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.
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.
|Unruh, Dusten; ÃœnlÃ¼, BetÃ¼l; Lewis, Clayton S et al. (2016) Antibody-based targeting of alternatively spliced tissue factor: a new approach to impede the primary growth and spread of pancreatic ductal adenocarcinoma. Oncotarget 7:25264-75|
|Ramchandani, Divya; Unruh, Dusten; Lewis, Clayton S et al. (2016) Activation of carbonic anhydrase IX by alternatively spliced tissue factor under late-stage tumor conditions. Lab Invest 96:1234-1245|
|KocatÃ¼rk, B; Tieken, C; Vreeken, D et al. (2015) Alternatively spliced tissue factor synergizes with the estrogen receptor pathway in promoting breast cancer progression. J Thromb Haemost 13:1683-93|
|Bogdanov, Vladimir Y; Versteeg, Henri H (2015) "Soluble Tissue Factor" in the 21st Century: Definitions, Biochemistry, and Pathophysiological Role in Thrombus Formation. Semin Thromb Hemost 41:700-7|
|Unruh, Dusten; Turner, Kevin; Srinivasan, Ramprasad et al. (2014) Alternatively spliced tissue factor contributes to tumor spread and activation of coagulation in pancreatic ductal adenocarcinoma. Int J Cancer 134:9-20|
|Davila, M; Robles-Carrillo, L; Unruh, D et al. (2014) Microparticle association and heterogeneity of tumor-derived tissue factor in plasma: is it important for coagulation activation? J Thromb Haemost 12:186-96|
|KocatÃ¼rk, BegÃ¼m; Van den Berg, Yascha W; Tieken, Chris et al. (2013) Alternatively spliced tissue factor promotes breast cancer growth in a Î²1 integrin-dependent manner. Proc Natl Acad Sci U S A 110:11517-22|