Oncogenic events regulate tissue factor expression in colorectal cancer cells: implications for tumor progression and angiogenesis

Abstract

Tissue factor (TF) is the primary cellular initiator of blood coagulation and a modulator of angiogenesis and metastasis in cancer. Indeed, systemic hypercoagulability in patients with cancer and TF overexpression by cancer cells are both closely associated with tumor progression, but their causes have been elusive. We now report that in human colorectal cancer cells, TF expression is under control of 2 major transforming events driving disease progression (activation of K-ras oncogene and inactivation of the p53 tumor suppressor), in a manner dependent on MEK/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K). Furthermore, the levels of cell-associated as well as circulating (microvesicle-associated) TF activity are linked to the genetic status of cancer cells. Finally, RNA interference experiments suggest that TF expression is an important effector of the K-ras-dependent tumorigenic and angiogenic phenotype in vivo. Thus, this study establishes a causal link between cancer coagulopathy, angiogenesis, and genetic tumor progression. ( IntroductionCancer is believed to arise and progress toward increasing malignancy as a result of cumulative genetic "hits" sustained by the tumor cell genome. Paradigmatic in this regard is the development of colorectal carcinoma (CRC), where sequential transition through clinical stages of the disease is paralleled by a series of well-characterized alterations in proto-oncogenes and tumor suppressor genes. 1 In this tumor type, activation of mutant K-ras and subsequent inactivation/loss of p53 are key changes, which drive many interrelated aspects of the malignant phenotype including aberrant mitogenesis and survival. 2 Moreover, both of these genetic alterations are thought to contribute to proangiogenic properties of affected cancer cells, 3,4 and thereby enable them to exploit the host vascular system to advance malignant growth and metastasize in vivo. 5 The involvement of the vascular system in malignancy encompasses not only angiogenesis but also systemic hypercoagulability. Blood clotting abnormalities are detected in up to 90% of patients with metastatic disease, and thrombosis represents the second most frequent cause of cancer-related mortality. 6 Cancer coagulopathy is often linked to up-regulation of tissue factor (TF), the primary cellular initiator of the blood coagulation cascade. 7,8 Interaction of coagulation factor VIIa with TF on the cell surface leads to activation of factor X and generation of thrombin, with subsequent involvement of platelets and formation of a fibrin clot. 9 Remarkably, as a member of the class II cytokine receptor family, TF is also capable of transducing intracellular signals and regulating gene expression. 10,11 Interestingly, elements of the coagulation/fibrinolytic system in general, 12 and TF in particular, have been implicated in regulation of angiogenesis, 13,14 as well as tumor growth 15 and metastasis 16 in various experimental settings. This is consistent with the observed up-regulation of TF in huma...

Modulation of the oncogene-dependent tissue factor expression by kinase suppressor of ras 1

“…The cancer related increase in TF levels is attributed to the regulatory influences of oncogenic signalling pathways [6,7], which may be coupled with the modulating role of the tumour microenvironment [8][9][10]. Activated K-ras and members of the ErbB family, such as epidermal growth factor receptor (EGFR) and EGFRvIII all upregulate TF on the surface of cancer cells [10][11][12] and trigger the release of TF-containing microvesicles into the circulation of tumour bearing mice [7,10].…”

“…Activated K-ras and members of the ErbB family, such as epidermal growth factor receptor (EGFR) and EGFRvIII all upregulate TF on the surface of cancer cells [10][11][12] and trigger the release of TF-containing microvesicles into the circulation of tumour bearing mice [7,10]. These events contribute to tumour formation and angiogenesis by causing changes in the expression of angiogenic factors [7], or through their impact on tumour initiation [10].…”

“…The EGFR inhibitor, AG1478 (Biomol Research Laboratories, Plymouth Meeting, PA) was used at concentrations between 1 and 10 µM, while Herceptin (Genentech, San Francisco, CA) was used at 20 µg/ml, for time intervals indicated. Both agents were prepared in phosphate buffered saline from concentrated stocks, as described earlier [7,10]. CI-1033 was made available by Pfizer Inc., Montreal courtesy of Dr. Luc Levesque.…”

“…Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer. Northern blotting was performed as described previously [7,9] using a 600bp cDNA fragment for human tissue factor as a probe (a gift from Dr. Nigel Mackman, University of North Carolina). Changes in TF mRNA were quantified following normalisation to the 28 S rRNA, as indicated.…”

“…Tumour cells were harvested using 2mM EDTA and processed as described previously [7,10]. Briefly, the single cell suspension was incubated with the sheep antibody against human TF (Affinity Biologicals Ancaster, ON, Canada) and Alexa Fluor 488 donkey anti-sheep secondary antibody (Molecular Probes, Eugene, OR), and analyzed (FACScalibur; BD Biosciences, Mississauga, ON, Canada) [10].…”