Joanne Yu

Possible mechanisms of acquired resistance to anti-angiogenic drugs: implications for the use of combination therapy approaches.

The ultimate target of anti-angiogenic drugs is the genetically stable, activated endothelial cell of a newly forming tumor blood vessel, rather than the genetically unstable tumor cell population per se. This led to the notion that acquired resistance to such drugs may not develop as readily, if at all. While there is some evidence that this lack of resistance development may be the case for some direct-acting angiogenesis inhibitors, it is becoming apparent that resistance can develop over time to many types of angiogenesis inhibitors including, possibly, some direct inhibitors, especially when used as monotherapies. Possible mechanisms for such acquired or induced resistance include: (i) redundancy of pro-angiogenic growth factors when the drug used targets a single such growth factor or its cognate endothelial cell-associated receptor tyrosine kinase; (ii) the anti-apoptotic/pro-survival function of growth factors such as VEGF, which, in high local concentrations, can antagonize the pro-apoptotic effects of various angiogenesis inhibitors; (iii) epigenetic, transient upregulation, or induction, of various anti-apoptotic effector molecules in host-endothelial cells; and (iv) heterogeneous vascular dependence of tumor cell populations. It is suggested that long-term disease control with anti-angiogenic drugs can be best achieved by judicious combination therapy. In this regard, the great molecular diversity of anti-angiogenic drug targets, in contrast to chemotherapy, makes this a particularly attractive therapeutic option, especially when approved, commercially available drugs considered to have anti-angiogenic effects are used in such combination treatment strategies.

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.

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.

Tissue factor in tumour progression

The linkage between activation of the coagulation system and cancer is well established, as is deregulation of tissue factor (TF) by cancer cells, their vascular stroma and cancer-associated inflammatory cells. TF is no longer perceived as an alternative coagulation factor, but rather as a central trigger of the coagulation cascade and an important cell-associated signalling receptor activated by factor VIIa, and interacting with several other regulatory entities, most notably protease-activated receptors (PAR-1 and PAR-2). Preclinical studies revealed the role of oncogenic transformation and tumour micro-environment as TF regulators in cancer, along with the impact of this receptor on gene expression, tumour growth, metastasis, angiogenesis and, possibly, formation of the cancer stem cell niche. Increasing interest surrounds the shedding of TF-containing microvesicles from cancer cells, their entry into the circulation and their role in the intercellular transfer of TF activity, cancer coagulopathy and other processes. Recent data also suggest differential roles of cell autonomous versus global effects of TF in various settings. Questions are raised regarding the consequences of TF expression by tumour cells themselves and by their associated host stroma. Progress in these areas may soon begin to impact on clinical practice and, as such, raises several important questions. Can TF be exploited as a therapeutic target in cancer? Where and when may this be safe and beneficial? Is expression of TF in various disease settings useful as a biomarker of cancer progression or the associated hypercoagulability? What clinical questions related to TF are especially worthy of further exploration, at present and in the near future? Some of these developments and questions will be discussed in this chapter.

Role of the tissue factor pathway in the biology of tumor initiating cells.

Oncogenic transformation and aberrant cellular differentiation are regarded as key processes leading to malignancy. They produce heterogenous cellular populations including subsets of tumour initiating cells (TICs), also known as cancer stem cells (CSCs). Intracellular events involved in these changes profoundly impact the extracellular and systemic constituents of cancer progression, including those dependent on the vascular system. This includes angiogenesis, vasculogenesis, activation of the coagulation system and formation of CSC-related and premetastatic niches. Tissue factor (TF) is a unique cell-associated receptor for coagulation factor VIIa, initiator of blood coagulation, and mediator of cellular signalling, all of which influence vascular homeostasis. Our studies established a link between oncogenic events, angiogenesis and the elevated expression of TF in several types of cancer cells. The latter suggests that cancer coagulopathy and cellular events attributed to the coagulation system may have cancer-specific and genetic causes. Indeed, in human glioma cells, a transforming mutant of the epidermal growth factor receptor (EGFRvIII) triggers not only the expression of TF, but also of its ligand (factor VII) and protease activated receptors (PAR-1 and PAR-2). Consequently, tumour cells expressing EGFRvIII become hypersensitive to contact with blood borne proteases (VIIa, thrombin), which upregulate their production of angiogenic factors (VEGF and IL-8), and contribute to formation of the growth promoting microenvironment (niche). Moreover, TF overexpression accompanies features of cellular aggressiveness such as markers of CSCs (CD133), epithelial-to-mesenchymal transition (EMT) and expression of the angiogenic and prometastatic phenotype. Conversely, TF blocking antibodies inhibit tumour growth, angiogenesis, and especially tumour initiation upon injection of threshold numbers of tumourigenic cells. Likewise, TF depletion in the host compartment (e.g. in low-TF mice) perturbs tumour initiation. These observations suggest that both cancer cells and their adjacent host stroma contribute TF activity to the tumour microenvironment. We postulate that the TF pathway may play an important role in formation of the vascular niche for tumour initiating CSCs, through its procoagulant and signalling effects. Therapeutic blockade of these mechanisms could hamper tumour initiation processes, which are dependent on CSCs and participate in tumour onset, recurrence, drug resistance and metastasis.

