Apoptosis of Invasive Breast Cancer Cells by Inhibitors of Intracellular uPA

描述（由申请人提供）：uPA-uPAR通路组分的过表达是许多不同类型癌症患者疾病复发和总体生存的强预后。由于uPA直接参与肿瘤的恶性程度，人们对了解乳腺肿瘤细胞利用uPA系统促进肿瘤复发和转移的机制以及开发能够干扰uPA活性的治疗药物产生了相当大的兴趣。我们最近和意想不到的观察表明，uPA成为酶激活细胞内。已经开发了一系列的十二种基于阿米洛利的化合物，其能够抑制乳腺和脑肿瘤细胞中的细胞内或细胞外uPA活性。令人惊讶的是，被排除在细胞内部并仅作用于表面uPA的药物形式是细胞抑制性的，而它们的能够穿过质膜并作用于细胞内uPA的同类物诱导侵袭性肿瘤细胞的凋亡，但不影响正常组织。这些观察结果指出了恶性肿瘤细胞可以依赖于细胞内uPA存活的新概念。此外，由于在正常组织中的细胞内未观察到细胞内uPA活化，因此这些观察结果表明细胞内uPA是恶性乳腺肿瘤细胞的阿喀琉斯之踵，其可被靶向用于治疗干预。因此，提出细胞渗透性的基于阿米洛利的uPA抑制剂通过破坏uPA信号系统的自分泌调节的新的细胞内机制引起增殖性和浸润性乳腺癌细胞的凋亡。此外，将这些化合物施用至复发性乳腺癌的转基因小鼠模型应导致肿瘤转移和复发的抑制。目的1：分析药物诱导乳腺癌细胞凋亡的分子机制。目的2：确定uPA抑制剂对非转基因小鼠乳腺癌模型中肿瘤发展、生长、转移和复发的影响。 公共卫生相关性：开发新型有效的乳腺癌治疗方法的主要挑战是确定在乳腺肿瘤恶性程度中起主要作用但在其他生理过程中起次要作用的可药用靶标。对这些靶点的治疗性抑制将特异性地阻止肿瘤细胞，同时引起最小的患者副作用。由于细胞内uPA激活是恶性肿瘤细胞所特有的，并且由于肿瘤细胞存活似乎依赖于这种机制，因此细胞内uPA抑制剂在治疗最具侵袭性形式的乳腺癌方面具有巨大的潜力。

DESCRIPTION (provided by applicant): Overexpression of uPA-uPAR pathway components is strongly prognostic of disease recurrence and overall survival of patients with many different kinds of cancer. Because of its direct involvement in tumor malignancy, considerable interest has developed in understanding the mechanisms by which breast tumor cells exploit the uPA system to promote tumor recurrence and metastasis, and in the development of therapeutic agents that can interfere with uPA activity. Our recent and unexpected observations suggest that uPA becomes enzymatically activated intracellularly. A series of hwo dozen amiloride-based compounds have been developed capable of inhibiting intracellular or extracellular uPA activity in breast and brain tumor cells. Surprisingly, forms of the drugs that are excluded from the cell interior and act only on surface uPA are cytostatic, while their congeners capable of crossing the plasma membrane and acting on intracellular uPA induce the apoptosis of invasive tumor cells but do not affect normal tissue. These observations point to the novel notion that malignant tumor cells can become reliant on intracellular uPA for their survival. Moreover, since intracellular uPA activation is not observed within cells in normal tissue, these observations suggest that intracellular uPA is an Achilles' heel of malignant breast tumor cells that can be targeted for therapeutic intervention. Therefore, it is proposed that cell permeant, amiloride-based uPA inhibitors cause apoptosis of proliferative and infiltrative breast cancer cells by a novel intracellular mechanism that disrupts autocrine regulation ofthe uPA signaling system. Furthermore, the administration of these compounds to a transgenic mouse model of recurrent breast cancer should result in a suppression of tumor metastasis and recurrence. Aim 1: Analyze molecular mechanisms underlying drug-induced apoptosis in cultured breast cancer cells. Aim 2: Determine the impact of uPA inhibitors on tumor development, growth, metastasis and recurrence in atransgenic mouse model of breast cancer. Public Health Relevance: The major challenge in developing novel and effective treatments for breast cancer is the identification of druggable targets that play major roles in the malignancy of breast tumors, but play minor roles in other physiological processes. Therapeutic inhibition of such targets will specifically thwart tumor cells while eliciting minimal patient side effects. Since intracellular uPA activation is unique to malignant tumor cells, and because tumor cell survival appears to be dependent on this mechanism, inhibitors of intracellular uPA offer tremendous potential in the treatment of the most aggressive forms of breast cancer.