Chloe Milsom

Tissue Factor Regulation by Epidermal Growth Factor Receptor and Epithelial-to-Mesenchymal Transitions: Effect on Tumor Initiation and Angiogenesis

ErbB oncogenes drive the progression of several human cancers. Our study shows that in human carcinoma (A431) and glioma (U373) cells, the oncogenic forms of epidermal growth factor receptor (EGFR; including EGFRvIII) trigger the up-regulation of tissue factor (TF), the transmembrane protein responsible for initiating blood coagulation and signaling through interaction with coagulation factor VIIa. We show that A431 cancer cells in culture exhibit a uniform TF expression profile; however, these same cells in vivo exhibit a heterogeneous TF expression and show signs of E-cadherin inactivation, which is coupled with multilineage (epithelial and mesenchymal) differentiation. Blockade of E-cadherin in vitro, leads to the acquisition of spindle morphology and de novo expression of vimentin, features consistent with epithelial-to-mesenchymal transition. These changes were associated with an increase in EGFR-dependent TF expression, and with enhanced stimulation of vascular endothelial growth factor production, particularly following cancer cell treatment with coagulation factor VIIa. In vivo, cells undergoing epithelial-to-mesenchymal transition exhibited an increased metastatic potential. Furthermore, injections of the TF-blocking antibody (CNTO 859) delayed the initiation of A431 tumors in immunodeficient mice, and reduced tumor growth, vascularization, and vascular endothelial growth factor expression. Collectively, our data suggest that TF is regulated by both oncogenic and differentiation pathways, and that it functions in tumor initiation, tumor growth, angiogenesis, and metastasis. Thus, TF could serve as a therapeutic target in EGFR-dependent malignancies.

Contribution of Host-Derived Tissue Factor to Tumor Neovascularization

Objective—The role of host-derived tissue factor (TF) in tumor growth, angiogenesis, and metastasis has hitherto been unclear and was investigated in this study. Methods and Results—We compared tumor growth, vascularity, and responses to cyclophosphamide (CTX) of tumors in wild-type (wt) mice, or in animals with TF levels reduced by 99% (low-TF mice). Global growth rate of 3 different types of transplantable tumors (LLC, B16F1, and ES teratoma) or metastasis were unchanged in low-TF mice. However, several unexpected tumor/context-specific alterations were observed in these mice, including: (1) reduced tumor blood vessel size in B16F1 tumors; (2) larger spleen size and greater tolerance to CTX toxicity in the LLC model; (3) aborted tumor growth after inoculation of TF-deficient tumor cells (ES TF−/−) in low-TF mice. TF-deficient tumor cells grew readily in mice with normal TF levels and attracted exclusively host-related blood vessels (without vasculogenic mimicry). We postulate that this complementarity may result from tumor-vascular transfer of TF-containing microvesicles, as we observed such transfer using human cancer cells (A431) and mouse endothelial cells, both in vitro and in vivo. Conclusions—Our study points to an important but context-dependent role of host TF in tumor formation, angiogenesis and therapy.

Cancer Cells Induced to Express Mesenchymal Phenotype Release Exosome-like Extracellular Vesicles Carrying Tissue Factor

Background: Cross-talk of oncogenic and differentiation pathways in cancer coagulopathy is poorly understood. Results: EGFR activation and blockade of E-cadherin in cancer cell lines induce mesenchymal phenotype and tissue factor (TF) shedding, as exosomes, capable of transferring procoagulant activity to endothelium. Conclusion: Mesenchymal and procoagulant phenotypes are linked in cancer. Significance: Epithelial-to-mesenchymal transition (EMT) may influence tumor-vascular interactions via TF-containing exosomes. Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes has not been studied. EVs are heterogeneous membrane structures that originate either at the surface (microparticles), or within (exosomes) activated or transformed cells, and are involved in intercellular trafficking of bioactive molecules. Here, we show that epithelial cancer cells (A431, DLD-1) adopt mesenchymal features (epithelial-to-mesenchymal transition-like state) upon activation of epidermal growth factor receptor (EGFR) coupled with blockade of E-cadherin. This treatment leads to a coordinated loss of EGFR and tissue factor (TF) from the plasma membrane and coincides with a surge in emission of small, exosome-like EVs containing both receptors. TF (but not EGFR) is selectively up-regulated in EVs produced by mesenchymal-like cancer cells and can be transferred to cultured endothelial cells rendering them highly procoagulant. We postulate that epithelial-to-mesenchymal transition-like changes may alter cancer cell interactions with the vascular systems through altered vesiculation and TF shedding.