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.

Vascular determinants of cancer stem cell dormancy--do age and coagulation system play a role?

The inability of tumour‐initiating cancer stem cells (CSCs) to bring about a net increase in tumour mass could be described as a source of tumour dormancy. While CSCs may be intrinsically capable of driving malignant growth, to do so they require compatible surroundings of supportive cells, growth factors, adhesion molecules and energy sources (e.g. glucose and oxygen), all of which constitute what may be referred to as a ‘permissive CSC niche. However, in some circumstances, the configuration of these factors could be incompatible with CSC growth (a ‘non‐permissive niche) and lead to their death or dormancy. CSCs and their niches may also differ between adult and paediatric cancers. In this regard the various facets of the tumour‐vascular interface could serve as elements of the CSC niche. Indeed, transformed cells with an increased tumour‐initiating capability may preferentially reside in specific zones adjacent to tumour blood vessels, or alternatively originate from poorly perfused and hypoxic areas, to which they have adapted. CSCs themselves may produce increased amounts of angiogenic factors, or rely for this on their progeny or activated host stromal cells. It is likely that ‘vascular properties of tumour‐initiating cells and those of their niches may diversify and evolve with tumour progression. The emerging themes in this area include the role of vascular (and bone marrow) aging, vascular and metabolic comorbidities (e.g. atherosclerosis) and the effects of the coagulation system (both at the local and systemic levels), all of which could impact the functionality of CSCs and their niches and affect tumour growth, dormancy and formation of occult as well as overt metastases. In this article we will discuss some of the vascular properties of CSCs relevant to tumour dormancy and progression, including: (i) the role of CSCs in regulating tumour vascular supply, i.e the onset and maintenance of tumour angiogenesis; (ii) the consequences of changing vascular demand (vascular dependence) of CSC and their progeny; (iii) the interplay between CSCs and the vascular system during the process of metastasis, and especially (iv) the impact of the coagulation system on the properties of CSC and their niches. We will use the oncogene‐driven expression of tissue factor (TF) in cancer cells as a paradigm in this regard, as TF represents a common denominator of several vascular processes that commonly occur in cancer, most notably coagulation and angiogenesis. In so doing we will explore the therapeutic implications of targeting TF and the coagulation system to modulate the dynamics of tumour growth and tumour dormancy.

Diverse roles of tissue factor-expressing cell subsets in tumor progression.

Oncogenic upregulation of tissue factor (TF) and release of TF-containing microvesicles play an important role in cancer-related coagulopathy (Trousseaus syndrome), angiogenesis, and disease progression. In addition, certain types of host cells (stromal cells, inflammatory cells, activated endothelium) may also express TF. Although the relative contribution of host-related versus tumor-related TF to tumor progression is not known, our recent studies indicate that the role of both sources of TF in tumor formation is complex and context-dependent. Disruption of TF expression/activity in cancer cells leads to tumor growth inhibition in immunodeficient mice, even in cases where TF overexpression is driven by potent oncogenes ( K-RAS or EGFR). Interestingly, TF expression in vivo appears to be influenced by many factors, including the level of oncogenic transformation, tumor microenvironment, and differentiation from cancer stem-like cells. We postulate that activation of TF signaling and coagulation 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.

Oncogene-Driven Hemostatic Changes in Cancer

A common feature in progression of multiple human malignancies is the protracted deregulation of the coagulation system, often referred to as cancer coagulopathy. The genesis of this syndrome can be traced to changes in the tumor vascular interface, formed through vascular invasion, angiogenesis, and metastasis. The resulting contact between cancer cells and the circulating components of the coagulation system compromise the regulatory barriers that normally control physiological hemostasis. In addition, cancer cells and their stroma often exhibit procoagulant properties. While these changes have long been thought to be unspecific in nature, evidence now exists to suggest that cancer coagulopathy and the related Trousseau syndrome are a function of the genetic tumor progression. Indeed, the expression of several effector molecules of the coagulation and fibrinolytic systems, including: tissue factor (TF), plasminogen activator inhibitor 1 (PAI-1), cyclooxygenase 2 (COX-2), or urokinase (uPA) are often direct regulatory targets of oncogenes (K-ras, EGFR, HER-2, c-MET) and tumor suppressors (p53, PTEN). Moreover, oncogenic alterations act on coagulation indirectly by driving formation of leaky vessels, metastasis, or inflammation. These procoagulant influences of oncogenic pathways are modulated by hypoxia, stress responses and cellular differentiation, the latter involving formation of cancer stem cells and their niches to which coagulation factors may contribute. It is possible that targeting cancer-related coagulopathy may require more cancer-specific measures to reduce thrombosis burden and improve overall survival.