Inter-Cellular Signaling of Invastion Receptors in the Tumor Microenvironment

描述（由申请人提供）：G蛋白偶联受体（GPCR）是细胞表面受体的最大家族，有超过1000个成员，但只有少数GPCR被发现是致癌基因。 其中，蛋白酶激活受体1（PAR 1）已被确定为一种有效的致癌基因，并赋予癌前乳腺细胞的侵袭行为。 在这项研究中，我们验证了PAR 1通过刺激肿瘤环境中旁分泌和血管生成因子的产生对肿瘤-宿主通讯至关重要的假设。 我们最近发现宿主成纤维细胞来源的基质金属蛋白酶-1（MMP-1）作为一种新的激动剂，可以切割和激活PAR 1在乳腺和卵巢肿瘤。 MMP-1在基质细胞中高度表达，是乳腺、结直肠和其他肿瘤预后不良的预测标志物。 用本文所述的新型细胞穿透肽蛋白靶向PAR 1阻断MMP-1和受体下游的通路，抑制癌症生长和侵袭。 PAR 1肽蛋白还导致乳腺癌和卵巢癌的基质浸润和血管生成的显著减少。 我们将检验基质来源的PAR 1在肿瘤生物学和刺激促进侵袭和血管生成的旁分泌因子中可能具有与癌细胞来源的PAR 1不同的作用的假设。 我们将利用两种癌症-基质共培养模型系统和体内癌症-基质共植入异种移植物。 PAR 1（和PAR 2）的激活已显示可产生IL-8、Gro-？、VEGF、IL-6和GM-CSF在包括前列腺癌在内的多种细胞类型中的作用，但PAR 1在癌症中的旁分泌通讯中的作用尚未得到直接解决。 我们假设癌细胞上PAR 1的激活导致产生趋化因子，其刺激内皮细胞，导致血管生成和肿瘤生长增加。 Pepducin技术将用于确定内皮趋化因子CXCR 1和CXCR 2受体在血管形成中的作用，并验证我们的初步数据，即PAR 1和潜在的CXCR 1/2 pepducins可以阻断血管生成并延长卵巢和乳腺异种移植动物模型的存活期。 最后，我们将跟进我们最近发现的一种新的PAR 1效应子BicD 1，它可以抑制PAR 1依赖的乳腺癌细胞的迁移和侵袭。 BicD 1表达的敲低极大地抑制了丝裂原活化激酶信号传导，表明BicD 1可能调节乳腺癌细胞中MAPK磷酸酶（MKP）的活性。 我们将测试的假设，上调PAR 1的表达或刺激PAR 1的MMP-1或凝血酶调节BicD 1和MKP的表达，这些下游效应反过来控制MAPK依赖性的乳腺癌的侵袭和增殖。 pepducin方法有前景显著改变我们对PAR 1和CXCR 1/2等受体之间的串扰在癌症生长和血管形成中的作用的理解。 正如所设想的那样，这些研究将开发这些侵袭和趋化因子受体的第一种抑制剂，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.