Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma ...(PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF‐overexpressing high‐grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell‐derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the procoagulant potential of PDAC cells and cell‐derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.
What's new?
Alternatively spliced tissue factor (asTF) triggers the growth of new blood vessels and is present at elevated levels in certain cancers, indicating that it could play an important role in tumor growth and metastasis. Here, asTF was found to be abundant in human pancreatic ductal adenocarcinoma (PDAC) lesions and stromal monocytes, and its overexpression potentiated PDAC metastasis in vivo. Furthermore, asTF augmented EGFR‐linked signaling pathways in PDAC cells and contributed to the coagulant activity of PDAC cells and cell‐derived microparticles. The findings suggest that asTF may be a key target for stemming metastatic spread and complications in PDAC.
Essentials
Tissue factor (TF) isoforms are expressed in pancreatic neuroendocrine tumors (pNET).
TF knockdown inhibits proliferation of human pNET cells in vitro.
mTOR kinase inhibitor ...sapanisertib/MLN0128 suppresses TF expression in human pNET cells.
Sapanisertib suppresses TF expression and activity and reduces the growth of pNET tumors in vivo.
Summary
Background
Full‐length tissue factor (flTF) and alternatively spliced TF (asTF) contribute to growth and spread of pancreatic ductal adenocarcinoma. It is unknown, however, if flTF and/or asTF contribute to the pathobiology of pancreatic neuroendocrine tumors (pNETs).
Objective
To assess TF expression in pNETs and the effects of mTOR complex 1/2 (mTORC1/2) inhibition on pNET growth.
Methods
Human pNET specimens were immunostained for TF. Human pNET cell lines QGP1 and BON were evaluated for TF expression and responsiveness to mTOR inhibition. shRNA were used to knock down TF in BON. TF cofactor activity was assessed using a two‐step FXa generation assay. TF promoter activity was assessed using transient transfection of human TF promoter‐driven reporter constructs into cells. Mice bearing orthotopic BON tumors were treated with the mTORC1/2 ATP site competitive inhibitor sapanisertib/MLN0128 (3 mg kg−1, oral gavage) for 34 days.
Results
Immunostaining of pNET tissue revealed flTF and asTF expression. BON and QGP1 expressed both TF isoforms, with BON exhibiting higher levels. shRNA directed against TF suppressed BON proliferation in vitro. Treatment of BON with sapanisertib inhibited mTOR signaling and suppressed TF levels. BON tumors grown in mice treated with sapanisertib had significantly less TF protein and cofactor activity, and were smaller compared with tumors grown in control mice.
Conclusions
TF isoforms are expressed in pNETs. Sapanisertib suppresses TF mRNA and protein expression as well as TF cofactor activity in vitro and in vivo. Thus, further studies are warranted to evaluate the clinical utility of TF‐suppressing mTORC1/2 inhibitor sapanisertib in pNET management.
Abstract
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.
The stability of prostacyclin (PGI2) in whole blood and plasma was studied in vitro by measuring the disappearance rate of labeled prostacyclin during a 37 degrees C incubation. Prostacyclin was ...assayed using a quantitative chromatographic method. The half-life of PGI2 was 6.3 +/- 0.8 minutes (mean +/- s.d., n = 6) in citrated human whole blood, significantly shorter (p less than 0.001) than the 10.7 +/- 2.3 minute half-life in citrated human plasma (n = 7). Prior freezing and thawing of plasma did not affect the rate of PGI2 hydrolysis. These values, including the prolonged half-life in plasma, were similar in the blood (5.4 +/- 1.8 min, n = 7) and plasma (9.0 +/- 1.9 min, n = 14) of diabetic patients. In plasma samples from patients with thrombotic thrombocytopenic purpura, the half-life of prostacyclin (4.9 +/- 1.0 min, n = 4) was significantly shortened (p less than 0.001) compared to that in plasma from normal volunteers. The stability of prostacyclin in rabbit blood and plasma was also quantified. The PGI2 half-life in citrated rabbit plasma (10.8 +/- 1.1 min, n = 3) was similar to that in citrated human plasma from control subjects. In contrast to the findings in human blood, the half-life of PGI2 in citrated rabbit whole blood (11.7 +/- 3.3 min, n = 4) was not different from the rabbit plasma value. Substitution of EDTA for citrate did not affect the half-life in rabbit blood or plasma.