Metronomic oral topotecan prolongs survival and reduces liver metastasis in improved preclinical orthotopic and adjuvant therapy colon cancer models

Objective Advanced and recurrent diseases are the major causes of death in colon cancer. No standard preclinical model addresses advanced disease and spontaneous metastasis after orthotopic tumour growth. In this study, the authors report the establishment of such standardised orthotopic mouse models of colon cancer and their use in evaluating metronomic topotecan alone or in combination with standard chemotherapy. Design Human colon cancer cell lines, transfected with human chorionic gonadotropin and luciferase, were injected orthotopically into the caecal wall of severe combined immunodeficient mice, intrasplenically or subcutaneously. For adjuvant therapy, caecal resections were performed 3–5 weeks after tumour cell injection. Chemotherapy drugs tested included uracil/tegafur, folinic acid, oxaliplatin, topotecan, pazopanib and various combinations. Results Subcutaneous tumours showed exaggerated sensitivity to treatment by delayed tumour growth (p=0.002) and increased survival (p=0.0064), but no metastatic spread. Intrasplenic cell injection resulted in rapid and extensive but artefactual metastasis without treatment effect. Intracaecal cell injection with tumour take rates of 87.5–100% showed spontaneous metastases at clinically relevant rates. Metronomic topotecan significantly polonged survival and reduced metastasis. In the adjuvant setting, metronomic maintenance therapy (after FOLFOX-like induction) prolonged survival compared with vehicle controls (p=0.0003), control followed by topotecan (p=0.0161) or FOLFOX-like therapy (p=0.0003). Conclusion The refined orthotopic implantation technique proved to be a clinically relevant model for metastasis and therapy studies. Furthermore, metronomic therapy with oral topotecan may be promising to consider for clinical trials of metastatic colon cancer and long-term adjuvant maintenance therapy of colon cancer.

Oncogenes and Angiogenesis: Down-regulation of Thrombospondin-1 in Normal Fibroblasts Exposed to Factors from Cancer Cells Harboring Mutant Ras

The onset of angiogenesis in cancer often involves down-regulation of endogenous angiogenesis inhibitors, of which thrombospondin-1 (TSP-1) is a paradigm. As this effect is thought to occur under the influence of transforming genetic lesions (e.g., expression of the mutant ras oncogene), its nature is regarded as intrinsic to cancer cells themselves. Here, we show that ras-transformed cancer cells can also induce TSP-1 down-regulation in their adjacent nontransformed stromal fibroblasts, but not in endothelial cells, in a paracrine and distance-dependent manner. Indeed, several H-ras-expressing fibrosarcoma (528ras1, B6ras, and NIH3T3Ras) and carcinoma (DLD-1 and IEC18Ras3) cells were found to release soluble factors capable of suppressing TSP-1 protein, mRNA, and promoter activity in nontumorigenic, immortalized dermal fibroblastic cell lines in culture (e.g., in fibroblasts expressing enhanced green fluorescent protein/TSP-1 reporter). This effect was abrogated in Id1-/- fibroblasts. At least two low molecular weight (<3 kDa), heat-labile, and trypsin-resistant mediators of TSP-1 suppression were found to be released from 528ras1 cells. Their effects on normal fibroblasts were inhibited (albeit to different extents) by pertussis toxin and, in one case, by dimethylsphingosine, none of which affected TSP-1 expression by 528ras1 cells. Collectively, our study suggests that the effect of mutant ras on tumor neovascularization is not limited to changes in angiogenic properties of cancer cells themselves. Rather, mutant ras, through a different signaling mechanism, may modulate the properties of the adjacent normal stroma, thus eliciting a proangiogenic field effect.

Tissue Factor and Cancer

Tissue factor (TF), the key regulator of haemostasis and angiogenesis, is also involved in the pathology of several diseases, including cardiovascular, inflammatory and neoplastic conditions. In the latter, TF is upregulated by cancer cells, as well as by certain host cells, and it is the interactions between these distinct pools of TF-expressing cells that likely influence tumour progression in several ways. Furthermore, the release of TF microparticles into the circulation is thought to contribute to the systemic coagulopathies commonly observed in cancer patients. The direct regulation of TF by oncogenic events has provided a plausible explanation for the relatively common overexpression of TF in various cancers and its involvement in tumour growth, angiogenesis, metastasis and coagulopathy. However, this constitutive influence is modified by the tumour microenvironment, cellular interactions and host factors rendering TF expression patterns complex and heterogeneous. It appears that in many biological contexts TF plays a central role in disease progression and thereby potentially constitutes an attractive therapeutic target, especially in scenarios where the risk of bleeding can be avoided by selecting appropriate medications, refined dosing or by targeting the signalling component of TF activity. The efficacy and safety of such approaches still awaits clinical verification.

