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活化蛋白（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%的所有人类上皮来源的癌症中在癌症相关成纤维细胞（CAF）中表达，但不在正常或转化（恶性/肿瘤）上皮细胞中表达。重要的是，我们实验室未发表的工作（J.Tchou和E. Puri）显示FAP可用作人乳腺癌中的稳健的泛CAF标记物，其在选择性和特异性方面上级其它CAF标记物。CAFs在ECM重塑、血管生成和基质形成中发挥作用，并且在肿瘤生长和转移中起重要作用。关于FAP活性的大多数报道描述了FAP表达与不良预后之间的相关性，使得FAP抑制成为癌症治疗的有吸引力的潜在靶标。我们的实验室最近证明，FAP促进突变K-ras驱动的肺癌的自体小鼠模型和结肠癌和胰腺癌的同基因移植模型中的肿瘤发生，并且FAP缺陷小鼠中肿瘤生长的减少与无序胶原蛋白的积累增加、异常整合素信号传导以及基质形成和血管生成减少有关。然而，FAP促进肿瘤发生的机制尚未确定，其在转移中的作用尚未在任何肿瘤类型中研究。我们的初步数据表明，FAP介导的胶原蛋白酶介导的切割产生的胶原蛋白的特征性3/4和1/4片段的蛋白水解加工（例如，MMP-1）。该提议的中心假设是FAP通过其蛋白酶活性在ECM重塑中发挥关键作用来促进肿瘤发生。具体来说，我假设，通过靶向胶原蛋白的降解和周转，FAP调节肿瘤细胞增殖，基质形成，血管生成和转移的基质依赖性信号。为了检验这些假设，我将比较FAP野生型小鼠、FAP敲除小鼠（表达荧光素酶代替FAP基因的FAP敲入）和表达酶促死亡FAP突变体（FAPS 624 A）或保留DPP活性但缺乏内肽酶/胶原酶活性的FAP突变体（FAPA 657 S）的敲入小鼠中MMTV-Neu驱动的自体乳腺肿瘤的肿瘤发生和转移。因此，通过利用这些酶缺陷的FAP小鼠，本研究还将研究FAP作用于ECM重塑的机制。这些新的FAP小鼠品系已经在实验室中产生，目前正在繁殖，而MMTV-Neu小鼠是市售的。! 公共卫生相关性：乳腺癌是当今女性癌症死亡的第二大原因。癌症的成本对被诊断患有癌症的人、他们的家庭和整个社会来说都是经济和精神上的损失。2005年，美国乳腺癌的负担是120.96亿美元的损失时间和经济生产力（NCI，2009/2010），因此，必须确定新的分子靶点，用于早期预后，治疗和根除这种毁灭性疾病。 本研究旨在通过研究乳腺肿瘤细胞与其微环境之间的关系，更好地了解肿瘤发生、发展和转移的原因和机制。这项研究将确定肿瘤基质细胞蛋白酶和细胞外基质重塑在原位乳腺癌模型中的促肿瘤活性。拟议研究的结果将揭示这种蛋白酶调节肿瘤发展的新机制，从而揭示其作为癌症治疗靶点的价值。此外，我们的研究可能会导致早期乳腺癌诊断生物标志物，可用于临床，以节省时间，资源，最重要的是，生命。!

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 Importantly, unpublished work from our lab (J.Tchou and E. Puri) shows that FAP can be used as a robust pan-CAF marker in human breast cancer, it being superior to other CAF markers in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. !