Angiogenesis and MMPs / TIMPs

2.2.6. Role of MMPs and TIMPs in invasion and metastasis

Metastases are the major cause of death from cancer whereas local lesions due to the primary tumor are a minor issue (YOON et al., 2003). In addition, studies performed over the last decades reveal the pivotal role of MMPs in tumor metastasis (WESTERMARCK and KAHARI, 1999).

The first evidence for a role of MMPs in metastasis came from the antimetastatic effect of TIMPs on murine melanoma cells (SCHULTZ et al., 1988). Among the previously reported human MMPs, MMP-2 and MMP-9 are the key proteins involved in tumor invasion and metastasis and they are abundantly expressed in various malignant tumors (JOHNSEN et al., 1998). Metastasis is an extremely complex process and involves detachment of the cell from the neoplastic tissue, invasion of the surrounding stroma and spread through blood vessels or lymphatics (EGEBLAD and WERB, 2002). Cleavage of laminin-5 (ln-5), an extracellular matrix substrate for cell adhesion found in epithelial basement membranes, by MMP-14 and MMP-2 can trigger cell migration and thus may play a role in the early phases of tumor invasion (KOSHIKAWA et al., 2000). MMP-14 promotes cell migration through the cleavage of CD44, a major receptor for hyaluronan (KAJITA et al., 2001). Moreover, CD44 also binds MMP-9 on the surface of mouse mammary carcinoma and human melanoma cells. This localization promotes collagen IV degradation and mediates tumor cell invasion (YU and STAMENKOVIC, 1999). MMPs have also important roles in the late events of metastasis which involve the intravasation into blood vessels or lymphatics, extravasation and establishment of a secondary growth site (CHAMBERS and MATRISIAN, 1997). MMP-9 is required for the intravasation of tumor cells and MMP-14 enhances the survival rate of tumor cells in an experimental pulmonary metastasis assay (TSUNEZUKA et al., 1996; KIM et al., 1998) (Fig.2 b).

2.2.7. Angiogenesis and MMPs / TIMPs

Pathological angiogenesis is a hallmark of cancer and it is necessary for persistent tumor growth, because it provides the tumor cells with the necessary gas exchange and nutrients (CHANG and WERB, 2001). Many studies demonstrate the significant role of MMPs in the

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induction of angiogenesis by targeting these MMPs using endogenous or synthetic MMP inhibitors (MARTIN et al., 1999; LI et al., 2001; OH et al., 2001a; RODRIGUEZ-MANZANEQUE et al., 2001; YOSHIDA et al., 2002). In addition, MMP-14- and MMP-9-deficient mice have severe defects in angiogenesis, demonstrating that MMP-14 and MMP-9 are implicated directly in the regulation of angiogenesis (VU et al., 1998; ZHOU et al., 2000).

MMPs may contribute in the angiogenesis process through the cleavage of individual matrix components, such as type I collagen (SEANDEL et al., 2001). In a rat corneal micropocket angiogenesis assay, cleavage of collagen type IV results in the exposure of an important cryptic site within its triple helical structure and blockage of this site disrupts integrin-dependent endothelial cell interaction and inhibits angiogenesis (XU et al., 2001). Another possibility is that MMPs, specifically MMP-9, are important for angiogenesis by releasing pro-angiogenic growth factors, such as VEGF during carcinogenesis of pancreatic islets in mice (BERGERS et al., 2000). Using an in vitro wound healing migration model, MMP-14 has been shown to play an important role in angiogenesis through regulating endothelial migration, invasion and formation of capillary tubes during angiogenic response and blocking the catalytic domain of MMP-14 inhibits these processes (GALVEZ et al., 2001). Moreover, MMP-14 can regulate neovascularization by acting as a pericellular fibrinolysin, thereby potentially allowing endothelial cells to invade further into the tumor tissue (HIRAOKA et al., 1998) (Fig. 2 c).

On the other hand, MMPs, specifically MMP-2, MMP-9 and MMP-12, may act anti-angiogenetically through cleavage of plasminogen to generate angiostatin which inhibits endothelial cell proliferation and thus diminishes metastatic tumor cell growth (DONG et al., 1997; CORNELIUS et al., 1998). In murine hemangioendothelioma cell cultures, MMP-3, MMP-9, MMP-13 and MMP-20 were found to have important roles in the generation of endostatin, a C-terminal fragment of the basement membrane collagen type XVIII, which is able to inhibit endothelial cell proliferation, migration in addition to angiogenesis and tumor growth (FERRERAS et al., 2000) (Fig. 2 c).

