Suppression of Prostate Tumor Growth and Metastasis by Inhibition of Vimentin

 描述（由申请人提供）：晚期前列腺肿瘤通常转移至肺、骨和其他重要组织，对常规治疗有抵抗力。前列腺凋亡反应-4（Par-4）是一种肿瘤抑制因子，可导致治疗抵抗性前列腺癌细胞凋亡。尽管细胞分泌的Par-4的基线水平不足以诱导细胞凋亡，但可以诱导正常细胞产生以旁分泌方式靶向癌细胞的Par-4蛋白的稳健分泌。因此，分泌的Par-4在体内是全身可用的，并与癌细胞中的其细胞表面受体GRP 78结合以触发原发性和转移性肿瘤中的细胞凋亡。重要的是，分泌的Par-4诱导癌症特异性细胞凋亡，但它不诱导正常细胞中的细胞凋亡，因为GRP 78仅在癌细胞的表面上表达，而不在正常细胞上表达。因此，有效和选择性地诱导Par-4分泌而不杀死正常细胞的“小分子”药物将代表重要的治疗进展。在初步研究中，我们合成并筛选了新开发的3-芳基喹啉类以及相关的喹诺酮类和喹硫酮类，用于从正常小鼠和人成纤维细胞和上皮细胞中分泌Par-4蛋白。该筛选导致鉴定出2-氨基-7-（N，N-二甲基氨基）-3-（2 '-氟苯基）喹啉（称为“Arylquin-1”）作为一类新的药剂的成员，所述药剂以纳摩尔无毒浓度触发Par-4从正常细胞的稳健分泌。正如预期的那样，从这些正常细胞分泌的Par-4在不同的前列腺和肺癌细胞培养物中诱导旁分泌凋亡，但在正常细胞培养物中不诱导。使用 作为Arylquin-1的生物素化的生物活性形式，我们鉴定了波形蛋白，一种与运动、侵袭、EMT和转移相关的蛋白，作为Arylquin-1的分子靶点，进而产生Par-4分泌。在这项研究中，我们将确定Arylquin-1对正常细胞Par-4分泌和前列腺癌细胞培养中的细胞凋亡以及体内前列腺癌模型中肿瘤生长的影响。计算动力学表明，Arylquin-1与波形蛋白上的疏水口袋形成稳定的相互作用。因此，我们将确定相互结合的Par-4和波形蛋白的结构域，以阐明波形蛋白抑制Par-4分泌和Arylquin-1释放的潜在机制。与Arylquin-1与波形蛋白的相互作用一致，我们的初步研究还表明，Arylquin-1通过非肿瘤依赖性机制抑制前列腺癌细胞的运动和侵袭。因此，我们将研究Arylquin-1是否通过波形蛋白依赖性机制抑制前列腺癌小鼠模型中细胞培养和转移中的运动性、侵袭性和EMT。开发选择性促进正常细胞Par-4分泌、诱导癌细胞凋亡和阻止前列腺癌转移过程的药物将具有非凡的学术和治疗价值。

 DESCRIPTION (provided by applicant): Advanced prostate tumors that often metastasize to the lung, bone, and other vital tissues, are resistant to conventional therapy. Prostate apoptosis response-4 (Par-4) is a tumor suppressor that causes apoptosis in therapy-resistant prostate cancer cells. Although baseline levels of Par-4 secreted by cells are inadequate to induce apoptosis, normal cells can be induced to produce robust secretion of Par-4 protein that targets cancer cells in a paracrine manner. Accordingly, secreted Par-4 is systemically available in vivo and binds to its cell surface receptor GRP78 in cancer cells to trigger apoptosis in primary and metastatic tumors. Importantly, secreted Par-4 induces cancer-specific apoptosis, but it does not induce apoptosis in normal cells as GRP78 is expressed only on the surface of cancer cells, not on normal cells. "Small-molecule" drugs that efficiently and selectively induce Par-4 secretion without killing the normal cells would, therefore, represent an important therapeutic advance. In preliminary studies, we synthesized and screened newly developed 3-arylquinolines as well as related quinolones and quinothiolones for the secretion of Par-4 protein from normal mouse and human fibroblast and epithelial cells. This screening led to the identification of 2-amino-7-(N,N-dimethylamino)-3-(2'-fluorophenyl)quinoline (called "Arylquin-1") as a member of a new class of agents that trigger robust secretion of Par-4 from normal cells at nanomolar, non-toxic concentrations. As expected, Par-4 secreted from these normal cells induced paracrine apoptosis in diverse prostate and lung cancer cell cultures, but not in normal cell cultures. Using a biotinylated, biologically active version of Arylquin-1, we identified vimentin, a protein linkedto motility, invasion, EMT and metastasis, as the molecular target of Arylquin-1 that in turn produces Par-4 secretion. In this study, we will determine the effect of Arylquin-1 on Par-4 secretion from normal cells and apoptosis in prostate cancer cell culture and in vivo, on tumor growth in prostate cancer models. Computational dynamics revealed that Arylquin-1 forms a stable interaction with a hydrophobic pocket on vimentin. We will, therefore, determine the domains of Par-4 and vimentin that bind to each other to elucidate the mechanism underlying inhibition of Par-4 secretion by vimentin and its release by Arylquin-1. Consistent with the interaction of Arylquin-1 with vimentin, our preliminary studies also indicated that Arylquin-1 inhibits motility and invasion in prostate cancer cells by an apoptosis-independent mechanism. We will, therefore, study whether Arylquin-1 inhibits motility, invasion and EMT in cell culture and metastasis in mouse models of prostate cancer via vimentin-dependent mechanisms. Developing agents that selectively promote Par-4 secretion from normal cells, induce apoptosis in cancer cells and halt metastatic processes in prostate cancer would be of extraordinary academic and therapeutic value.