Tissue Factor splicing and pancreatic tumor progression: pilot studies

描述(申请人提供)：我们将确定选择性剪接组织因子(AsTF)的过度表达是否促进胰腺肿瘤的发生，以及asTF表达的降低是否抑制胰腺肿瘤的发生。长期以来，在胰腺导管腺癌(PDAC)中一直观察到高TF活性。直到最近，在PDAC和其他形式的实体癌中高表达Tf的功能意义被认为与全长Tf的蛋白分解功能是分不开的。全长Tf是一种完整的膜蛋白，是丝氨酸蛋白酶VIIa的辅助因子。我们发现，asTF-分泌的选择性剪接的TF变体-非蛋白水解性通过连接整合素？6？1激活与癌症相关的信号通路，整合素？6？1是PDAC原发肿瘤生长和转移扩散的主要贡献者。？6？1在PDAC细胞上的表达与转移率相关；因此，由于TF-？6？1的相互作用可能会以一种不依赖血管生成的、细胞自主的方式促进转移扩散。我们的最新数据表明，asTF蛋白在PDAC病变中含量丰富，但在正常导管中不存在，并通过上调内皮细胞上的细胞黏附分子促进单核细胞募集。选择性靶向asTF的化合物可能对癌症进展有重大影响，而对止血影响最小。我们将进行体外研究，以及WT/低水平裸鼠SCID小鼠和表达asTF的人PDAC细胞株的原位模型。为了评估asTF表达升高的影响，我们将产生对多西环素反应的(过表达asTF的)PDAC细胞的亚克隆。为了评估降低asTF水平的效果，我们将用表达高效和特异的抗asTF shRNA的慢病毒转导PDAC细胞。定期进行肿瘤细胞和肿瘤血管的活体成像，处死动物，采集组织标本，评价血管形成程度/微血管密度/单核细胞浸润程度以及增殖和凋亡指数。分析的中心目标：1.服用多西环素后，i)我们观察到癌症进展加快和/或生存率降低吗？Ii)原发肿瘤生长增强了吗？3)转移发生率增加了吗？2.asTF水平降低是否抑制肿瘤生长和/或扩散？为了探讨肿瘤转移因子水平与疾病进展相关的可能性，将利用切除的PDAC组织进行临床病理研究。我们的发现将包括在理解asTF在胰腺癌病理生物学中的作用方面取得质的进展。为了产生asTF(过度)表达的PDAC细胞，我们将使用我们实验室开发的创新的微型基因。体内研究将使用尖端实时成像方法-标记的SapC(H2)-DOPS囊泡。与其他方法相比，SapC结合的囊泡具有优势，因为它们选择性地靶向肿瘤血管和富含磷脂酰丝氨酸的肿瘤细胞，并允许在体内进行快速评估，同时与传统的免疫组织化学技术完全兼容。 公共卫生相关性：该项目将调查是否可以开发一种主要的新策略来治疗胰腺癌--胰腺癌是西方世界癌症死亡的第四大原因。我们的研究重点是选择性剪接组织因子，这是一种最近发现的由胰腺癌细胞表达的分子，它可以诱导新血管的形成，从而促进肿瘤的生长和转移。如果成功，我们的研究将带头扩大治疗胰腺癌的方法，并拓宽对肿瘤生物学的一般理解。

DESCRIPTION (provided by applicant): We will ascertain whether (over)expression of alternatively spliced Tissue Factor (asTF) promotes pancreatic tumorigenesis, and whether a decrease in asTF expression suppresses pancreatic tumorigenesis. High TF activity has been long observed in pancreatic ductal adenocarcinoma (PDAC). Until recently, the functional significance of high TF expression in PDAC and other forms of solid cancer was thought to be inseparable from the proteolytic function of full-length TF, an integral membrane protein that serves as a co-factor for the serine protease VIIa. We found that asTF - the secreted alternatively spliced TF variant - non-proteolytically activates cancer-associated signaling pathways via ligation of integrin ?6?1, the major contributor to primary tumor growth and metastatic spread in PDAC. ?6?1 expression on PDAC cells correlates with the rate of metastases; thus, asTF-?6?1interactions may enhance metastatic spread in an angiogenesis-independent, cell-autonomous fashion. Our newest data indicate that asTF protein is abundant in PDAC lesions, but not normal ducts, and promotes monocyte recruitment via upregulation of cell adhesion molecules on endothelial cells. Compounds selectively targeting asTF may have a major impact on cancer progression while having minimal impact on hemostasis. We will perform in vitro studies, as well as the orthotopic model in athymic nude/SCID mice with WT/low levels on endogenous TF and human PDAC cell lines expressing asTF. To evaluate the effects of elevated asTF expression, we will generate subclones of PDAC cells (over)expressing asTF in response to doxycycline. To evaluate the effects of lowering asTF levels, we will transduce PDAC cells with a lentivirus expressing a highly effective and specific anti-asTF shRNA. In vivo imaging of cancer cells and tumor vessels will be carried out at regular intervals; the animals wil be sacrificed, tissue specimens collected, and the degree of vascularization / microvessel density / monocyte infiltration evaluated along with the proliferation and apoptosis indices. The central objectives of the analysis: 1. Following doxycycline administration, i) do we observe enhanced cancer progression and/or decreased survival? ii) Is the primary tumor growth augmented? iii) Is there an increased incidence of metastases? 2. Does a decrease in asTF levels inhibit tumor growth and/or spread? To explore the possibility that the levels of tumor asTF correlate with disease progression, clinicopathological studies utilizing resected PDAC tissue will be performed. Our findings will comprise a qualitative gain in the understanding of asTF's contribution to the pathobiology of pancreatic cancer. To generate the asTF-(over)expressing PDAC cells, we will employ an innovative mini-gene developed in our laboratory. In vivo studies will be carried out using cutting-edge real-time imaging methodology - labeled SapC(H2)-DOPS vesicles. SapC-conjugated vesicles present advantage over other methodologies as they selectively target tumor vasculature and phosphatidylserine-enriched tumor cells, and allow rapid in vivo evaluation while being fully compatible with conventional immunohistochemical techniques. PUBLIC HEALTH RELEVANCE: This project will investigate whether a principally novel strategy can be developed to treat pancreatic cancer - the fourth leading cause of cancer death in the Western world. Our research focuses on alternatively spliced Tissue Factor, a recently discovered molecule expressed by pancreatic cancer cells that induces formation of new vessels fueling tumor growth and metastases. If successful, our study will spearhead expansion of the arsenal of approaches to treat pancreatic cancer, and broaden the general understanding of tumor biology.