The TIMP family plays an important role in angiogenesis (SANG, 1998). TIMPs can prevent angiogenesis by the inhibition of MMPs (HANDSLEY and EDWARDS, 2005). Another possibility is the activation of MMP inhibitors, such as RECK, which in turn, inhibits angiogenesis (OH et al., 2004). In addition, TIMPs exhibit anti-angiogenic activity that is independent of their MMP inhibitory activities. Using in vitro cell culture and a syngeneic murine tumor model of colon cancer, the over-expression of TIMP-2 can inhibit tumor growth and angiogenesis by up-regulating mitogen-activated protein kinase phosphatase 1 which

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dephosphorylates p38 mitogen-activated protein kinase, a molecule involved in endothelial cell proliferation and migration (FELDMAN et al., 2004). Moreover, TIMP-1 inhibits endothelial cell invasion of human amniotic membranes (MIGNATTI et al., 1986). A TIMP-related protein isolated from bovine articular cartilage blocks endothelial cell proliferation and angiogenesis (MOSES et al., 1990). In addition, TIMP-1 and TIMP-2 inhibit chick yolk sac vessel morphogenesis in response to polyamines (TAKIGAWA et al., 1990).

TIMPs may also enhance tumor angiogenesis by inhibiting MMPs which play a critical role in the generation of potent inhibitors of angiogenesis. For example MMP-12 can generate angiostatin by cancer-mediated proteolysis of plasminogen (CORNELIUS et al., 1998). In addition, other members of the MMP family, such as MMP-3, -7, -9, -13, -20 can release endostatin, another well-known anti-angiogenic factor from collagen XVIII (HELJASVAARA et al., 2005). Therefore, by inhibiting these MMPs, TIMPs may enhance tumor angiogenesis (CORNELIUS et al., 1998; WEN et al., 1999). Supporting this idea, mammary carcinoma over-expressing TIMP-1, showed enhanced VEGF expression and enhanced growth rate (YOSHIJI et al., 1998).

19 Fig. 2: Role of MMPs in tumor progression (modified from EGEBLAD and WERB, 2002)

a. Matrix metalloproteinases (MMPs) promote tumor growth by cleaving insulin-growth-factor-binding protein (IGF-BP), thereby liberating IGF, by shedding precursors of growth factors including transforming growth factor-α (TGF-α) and by regulating extracellular matrix (ECM), which promotes growth indirectly through interactions between ECM molecules and integrins.

b. MMPs promote invasion and migration by degrading structural ECM components, such as laminin 5 (Lam-5). In addition, MMPs promote cell migration through cleavage of CD44.

c. MMPs promote angiogenesis by increasing the bioavailability of pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF). In addition, MMPs promote invasion of endothelial cells by cleaving structural components of the ECM, such as collagen types I (Col-I) and IV (Col-IV) and fibrin. Cleaved Col-IV acts pro-angiogenetically by binding to αvβ3 integrin. MMPs may act anti-pro-angiogenetically through cleavage of plasminogen and Col-XVIII resulting in the generation of anti-angiogenic factors such as angiostatin and endostatin.

20 2.3. Inflammation and cancer

In 1863, Rudolf Virchow was the first to describe the association of inflammation and cancer when he noted leukocytes in neoplastic tissue and suggested that the ‘‘lymphoreticular infiltrate’’ reflected the origin of cancer at sites of chronic inflammation (VIDAL-VANACLOCHA, 2009). In addition, previous epidemiological studies in humans have shown that chronic inflammation is a cofactor in many carcinogeneses, such as gastric cancer and gastric mucosal lymphoma which is linked to Helicobacter pylori, and other malignancies caused by chemical and physical agents and autoimmune reactions (EKBOM et al., 1990;

MANTOVANI et al., 2008).

2.3.1. Role of inflammation in cancer

Recent studies revealed that cancer and inflammation are connected by two pathways. The first pathway is called extrinsic, in which inflammatory or infectious diseases increase the risk for development of cancer (MANTOVANI et al., 2008). The second pathway is called the intrinsic one. This pathway has been addressed recently when evidence of inflammatory cells and mediators were noted within the tumor microenvironment without the presence of a usual cause of inflammation such as the presence of infectious agents (MANTOVANI et al., 2008).

During this pathway, the genetic alterations that cause neoplasia are responsible for generating the inflammatory changes (MANTOVANI et al., 2008). The two pathways then activate transcription factors, such as nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) which induce the production of inflammatory cytokines and chemokines which recruit inflammatory cell infiltration and augment the inflammation within the tumor microenvironment (MANTOVANI et al., 2008).

The inflammatory reaction and its components within the tumor microenvironment contribute to every aspect of carcinogenesis (GRIVENNIKOV et al., 2010). However, their detailed role in tumor development is still controversial (CHOW et al., 2012). Initial studies found that animals with impaired immune response are more susceptible to virally or chemically induced neoplasms (NOMOTO and TAKEYA, 1969; SANFORD et al., 1973; STUTMAN, 1974).