Ischemic colitis with colonic necrosis is one of the uncommon gastrointestinal complications of systemic lupus erythematosus. In the few reported cases, only the abdominal part of the colon was ...involved with rectal sparing. This is the first report of gangrenous ischemic colitis isolated to the rectum, due to systemic lupus erythematosus vasculitis.
An ability of intravenous nitroglycerin to interfere with the anticoagulant properties of intravenous heparin would have profound clinical implications. To investigation nitroglycerin-heparin ...interactions, the following pilot study was performed. Patients (N = 18) admitted to the coronary care unit with a diagnosis of either acute myocardial infarction or unstable angina were divided into four treatment groups: (1) intravenous nitroglycerin and intravenous heparin; (2) intravenous nitroglycerin alone; (3) intravenous heparin alone; or (4) neither intravenous nitroglycerin nor intravenous heparin. Serial determinations of activated partial thromboplastin time (APTT), serum heparin concentration, antithrombin III (ATIII) antigen (ATA), and ATIII activity (ATC) were obtained over a 72-hour period. Overall, patients receiving intravenous nitroglycerin did not differ significantly from other patients in APTT, heparin dose, heparin concentration, ATA, ATC, or ATA/ATC ratio (ATR). However, patients receiving intravenous nitroglycerin at a rate exceeding 350 micrograms per minute had a lower APTT (p less than 0.05), lower ATC (p = 0.02), higher ATR (p = 0.004), and a larger heparin dose requirement than patients receiving lower infusion rates. ATR correlated directly (r = 0.91; p less than 0.05) and ATC inversely (r = -0.78; p less than 0.05) with the intravenous nitroglycerin dose. Serum heparin concentration did not correlate with the intravenous nitroglycerin dose. Intravenous nitroglycerin-induced heparin resistance occurs at a critical nitroglycerin dose. A nitroglycerin-induced qualitative ATIII abnormality may be the underlying mechanism.
Twenty-two patients were selected from a group of 33 patients who underwent recombinant human tissue-type plasminogen activator (rt-PA) thrombolysis for thrombosed infrainguinal bypass grafts of the ...lower extremity and were compared with 38 matched patients who had undergone surgical thrombectomy during the same period. The proportion of persons with diabetes mellitus, smokers, and types of bypass grafts was similar in both groups. More patients in the rt-PA—treated group had hypertension (p = 0.01). To evaluate the different lengths of follow-up, Kaplan-Meier survival analysis was used with a log-rank test to compare the proportion of persons with patent grafts in the two treatment groups. At 30 days, 86% of the rt-PA—treated grafts were still patent compared with 42% of the surgically treated grafts (p = 0.001). When risk factors on the Kaplan-Meier curves were compared, there was no statistical difference with regard to graft patency among the groups. According to simultaneous Cox regression analysis, no risk factor was significantly associated with graft patency. When amputation was evaluated between treatment groups simultaneously with other risk factors in a logistic regression analysis, smoking and age of the graft were marginally significant (p = 0.07), whereas all other factors were clearly not significant. In 91% of the rt-PA—treated patients, a secondary surgical procedure was required to maintain patency of the graft segment. Eighty-nine percent of the surgically treated patients required similar graft revisions. Two patients in the surgical group and one patient in the rt-PA—treated group had major complications. This short clinical experience suggests that better 30-day patency rates and a lower risk of amputation can be achieved when thrombolysis is associated with surgical revision compared with surgical revision alone.
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