Divalent cation activation of the integrin-mediated malignant phenotype in pancre

描述(申请人提供)：最近的研究表明，I型胶原，一种在胰腺癌中高度上调的ECM蛋白，在体外和体内都促进了恶性表型。我们最近已经证明，a2？1整合素特异性地介导胰腺癌细胞与I型胶原的相互作用，并且这种整合素-ECM相互作用是依赖于镁离子的，并被钙离子抑制。这些观察结果类似于伤口愈合，这与胰腺癌的范例非常相似。在创面愈合过程中，细胞外镁离子和钙离子浓度的变化发生在愈合过程的早期，激活了a2？1整合素介导的各种细胞类型在I型胶原上的迁移，包括角质形成细胞。这种二价阳离子漂移的特征是增加了镁离子，降低了钙离子。胰腺癌也可能出现类似的变化，因为胰液中含有的镁离子是钙离子的1200倍。由于胰腺癌的导管基底膜不连续或不存在，预计胰液将渗入肿瘤微环境。此外，与伤口愈合类似，实体肿瘤的特点是镁离子负荷增加。综上所述，这些数据提出了一种假说，即在胰腺癌中，镁离子和钙离子浓度的病理生理变化激活了a2？1整合素介导的恶性表型。我们已发表的和初步的数据表明，当镁离子大于约1 mM时，胰腺癌细胞最大限度地附着在I型胶原上，而增加钙离子的加入减少了这种a2？1整合素介导的黏附。这些二价阳离子介导的效应是可逆的，因为以前细胞与镁离子中的I型胶原的附着可以通过添加钙离子来逆转，反之亦然。我们还证明了细胞仅在镁离子中迁移和增殖在I型胶原上，但当镁离子与钙离子的比率大于1时，细胞迁移和增殖最大。在这项提案中，我们将追求以下具体目标：我们将利用我们的红色荧光蛋白原位小鼠模型，在该模型中，癌细胞被RFP标记，以考察二价阳离子在胰腺癌切除术中作为手术辅助治疗以及在胰腺癌治疗中的应用。我们将在我们建立的RFP原位小鼠胰腺癌模型中进行体内实验，这是对人类疾病的概括。这些实验旨在检测二价阳离子在手术切除已确定的人胰腺癌肿瘤时作为辅助治疗的抑制作用，以及作为可能的治疗方法。我们还将使用针对特定整合素亚基的功能阻断单抗进行体内抑制研究，以确定哪些整合素受到二价阳离子治疗的特异性影响。这些研究有可能提高胰腺癌手术切除后的存活率，也为这种顽固性疾病提供了一种新的治疗方法。公共卫生相关性：每年将诊断出约30,000例新的胰腺癌病例，这是美国癌症相关死亡的第四大原因，仅有1%至4%的患者在首次诊断后5年内存活。手术切除、放射治疗和吉西他滨化疗通常只能延长几个月的生命。迫切需要改善胰腺癌患者的治疗方法。

DESCRIPTION (provided by applicant): Recent studies indicate that type I collagen, an ECM protein shown to be highly up-regulated in pancreatic cancer, promotes the malignant phenotype in vitro and in vivo. We have recently demonstrated that the a2¿1 integrin specifically mediates pancreatic cancer cell interactions with type I collagen, and that this integrin-ECM interaction is Mg2+-dependent and inhibited by Ca2+. These observations are similar to wound healing, which strongly parallels the pancreatic cancer paradigm. During wound healing, shifts in the concentrations of extracellular Mg2+ and Ca2+ occur early in the process, activating the a2¿1 integrin-mediated migration of various cell types on type I collagen, including keratinocytes. This divalent cation shift is characterized by increased Mg2+ and decreased Ca2+. Similar shifts are also likely in pancreatic cancer, as pancreas juice contains 1200-fold more Mg2+ than Ca2+. With ductal basement membranes either discontinuous or absent in pancreatic cancer, pancreas juice would be expected to leak into the tumor microenvironment. Additionally and similar to wound healing, solid tumors are characterized by increased Mg2+ load. Collectively, these data suggest the hypothesis where pathophysiological changes in the concentrations of Mg2+ and Ca2+ activate the a2¿1 integrin-mediated malignant phenotype in pancreatic cancer. Our published and preliminary data indicate that pancreatic cancer cells attach to type I collagen maximally when Mg2+ is greater than about 1mM, and that addition of increasing Ca2+ reduces this a2¿1 integrin-mediated adhesion. These divalent cation-mediated effects are reversible in that previous cell attachment to type I collagen in Mg2+ can be reversed by the addition of Ca2+, and vice versa. We also demonstrate that cells migrate and proliferate on type I collagen in Mg2+ alone, but maximally when the Mg2+ to Ca2+ ratio is greater than one. In this proposal, we will pursue the following Specific Aim: We will utilize our red fluorescent protein orthotopic mouse model, in which cancer cells are labeled with RFP, to examine the use of divalent cations as surgical adjuvant therapy during pancreatic cancer resection and as therapy in the treatment of pancreatic cancer. We will conduct in vivo experiments in our established RFP orthotopic mouse model of pancreatic cancer, which recapitulates human disease. These experiments are designed to examine the inhibitory effects of divalent cations used as adjuvant therapy during surgical resection of established human pancreatic cancer tumors, and also as possible therapy. We will also conduct in vivo inhibition studies using function-blocking monoclonal antibodies directed against specific integrin subunits to determine which integrins are specifically impacted by divalent cation therapy. These studies have the potential to improve survival rates for pancreatic cancer following surgical resection, and also provide a novel therapeutic approach to this recalcitrant disease. PUBLIC HEALTH RELEVANCE: Approximately 30,000 new cases of adenocarcinoma of the pancreas, the fourth leading cause of cancer related mortality in the United States, will be diagnosed each year with only 1 to 4% of patients surviving 5 years after their initial diagnosis. Surgical resection, radiation therapy and gemcitabine chemotherapy generally result in only a few months of extended life. The need for improved therapy for pancreatic cancer patients is urgently needed.