Abstract A39: Expression of alternatively spliced tissue factor in pancreatic neuroendocrine tumors
Authors: Lewis Clayton S. , Thomas Hala Elnakat , Lucas Fred V. , Bogdanov Vladimir Y. 
Published: December 2016

Background: Pancreatic cancer remains extremely difficult to treat, resulting in an 80% 1-year mortality rate. Tissue Factor (TF) serves as a co-factor for serine protease factor VIIa and the full-length (fl)TF/VIIa complex initiates blood coagulation. Alternatively spliced isoform of TF (asTF) occurs naturally, is minimally coagulant and, in contrast to flTF, dispensable for normal hemostasis. flTF is overexpressed in many forms of cancer and contributes to malignant growth; however, targeting flTF is risky due to the possibility of bleeding complications. We previously described that asTF is highly expressed in pancreatic ductal adenocarcinoma (PDAC) which account for ~75% of pancreatic cancer cases. Through non-canonical interactions with β1 integrins, asTF promotes activation of Akt and p42/44 MAPK, thus potentiating cancer cell migration and proliferation. We recently developed asTF-specific monoclonal antibody that inhibited growth and spread of PDAC in vivo in an orthotopic setting (Unruh et al, Oncotarget, in press). At this time it is not known whether, aside from PDAC, asTF also contributes to pathobiology of other pancreatic tumors such as pancreatic neuroendocrine tumors (pNET). Although pNETs comprise a minority subset of pancreatic cancer cases, pNET incidence is rapidly increasing and new therapies are thus needed for patients who ultimately progress on second line therapies which include mTOR inhibitors.

Objective: To evaluate i) expression of asTF in pNET, and ii) potential utility of targeting intracellular pathways engaged by asTF in pNET.

Methods: A tissue microarray containing 6 human pNET specimens was immunostained for asTF. Two human pNET cell lines, QGP1 and BON (a kind gift of Dr. Courtney Townsend, UTMB), were evaluated for TF isoform expression and their responsiveness to mTOR inhibition using the dual PI3K/mTOR ATP-site competitive inhibitor BEZ235.

Results: All 6 pNET tissue specimens stained positive for asTF: 4 samples were highly positive and the remaining 2 moderately positive; normal pancreatic tissue specimens in the microarray exhibited minimal staining for asTF. qRT-PCR and western blotting analyses revealed that BON and QGP1 express flTF as well as asTF mRNA and protein, respectively. Compared to QGP1 cells, BON cells expressed ~3 fold more asTF and ~10 fold more flTF. Western blotting revealed that, compared to QGP1 cells, BON cells express significantly higher levels of β1 integrin. Treatment of QGP1 and BON cells with BEZ235 (400nM, 48 hours) markedly suppressed phosphorylation of mTOR targets including Akt, S6K1, 4E-BP1, and ULK1.

Conclusion: To our knowledge, this is the first study to report that flTF and asTF are expressed in pNET tumor tissue and cultured cells. In pNET cell lines QGP1 and BON, BEZ235 effectively reduced phosphorylation of Akt, a downstream target of the pathways activated by asTF:β1 interactions. Further studies are warranted to evaluate asTF as a potential target in pNET, specifically strategies aimed at concurrent inhibition of asTF:β1 interactions and Akt/S6K1 activity.

Citation Format: Clayton S. Lewis, Hala Elnakat Thomas, Fred V. Lucas, Vladimir Y. Bogdanov.{Authors}. Expression of alternatively spliced tissue factor in pancreatic neuroendocrine tumors. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A39.