Furthermore, other investigations demonstrate the importance of cytokines such as interferon γ (IFN- γ) in protecting the host from tumor development (DIGHE et al., 1994). On the other hand, chronic inflammation significantly contributes to tumor development through several mechanisms such as enhancing angiogenesis and tissue invasion (GRIVENNIKOV et al., 2010). However, some surveys suggest that both tumor-suppressing and tumor-induced inflammation can co-exist within the same tumor (SWANN et al., 2008).

21 2.3.2. Inflammatory cell components of tumors

Often a wide range of immune cells, such as monocytes, macrophages, dendritic cells, neutrophils, eosinophils, mast cells and lymphocytes infiltrate a tumor and establish an inflammatory microenvironment (MACARTHUR et al., 2004). The infiltrating immune cells may repress neoplastic growth or may act as tumor promoters (LU et al., 2006). All of these immune cells are capable of producing an assorted array of cytokines, other mediators such as reactive oxygen species, serine and cysteine proteases, MMPs, tumor necrosis factor α (TNF-α), interleukins and interferons which elicit innate immune response, tissue remodeling and angiogenesis (COUSSENS and WERB, 2002). Macrophages represent up to 50% of the tumor mass and are considered as the major players in the connection between inflammation and neoplasms (SOLINAS et al., 2009). Macrophages most often originate from blood-derived monocytes and can be activated in response to cytokines and other signals and differentiate into two distinct types, M1 (classically activated) and M2 (alternatively activated; SCHMID and VARNER, 2012). M1 polarization occurs upon stimulation with IFN-γ and lipopolysaccharides (LPS). M1 macrophages are characterized by production of high levels of IL-12, IL-23, TNF-α and CXC chemokine ligand 10 (CXCL10) and elicit cytotoxicity against microorganisms and neoplastic cells by production of reactive oxygen intermediates (ROI) and antigen presenting cell activity (SOLINAS et al., 2009). On the other hand, when macrophages are stimulated with IL-4, IL-10, IL-13 or corticosteroids, they differentiate into the M2 phenotype which is characterized by the production of IL-10, chemokine 17 ligands (CCL17), CCL22, interleukin-1 receptor antagonist (IL-1ra) and interleukin-1 receptor (IL-1R). M2 macrophages are involved in promotion of wound healing, angiogenesis, tumor progression and control of inflammatory responses (SOLINAS et al., 2009). It is now generally accepted that M2 macrophages are the predominant phenotype of tumor associated macrophages (TAM) within the tumor microenvironment (MANTOVANI and SICA, 2010). The role of TAMs in neoplasms is still controversially discussed in the literature. TAMs in a colorectal cancer model have been reported to act proinflammatory and inhibit the proliferation of tumor cells by producing chemokines and cytokines such as IFN-γ, IL-1, and IL-6 that attract T cells, stimulate proliferation of allogeneic T cells and activate T cells associated with anti-tumor immune responses (ONG et al., 2012). On the other hand, in most tumors such as breast, prostate, ovarian, cervical and lung carcinoma and cutaneous melanoma, TAMs are considered to be anti-inflammatory and correlated with a poor prognosis (HAO et al., 2012). TAMs contribute to tumor development in several ways. They can produce many growth factors which induce the growth and motility of tumor cells

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(POLLARD, 2004). Examples of these growth factors are fibroblast growth factor (FGF), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR)-family ligands, platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β;

POLLARD, 2004). TAMs may also release growth factors such as VEGF, PDGF, TGF-β, and members of the FGF family, which can promote angiogenesis in many tumors such as gliomas, squamous cell carcinoma of the esophagus, and breast, bladder and prostate carcinoma (TANAKA et al., 2002; SIVEEN and KUTTAN, 2009; SOLINAS et al., 2009). In addition to TAMs, other inflammatory cells such as neutrophils, mast cells, eosinophils and T lymphocytes similarly have the ability to contribute to tumor progression by releasing proangiogenic factors such as VEGF, IL-8 and proteases such as matrix metalloproteinases (BELLOCQ et al., 1998; SHAMAMIAN et al., 2001; CARUSO et al., 2002; COUSSENS and WERB, 2002; LIN and POLLARD, 2004).

2.3.3. Inflammation as a target for anti-cancer therapy

The best evidence for the role of inflammation in tumor progression originates from studies which showed the preventive and tumor effect of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) on colorectal cancer and probably other cancer types (BARON and SANDLER, 2000; GARCIA-RODRIGUEZ and HUERTA-ALVAREZ, 2001). Sulindac, a member of the NSAIDs is able to regress human adenomatous colorectal polyps via apoptosis (KELLER et al., 1999). In addition, NSAIDs are able to inhibit angiogenesis in cell culture and rodent models (JONES et al., 1999). These findings suggest that NSAIDs represent a promising platform for the development of anticancer agents (THUN et al., 2002).