Tissue factor in cancer

Purpose of reviewTissue factor is increasingly viewed as an integral part of the vicious circle that links the vascular system with cancer progression at multiple systemic, cellular and molecular levels. Recent findingsThe emerging tenet in this area is that oncogenic events/pathways driving the malignant process also stimulate the expression of tissue factor by cancer cells and promote the release of tissue factor-containing microvesicles into the circulation. The combined effects of these changes likely contribute to cancer coagulopathy, cessation of tumour dormancy, aggressive growth, angiogenesis and metastasis, notably through a combination of procoagulant and signalling effects set in motion by tissue factor. As certain tumour-associated host cell types (inflammatory cells, endothelium) may also express tissue factor their contribution is plausible, though poorly understood. Interestingly, tissue factor could be ‘shared’ between various subsets of cancer and host cells due to intercellular transfer of tissue factor-containing microvesicles. It has recently been proposed that tissue factor may influence the interactions between tumour initiating (stem) cells and their growth or prometastatic niches. SummaryWhereas targeting tissue factor in cancer is appealing, the prospects in this regard will depend on the identification of disease specific indications, active agents and their safe regimens.

Elevated tissue factor procoagulant activity in CD133‐positive cancer cells

Clinical and experimental evidence suggests a parallel between increasing cancer aggressiveness and procoagulant tendencies, which are often attributable to high levels of tissue factor (TF). TF is upregulated on the surface of cancer cells and their derived microvesicles due to a combined impact of oncogenic events and tumor microenvironment [1–4] and thereby becomes involved in cancer progression, both as the principal initiator of the blood coagulation cascade and as a signaling receptor [1,2,5,6]. These TF activities are implicated in tumor growth, angiogenesis and metastasis [1,2,5,6], a finding congruent with the anticancer effects of anticoagulation revealed in recent clinical trials [1,7]. It remains unclear which cancer cells harbor biologically relevant TF. It has recently come to light that the capacity of cancer cells to initiate tumor growth is not universal, but rather can only be executed by a small minority of specialized cancer cells (fewer than 1%), often referred to as cancer stem cells (CSCs) [8]. Identification of CSCs in several solid tumors has been made possible by the recent discovery of their molecular markers, of which expression of CD133 (prominin-1) represents one of the best-known paradigms [9–12]. CD133 belongs to a family of five-transmembrane cell-surface glycoproteins commonly localized to membrane protrusions of various progenitors [9,13]. While CD133-positive cells have been identified as CSCs in several solid tumors, e.g. of the brain [10], colon [12] and in melanoma [11], it is unclear whether CD133 expression plays a causative, contributive or correlative role in the formation of the CSC population. Still, CD133-expressing CSCs have been implicated in a number of key processes, including tumor repopulation, resistance to therapy [14], increased aggressiveness [10] and angiogenesis [15]. It is intriguing to note that various recent reports point either to cancer cells with increased expression of TF or to cancer cells harboring the CSC marker CD133 as particularly central to cancer progression [4,16]. We reasoned that this may signify a deeper interrelationship between these two properties. In order to examine this in more detail, we tested the expression of CD133 and TF in the highly tumorigenic squamous cell carcinoma cell line A431, which is known to express considerable procoagulant activity [17]. We used Caco-2 cells as a positive control due to their curiously high CD133 expression [coupled with a paradoxically poor tumorforming capacity and undetectable TF expression (our unpubl. obs.)]. Interestingly, whole cell lysates of the pooled A431 cell population were found to contain appreciable amounts of TF [3], but surprisingly little detectable CD133 (Fig. 1A). However, flow cytometry analysis revealed that A431 are heterogeneous in that a small subset of these cells (0.5%) stained strongly with the CD133 antibody (Fig. 1B). In order to better understand the significance of this heterogeneity, A431 cells were separated immunomagnetically into CD133-positive and CD133-negative fractions (using the CD133 MACS system, Miltenyi Biotech, Auburn, CA, USA), each of which was then tested for TF content. Interestingly, CD133-positive A431 cells expressed a 5to 6-fold greater amount of TF antigen on their surfaces than their CD133-negative counterparts did (Fig. 1C) [3]. This was paralleled by a corresponding increase in the TF-dependent procoagulant activity (TF-PCA) [3,18] of CD133-positive A431 cells relative to cells lacking this stem cell marker. This latter assay measures the ability of the TF/ factor (F) VIIa complex to generate FXa-dependent, prothrombin-activating proteolytic activity [18] on the surface of A431 cancer cells. Thus we found that in a subset of A431 cancer cells CD133 is coexpressed with high levels of TF. As the CD133-positive (CSC) cancer cell subset is thought to drive tumor initiation/ formation events, we asked whether TF was required for the manifestation of these properties in vivo. Immunodeficient (SCID) mice were injected with A431 cells and treated with CNTO 859, a neutralizing TF-directed antibody that blocks Correspondence: Janusz Rak, McGill University, Montreal Children s Hospital Research Institute, Place Toulon, 4060 Ste Catherine West, PT-232, Montreal, Quebec, H3Z 2Z3, Canada. Tel.: +1 514 412 4400 ext. 22342; fax: +1 514 412-4331; e-mail: janusz.rak@mcgill.ca

Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations

Significance Our study shows that the clotting protein tissue factor (TF) controls the state of tumor dormancy and does so in conjunction with recruitment of inflammatory cells and blood vessels. We show that indolent glioma cells remain harmless in mice unless rendered TF positive. Our work also demonstrates the ability of TF to indirectly influence the DNA of cancer cells by facilitating gene mutations and silencing. This ability is important because injury, cardiovascular disease, or other conditions may activate the clotting system and contribute to the awakening of occult cancer cells. This understanding also may suggest a prophylactic use of blood thinners in cases where dormant cancer cells and clotting are suspected to coexist (e.g., after surgery). The coagulation system links immediate (hemostatic) and late (inflammatory, angiogenic) tissue responses to injury, a continuum that often is subverted in cancer. Here we provide evidence that tumor dormancy is influenced by tissue factor (TF), the cancer cell-associated initiator of the coagulation system and a signaling receptor. Thus, indolent human glioma cells deficient for TF remain viable but permanently dormant at the injection site for nearly a year, whereas the expression of TF leads to a step-wise transition to latent and overt tumor growth phases, a process that is preceded by recruitment of vascular (CD105+) and myeloid (CD11b+ and F4/80+) cells. Importantly, the microenvironment orchestrated by TF expression drives permanent changes in the phenotype, gene-expression profile, DNA copy number, and DNA methylation state of the tumor cells that escape from dormancy. We postulate that procoagulant events in the tissue microenvironment (niche) may affect the fate of occult tumor cells, including their biological and genetic progression to initiate a full-blown malignancy.

The role of tumor-and host-related tissue factor pools in oncogene-driven tumor progression

Oncogenic events play an important role in cancer-related coagulopathy (Trousseau syndrome), angiogenesis and disease progression. This can, in part, be attributed to the up-regulation of tissue factor (TF) and release of TF-containing microvesicles into the pericellular milieu and the circulation. In addition, certain types of host cells (stromal cells, inflammatory cells, activated endothelium) may also express TF. At present, the relative contribution of host- vs tumor-related TF to tumor progression is not known. Our recent studies have indicated that the role of TF in tumor formation is complex and context-dependent. Genetic or pharmacological disruption of TF expression/activity in cancer cells leads to tumor growth inhibition in immunodeficient mice. This occurred even in the case of xenotransplants of human cancer cells, in which TF overexpression is driven by potent oncogenes (K-ras or EGFR). Interestingly, the expression of TF in vivo is not uniform and appears to be influenced by many factors, including the level of oncogenic transformation, tumor microenvironment, adhesion and the coexpression of markers of cancer stem cells (CSCs). Thus, minimally transformed, but tumorigenic embryonic stem (ES) cells were able to form malignant and angiogenic outgrowths in the absence of TF. However, these tumors were growth inhibited in hosts (mice) with dramatically reduced TF expression (low-TF mice). Depletion of host TF also resulted in changes affecting vascular patterning of some, but not all types of tumors. These observations suggest that TF may play different roles growth and angiogenesis of different tumors. Moreover, both tumor cell and host cell compartments may, in some circumstances, contribute to the functional TF pool. We postulate that activation of the coagulation system and TF signaling, may deliver growth-promoting stimuli (e.g. fibrin, thrombin, platelets) to dormant cancer stem cells (CSCs). Functionally, these influences may be tantamount to formation of a provisional (TF-dependent) cancer stem cell niche. As such these changes may contribute to the involvement of CSCs in tumor growth, angiogenesis and metastasis.