PDGF D AND PROSTATE CANCER BONE METASTASIS

描述（由申请人提供）：本研究申请的目的是揭示前列腺癌产生的血小板衍生生长因子（PDGF）调节破骨细胞骨吸收和新骨生长的分子和细胞机制，为转移性沉积创造有利的微环境。越来越多的证据表明 PDGF 受体信号传导（尤其是 2-PDGFR）在前列腺癌进展和骨转移中的重要性。然而，PDGF B 最初被认为是 2-PDGFR 的唯一配体，但在前列腺癌临床样本中却很少被发现。重要的是，我们最近对人类前列腺癌标本进行的免疫组织化学分析表明，PDGF D（一种新发现的 PDGF 受体-β (2-PDGFR) 配体）表达的增加与格里森评分和肿瘤分期的增加相关。经典的 PDGF 配体 A 和 B 作为活性二聚体分泌，而 PDGF D 包含 N 端 CUB 结构域和 C 端 PDGF 结构域，后者作为潜在二聚体分泌。 PDGF 结构域需要 CUB 结构域的细胞外蛋白水解裂解才能刺激 2-PDGFR。最近，我们有了一个新的发现，即人类前列腺癌细胞利用丝氨酸蛋白酶 uPA 和 matriptase 将潜在的 PDGF D 自动激活为 PDGF D 的活性生长因子结构域。与之前的报道一致，matriptase 在前列腺癌中经常过度表达，尤其是在转移样本中，我们的初步数据显示人类前列腺癌中 matriptase 表达增加。在胫骨注射模型中，PDGF D 显着增强 LNCaP 肿瘤的肿瘤摄取和生长速率，并增加溶骨和成骨细胞反应。此外，我们的体外研究揭示了 PDGF D 在独立于 RANKL/RANK 信号轴的破骨细胞分化调节以及涉及同源蛋白表达调节的成骨细胞分化中的令人兴奋的新作用。综上所述，我们假设前列腺癌产生的 PDGF D 由跨膜丝氨酸蛋白酶基质酶激活，诱导骨基质细胞中的旁分泌细胞信号传导，介导对骨微环境中前列腺癌生长至关重要的肿瘤-基质相互作用。为了检验这一假设，我们建议 (1) 研究 matriptase 介导的 PDGF D 蛋白水解过程； (2) 利用RAW264.7和MC3T3-E1细胞系体外模型探讨PDGF D诱导破骨细胞和成骨细胞分化的分子机制； (3)研究PDGF D及其功能调节蛋白基质酶在动物骨基质反应和前列腺肿瘤生长中的作用。考虑到 2-PDGFR 和 matriptase 在骨转移和原发性前列腺癌标本中均高度上调，完成拟定的 2-PDGFR 特异性激活剂 PDGF D 与 matriptase 之间相互作用的研究将有助于我们了解前列腺癌进展的分子基础。此外，拟议的研究可能提供具有治疗价值的重要信息，特别是考虑到 VEGF/PDGF 抑制剂目前正处于癌症患者的临床试验中。

DESCRIPTION (provided by applicant): The objective of this research application is to unveil the molecular and cellular mechanisms by which prostate carcinoma-produced platelet-derived growth factor (PDGF) regulates osteoclastic bone resorption and new bone growth, creating a favorable microenvironment for metastatic deposit. Increasing evidence indicate the significance of PDGF receptor signaling, especially 2-PDGFR, in prostate cancer progression and bone metastasis. PDGF B, however, originally thought to be the sole ligand for 2-PDGFR, has rarely been found in prostate cancer clinical samples. Importantly, our recent immunohistochemical analysis of human prostate carcinoma specimens showed that increased expression of PDGF D, a newly discovered ligand for PDGF receptor-beta (2-PDGFR), is associated with increased Gleason scores and tumor stages. Whereas the classic PDGF ligands A and B are secreted as active dimers, PDGF D contains an N-terminal CUB domain and a C terminal PDGF domain which is secreted as a latent dimer. Extracellular proteolytic cleavage of the CUB domain is required for the PDGF domain to stimulate 2-PDGFR. Recently, we made a novel finding that human prostate carcinoma cells auto-activate latent PDGF D into an active growth factor domain of PDGF D utilizing the serine proteases uPA and matriptase. Consistent with previous reports that matriptase is frequently overexpressed in prostate cancer, especially in metastatic samples, our preliminary data showed increased matriptase expression in human prostate cancer. In a tibiae-injection model, PDGF D significantly enhanced tumor take and growth rate of LNCaP tumors with increased osteolytic and osteoblastic responses. Additionally, our in vitro study unveiled exciting new roles of PDGF D in the regulation of osteoclast differentiation independent of the RANKL/RANK signaling axis as well as osteoblast differentiation involving modulation of homeoprotein expression. Taken together, we hypothesize that prostate carcinoma-produced PDGF D, activated by the transmembrane serine protease matriptase, induces paracrine cell signaling in bone stromal cells, mediating tumor-stromal interactions critical for prostate carcinoma growth in the bone microenvironment. To test this hypothesis, we propose (1) To investigate matriptase-mediated proteolytic processing of PDGF D; (2) To investigate the molecular mechanisms by which PDGF D induces osteoclast and osteoblast differentiation using in vitro models of RAW264.7 and MC3T3-E1 cell lines; and (3) To investigate the roles of PDGF D and its functional regulator matriptase in bone stromal responses and prostate tumor growth in animals. Considering that 2-PDGFR as well as matriptase is highly upregulated in both bone metastases and primary prostate cancer specimens, the completion of the proposed study of interplay between PDGF D, a specific activator of 2-PDGFR, and matriptase will help us in understanding the molecular basis for prostate cancer progression. In addition, the proposed study may provide important information with therapeutic value, especially given that VEGF/PDGF inhibitors are currently in clinical trials for cancer patients.