Inter-Cellular Signaling of Invastion Receptors in the Tumor Microenvironment

描述(申请人提供)：G蛋白偶联受体(GPCRs)是最大的细胞表面受体家族，有1000多个成员，但只有少数GPCRs被发现是癌基因。其中，蛋白水解酶激活受体1(PAR1)已被确定为一个有效的癌基因，并赋予癌前乳腺细胞侵袭行为。在这项拨款中，我们通过刺激肿瘤环境中旁分泌和血管生成因子的产生来检验PAR1对癌症-宿主沟通至关重要的假设。我们最近发现宿主成纤维细胞衍生的基质金属蛋白酶-1(MMP1)是一种新的激动剂，可以在乳腺和卵巢肿瘤中切割和激活PAR1。基质细胞基质金属蛋白酶-1高表达，是乳腺癌、结直肠肿瘤和其他肿瘤预后不良的预测指标。靶向PAR1的新型细胞穿透性多肽阻断了基质金属蛋白酶-1下游的途径和受体抑制癌症的生长和侵袭。PAR1肽还可显著减少乳腺癌和卵巢癌的间质浸润和血管生成。我们将验证这一假设，即间质来源的PAR1在肿瘤生物学和刺激促进侵袭和血管生成的旁分泌因子方面可能与癌细胞来源的PAR1具有不同的作用。我们将利用两个癌症-基质共培养模型系统和体内癌症-基质共植入异种移植。PAR1(和PAR2)的激活在包括前列腺癌在内的多种细胞类型中产生IL-8、Gro-β、VEGF、IL-6和GM-CSF，但PAR1在癌症中的旁分泌通讯中的作用尚未被直接研究。我们假设，癌细胞上PAR1的激活会导致趋化因子的产生，从而刺激内皮细胞，从而增加血管生成和肿瘤生长。Pepducin技术将用于确定内皮趋化因子CXCR1和CXCR2受体在血管形成中的作用，并验证我们的初步数据，即PAR1和潜在的CXCR1/2肽可以在卵巢和乳腺移植动物模型中阻断血管生成并延长存活时间。最后，我们将继续我们最近发现的一种新的PAR1效应器BicD1，它可以抑制依赖PAR1的乳腺癌细胞的迁移和侵袭。下调BicD1的表达可显著延长丝裂原激活的激酶信号转导时间，提示BicD1可能调节乳腺癌细胞MAPK磷酸酶(MKP)的活性。我们将验证这样一种假设，即上调PAR1的表达或由MMP-1或凝血酶刺激PAR1调节BicD1和MKP的表达，并反过来控制MAPK依赖的乳腺癌的侵袭和增殖。这种方法有望显著改变我们对PAR1和CXCR1/2等受体之间的串扰在肿瘤生长和血管形成中的作用的理解。正如预期的那样，这些研究将开发这些侵袭性和趋化因子受体的第一批抑制剂，用于晚期乳腺癌和卵巢癌的潜在治疗。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors with more than 1000 members yet only a few GPCRs have been found to be oncogenes. Among these, the protease-activated receptor 1 (PAR1) has been identified as a potent oncogene and confers invasive behavior to pre-cancerous breast cells. In this grant we test the hypothesis that PAR1 is critical for cancer-host communication by stimulating production of paracrine and angiogenesis factors in the tumor environment. We recently identified host fibroblast-derived matrix metalloprotease-1 (MMP-1) as a novel agonist that can cleave and activate PAR1 in breast and ovarian tumors. MMP-1 is highly expressed in stromal cells and is a predictive marker for poor prognosis in breast, colorectal and other tumors. Targeting PAR1 with the novel cell-penetrating pepducins described here blocks the pathway downstream of MMP-1 and receptor inhibiting cancer growth and invasion. PAR1 pepducins also resulted in pronounced reduction of stromal infiltration and angiogenesis of breast and ovarian cancers. We will test the hypothesis that stromal-derived PAR1 may have a distinct role from cancer cell-derived PAR1 in tumor biology and stimulation of paracrine factors that promote invasion and angiogenesis. We will utilize two cancer-stromal co-culturing model systems and in vivo cancer- stromal coimplantation xenografts. Activation of PAR1 (and PAR2) have been shown to produce IL-8, Gro-?, VEGF, IL-6 and GM-CSF in a variety of cell types including prostate cancer, but the role of PAR1 in paracrine communication in cancer has not been directly addressed. We hypothesize that activation of PAR1 on cancer cells leads to production of chemokines which stimulate endothelial cells resulting in increased angiogenesis and tumor growth. Pepducin technology will be used to define the role of endothelial chemokine CXCR1 and CXCR2 receptors in blood vessel formation and validate our preliminary data that PAR1 and potentially CXCR1/2 pepducins can block angiogenesis and extend survival in ovarian and breast xenograft animal models. Lastly, we will follow-up our recent discovery of a novel PAR1-effector, BicD1, which acts as a suppressor of PAR1-dependent migration and invasion of breast cancer cells. Knock-down of BicD1 expression greatly prolongs mitogen-activated kinase signaling suggesting that BicD1 may regulate MAPK phosphatase (MKP) activity in breast cancer cells. We will test the hypothesis that upregulation of PAR1 expression or stimulation of PAR1 by MMP-1 or thrombin regulates BicD1 and MKP expression and that these downstream effectors in turn control MAPK-dependent invasion and proliferation of breast cancer. The pepducin approach has the prospect to significantly change our understanding of the role of cross-talk between receptors such PAR1 and CXCR1/2 in cancer growth and blood vessel formation. As envisioned, these studies will develop the first inhibitors of these invasion and chemokine receptors for the potential treatment of advanced breast and ovarian cancers.