和E. Puri）未发表的工作表明，FAP可以作为人类乳腺癌中强大的泛CAF标记物，其选择性和特异性优于其他CAF标记物。CAFs在ECM重塑、血管生成和基质形成中发挥重要作用，是肿瘤生长和转移的重要参与者。大多数关于FAP活性的报道都描述了FAP表达与不良预后之间的相关性，这使得FAP抑制成为癌症治疗的一个有吸引力的潜在靶点。我们的实验室最近证明，FAP促进突变型K-ras驱动肺癌的本地小鼠模型和结肠癌和胰腺癌的同基因移植模型的肿瘤发生，并且在缺乏FAP的小鼠中，肿瘤生长的减少与无序胶原积累的增加、整合素信号的异常以及间质增生和血管生成的减少有关。然而，FAP促进肿瘤发生的机制尚未明确，其在任何肿瘤类型转移中的作用尚未被研究。我们的初步数据表明，FAP介导了由胶原酶介导的裂解(例如；金属蛋白酶- 1)。该建议的一个中心假设是FAP通过其蛋白酶活性促进肿瘤发生，在ECM重塑中发挥关键作用。具体来说，我假设FAP通过靶向胶原蛋白的降解和转换，调节肿瘤细胞增殖、基质形成、血管生成和转移的基质依赖信号。为了验证这些假设，我将比较MMTV-Neu驱动的自体乳腺肿瘤在FAP野生型小鼠、FAP缺失小鼠（FAP敲入蛋白表达荧光素酶代替FAP基因）和表达酶促死亡FAP突变体（FAPS624A）或FAP突变体（fap657s）中发生和转移的情况，后者保留了DPP活性，但缺乏内肽酶/胶原酶活性。因此，通过使用这些酶缺陷的FAP小鼠，本研究还将研究FAP对ECM重塑的作用机制。这些新的FAP小鼠品系已在实验室生产，目前正在繁殖，而MMTV-Neu小鼠已商品化。！

DESCRIPTION (provided by applicant): My research aims to understand the tumorigenic role of fibroblast activation protein (FAP), a membrane-bound serine protease with dipeptidyl peptidase (DPP) and endopeptidase/collagenase activities. FAP is unique, in that it is expressed in cancer associated fibroblasts (CAFs) in 90% of all human epithelial derived cancers, but not in normal or transformed (malignant/neoplastic) epithelial cells. Importantly, unpublished work from our lab (J.Tchou and E. Puri) shows that FAP can be used as a robust pan-CAF marker in human breast cancer, it being superior to other CAF markers in its selectivity and specificity. CAFs play roles in ECM remodeling, angiogenesis and stromagenesis, and are important players in tumor growth and metastasis. A wide majority of reports on FAP activity describe correlations between FAP expression and poor prognosis, making FAP inhibition an appealing potential target for cancer therapeutics. Our lab recently demonstrated that FAP promotes tumorigenesis in an autochthonous mouse model of mutant K-ras- driven lung cancer and syngeneic transplant models of colon and pancreatic cancer and that the reduction in tumor growth in FAP-deficient mice was associated with enhanced accumulation of disorganized collagen, aberrant integrin signaling and reduced stromagenesis and angiogenesis. However, the mechanisms by which FAP promotes tumorigenesis are yet to be defined and its role in metastasis has not yet been investigated in any tumor type. Our preliminary data indicate that FAP mediates the proteolytic processing of the characteristic 3/4 and 1/4 fragments of collagen generated by collagenase mediated cleavage (eg. MMP-1). A central hypothesis of this proposal is that FAP promotes tumorigenesis through its protease activity by playing a critical role in ECM remodeling. Specifically, I hypothesize that by targeting collagen for degradation and turnover, FAP regulates matrix-dependent signaling of tumor cell proliferation, stromagenesis, angiogenesis and metastasis. To test these hypotheses I will compare tumorigenesis and metastasis of MMTV-Neu driven autochthonous mammary tumors in FAP wild-type mice, FAP-null mice (FAP-knock-ins expressing luciferase in place of the FAP gene) and knock-in mice expressing an enzymatic dead FAP mutant (FAPS624A) or a FAP mutant (FAPA657S) that retains DPP activity but lacks endopeptidase/collagenase activity. Therefore, by making use of these enzymatically defective FAP mice, this study will also examine the mechanism by which FAP acts on ECM remodeling. These novel FAP mouse strains have been produced in the lab and are currently breeding, while the MMTV-Neu mouse is commercially available. ! PUBLIC HEALTH RELEVANCE: Breast cancer is the second leading cause of cancer deaths in women today. The costs of cancer are a financial and mental toll on people diagnosed with cancer, their families, and society as a whole. In 2005 the burden of breast cancer in the U.S. was $12.096 billion in lost time and economic productivity (NCI, 2009/2010), therefore it is imperative to identify new molecular targets for early prognosis, treatment and eradication of this devastating disease. This research aims to better understand the causes and mechanisms underlying tumor initiation, progression and metastasis by studying the relationship between mammary tumor cells and their microenvironment. The research proposed will define the protumorigenic activity of a tumor stromal cell protease and extracellular matrix remodeling in an autochthonous breast cancer model. Results of the proposed studies will reveal novel mechanisms through which this protease regulates tumor development and therefore its value as a target for cancer therapeutics. Further, our studies may lead to an early breast cancer diagnostic biomarker that could be used in the clinic to save time, resources and most importantly, life. !