Serine Protease Modulation of ErbB Receptors

描述（由申请人提供）：Her 2 +/uPA+乳腺癌在临床上是最具侵袭性的，比Her 2 +/uPA-、Her 2-/uPA+和Her 2-/uPA-亚型更具侵袭性。通过尿激酶型纤溶酶原激活剂（uPA）和一种新的细胞外膜丝氨酸蛋白酶网络的胞外结构域/胞外结构域（ECD）脱落对Her 2/neu/ErbB 2受体酪氨酸激酶信号传导的蛋白水解调节被假设在Her 2 +/uPA+乳腺癌的侵袭性行为中以及在使用单克隆抗体或小分子抗体的抗Her 2疗法的抗性中发挥机制作用。分子酪氨酸激酶抑制剂药物。因此，在Her 2 +/uPA+乳腺癌中同时靶向Her 2和uPA可能是治疗这些癌症的更有效策略;并且可能是所有Her 2+乳腺癌。这一假设将在本项目中得到检验。有了以下具体目标，我们将创建Her 2 +/uPA+乳腺癌的新型细胞系模型，并通过同时靶向Her 2和uPA来测试新型治疗策略的有效性。我们还将研究与乳腺癌和许多其他癌症相关的几种细胞外膜丝氨酸蛋白酶的新功能作用。具体目标1。建立Her 2 +/uPA+人乳腺癌细胞系，用于评估增强的侵袭性。我们将通过在Her 2扩增的SK-BR-3和BT-474细胞系中过表达uPA/uPAR或在uPA+但Her 2阴性（未扩增）的MDA-MB-231细胞系中过表达Her 2来建立Her 2 +/uPA+人乳腺癌细胞系;并在增殖、迁移、侵袭和锚定非依赖性生长中进行肿瘤侵袭性的体外评价。具体目标2。用赫赛汀、拉帕替尼和WX-UK 1处理Her 2 +/uPA+人乳腺癌细胞系，以评价同时靶向Her 2和uPA的有效性。利用我们的工程化Her 2 +/uPA+模型细胞系，将解决以下临床相关问题：1）。Her 2 +/uPA+乳腺癌是否对赫赛汀或甚至拉帕替尼呈现新生耐药性，以及用选择性uPA抑制剂WX-UK 1抑制uPA是否克服了耐药性; 2）Her 2 +/uPA+乳腺癌是否更积极地获得对抗Her 2疗法的耐药性，以及用WX-UK 1抑制uPA是否延迟了耐药性的获得。具体目标3。确定间质蛋白酶、前列腺素和uPA对Her 2 ECD的特异性作用。我们将在共转染实验中使用FT-293细胞系，以确定uPA是否是Her 2 ECD脱落酶，以及纤溶酶是否在Her 2 ECD脱落中起作用;纯化蛋白酶切割的Her 2以绘制切割位点;并确定蛋白酶切割的Her 2是否对赫赛汀诱导的周转有反应。我们将使用SK-BR-3细胞系来确定乳腺癌细胞中与uPA诱导相关的Her 2 ECD脱落是否也涉及或需要通过使用特异性抑制剂的间质蛋白酶和前列腺素。 公共卫生相关性：对靶向Her 2/neu/ErbB 2的化疗药物的耐药性限制了这些药物在选定的乳腺癌患者群体中的成功，并可能使获得性耐药的患者无法治疗。蛋白酶对Her 2细胞外结构域/胞外域（ECD）的蛋白水解脱落是这种抗性的机制。我们的初步研究已经确定了参与Her 2 ECD脱落的新的候选蛋白酶，拟议的研究将揭示是否应考虑使用特定的蛋白酶抑制剂进行联合治疗，以减少或预防抗Her 2药物的耐药性。

DESCRIPTION (provided by applicant): The Her2+/uPA+ breast cancers are clinically the most aggressive, more so than the Her2+/uPA-, Her2-/uPA+, and Her2-/uPA- subtypes. Proteolytic modulation of Her2/neu/ErbB2 receptor tyrosine kinase signaling via extracellular domain/ectodomain (ECD) shedding by the urokinase-type plasminogen activator (uPA) and a novel network of extracellular membrane serine proteases is hypothesized to play a mechanistic role in the aggressive behaviors of the Her2+/uPA+ breast cancers and in resistance to anti-Her2 therapies using monoclonal antibody or small-molecule tyrosine kinase inhibitor drugs. Targeting Her2 and uPA simultaneously in Her2+/uPA+ breast cancers could therefore be a more effective strategy of treating these cancers; and potentially all Her2+ breast cancers. This hypothesis will be tested in this project. With the following specific aims, we will be creating novel cell-line models of Her2+/uPA+ breast cancer and testing the effectiveness of a novel treatment strategy by targeting Her2 and uPA simultaneously. We will also be investigating novel functional roles of several extracellular membrane serine proteases associated with breast cancer, and many other cancers. Specific Aim 1. To establish Her2+/uPA+ human breast cancer cell lines for evaluation of enhanced aggressiveness. We will establish Her2+/uPA+ human breast cancer cell lines by over-expressing uPA/uPAR in the Her2-amplified SK-BR-3 and BT-474 cell lines or over-expressing Her2 in the uPA+ but Her2-negative (unamplified) MDA-MB-231 cell line; and perform in vitro evaluations of tumor aggressiveness in proliferation, migration, invasion, and anchorage-independent growth. Specific Aim 2. To treat Her2+/uPA+ human breast cancer cell lines with Herceptin, lapatinib, and WX-UK1 for evaluation of effectiveness of targeting Her2 and uPA together. With our engineered Her2+/uPA+ model cell lines, the following clinically relevant questions will be addressed: 1). whether Her2+/uPA+ breast cancers present de novo drug resistance to Herceptin or even lapatinib and whether uPA inhibition with a selective uPA inhibitor WX-UK1 overcomes the resistance; 2) whether Her2+/uPA+ breast cancers acquire resistance to anti- Her2 therapies more aggressively and whether uPA inhibition with WX-UK1 delays the acquisition of resistance. Specific Aim 3. To determine the specific actions of matriptase, prostasin, and uPA on Her2 ECD. We will use the FT-293 cell line in co-transfection experiments to determine if uPA is a Her2 ECD sheddase and if plasmin plays a role in Her2 ECD shedding; to purify protease-cleaved Her2 for mapping the cleavage sites; and to determine if the protease-cleaved Her2 is responsive to Herceptin-induced turnover. We will use the SK-BR-3 cell line to determine if the Her2 ECD shedding associated with uPA induction in breast cancer cells also involves or requires matriptase and prostasin by using specific inhibitors. PUBLIC HEALTH RELEVANCE: Resistance to chemotherapy drugs targeting Her2/neu/ErbB2 limits the success of these drugs on select groups of breast cancer patients and could render patients with acquired resistance untreatable. Proteolytic shedding of the Her2 extracellular domain/ectodomain (ECD) by proteases is a mechanism of this resistance. Our preliminary studies have identified new candidate proteases involved in Her2 ECD shedding and the proposed research will reveal if specific protease inhibitors should be considered for combination therapy to reduce or prevent the resistance to the anti-Her2 drugs.