Apoptosis of Invasive Breast Cancer Cells by Inhibitors of Intracellular uPA

描述(由申请人提供)：uPA-uPAR途径成分的过度表达是许多不同类型癌症患者疾病复发和总存活率的强烈预后。由于它直接参与肿瘤的恶性转化，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活性的治疗剂产生了极大的兴趣。我们最近意想不到的观察表明，uPA在细胞内被酶激活。已经开发出一系列基于阿米洛利的化合物，能够抑制乳腺和脑肿瘤细胞内或细胞外的uPA活性。令人惊讶的是，被排除在细胞内部、只作用于表面uPA的药物形式具有细胞抑制作用，而它们的同系物能够穿过质膜作用于细胞内uPA，诱导侵袭性肿瘤细胞凋亡，但不影响正常组织。这些观察指向了一种新的概念，即恶性肿瘤细胞可以依赖细胞内uPA生存。此外，由于在正常组织的细胞内没有观察到uPA的激活，这些观察表明细胞内的uPA是恶性乳腺肿瘤细胞的致命弱点，可以作为治疗干预的靶点。因此，有人认为，基于阿米洛利的细胞内uPA抑制剂通过一种新的细胞内机制破坏uPA信号系统的自分泌调节，从而导致增殖和浸润性乳腺癌细胞的凋亡。此外，将这些化合物应用于复发乳腺癌的转基因小鼠模型，应该会抑制肿瘤的转移和复发。目的1：分析药物诱导乳腺癌细胞凋亡的分子机制。目的：研究uPA抑制剂对裸鼠乳腺癌模型肿瘤发生、生长、转移和复发的影响。 公共卫生相关性：开发新的和有效的乳腺癌治疗方法的主要挑战是确定可用药靶点，这些靶点在乳腺肿瘤的恶性肿瘤中起主要作用，但在其他生理过程中起次要作用。对这些靶点的治疗抑制将特异性地挫败肿瘤细胞，同时产生最小的患者副作用。由于细胞内uPA的激活是恶性肿瘤细胞独有的，而且肿瘤细胞的存活似乎依赖于这一机制，因此细胞内uPA的抑制剂在治疗最具侵袭性的乳腺癌方面提供了巨大的潜力。

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.