Tumor necrosis factor (TNF) also represents another candidate for cancer treatment (VAN HORSSEN et al., 2006). The anti-cancer actions of TNF can be due to direct cytotoxic effects and / or indirectly by altering the host stroma namely the neoplastic vasculature or the generation of a specific, cell mediated antitumor immunity (PALLADINO et al., 1987;

SHIMOMURA et al., 1988; UEDA et al., 2013). In addition, TNF can potentiate its antitumor effect through induction of cytotoxicity-related proteins such as IFN-γ (MOCELLIN et al., 2005). Despite the history of TNF and promising pre-clinical studies, TNF needs further therapeutic evaluation due to its biological effects as a mediator of endotoxic shock and endogenous pyrogen (SELBY et al., 1987). Finally, MMPs have long been associated with tumor growth, invasion and metastasis and many studies show their importance in tumor pathogenesis (EGEBLAD and WERB, 2002). Therefore, MMP-inhibitors are also representing other candidates for tumor therapy (PURCELL et al., 2002).

23 2.4. Oncolytic virus therapy

2.4.1. History

The use of viruses in the treatment of cancer came from observations that, occasionally, cancer patients with viral infections went into short periods of clinical remission, for example leukemia patients infected during their disease with influenza virus have shown clinical improvement (DOCK, 1904; PELNER et al., 1958). In 1896, a 42-year-old woman with

‘‘myelogenous leukemia’’ showed remission after a presumed influenza infection. This woman had a greatly enlarged liver and spleen, which shrank to nearly normal size, and highly elevated leukocytes counts, which dropped after the infection (DOCK, 1904). In another case, a 4-year-old boy displayed an enlarged liver, spleen and cervical lymph nodes and severely elevated leukocyte counts. His spleen and liver size returned back to normal and his leucocytes count reverted to the reference range within a few days after he was infected with chickenpox (BIERMAN et al., 1953).

In 1949, clinical trials were performed using hepatitis virus which was administered to patients suffering from Hodgkin’s disease (HOSTER et al., 1949). Some of the patients showed improvement in clinical signs and laboratory findings for at least one month (HOSTER et al., 1949). A few years later, patients with acute monocytic leukemia were injected with Epstein-Barr virus taken from cases of glandular fever (TAYLOR, 1953).

Leukemia cases showed improvement with a sharp drop in leukocyte count (TAYLOR, 1953). After these early clinical studies, many human pathogens were used in therapeutic trials against a wide range of cancers in human patients and also in tumor xenograft models (KELLY and RUSSELL, 2007). Among the viruses used adenoviruses, paramyxoviruses, picornaviruses, and poxviruses gained more attention as viral agents of cancer treatment (KELLY and RUSSELL, 2007). In November 2005, the genetically modified adenovirus H101 was the first drug for viral oncolysis approved by the Chinese authorities (GARBER et al., 2006).

2.4.2. Mechanisms of viral oncolysis

The concept behind using oncolytic viruses for tumor therapy, based on their preferable intratumoral replication as well as their potent anti-tumor effects (PRESTWICH et al., 2008).

Some viruses possess natural inherent tumor selectivity and thus they can specifically target tumor cells. Examples of these viruses include Newcastle disease virus, vesicular stomatitis virus, reovirus and measles virus (ROBERTS et al., 2006). Another group of oncolytic viruses are attenuated viruses with deleted or modified critical genes to restrict their replication to

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tumor cells and enhance their oncolytic activity, such as adenovirus and herpes simplex virus type 1 (NETTELBECK and CURIEL, 2003; LIN and NEMUNAITIS, 2004). Following infection, oncolytic viruses mediate the destruction of tumor cells by several mechanisms.

The virus can infect and multiply within the tumor cell with subsequent lysis (SINGH et al., 2012). In addition progeny virions can infect adjacent cells and destroy them (MULLEN and TANABE, 2002; BELL and MCFADDEN, 2015). The mechanisms by which oncolytic viruses induce tumor cell death are poorly understood and may show some variation from the classical schemes of apoptosis and necrosis (WOLLER et al., 2014). A necrosis-like programmed cell death mechanism has been proposed for a class of oncolytic adenoviruses called conditionally replicating adenoviruses (CRAds; ABOU EL HASSAN et al., 2004). In addition, oncolytic vaccinia virus induces necrotic cell death of ovarian cancer cells through a programmed necrosis mechanism which shares limited apoptotic features (WHILDING et al., 2013).

Several oncolytic viruses encode cytotoxic proteins which can induce lysis of infected tumor cells. Examples of these proteins are the adenovirus death protein (ADP) and the adenovirus type 5 E4 open reading frame 4 (E4orf4) protein (TOLLEFSON et al., 1996; SHTRICHMAN and KLEINBERGER, 1998).

Another mechanism is via altering the tumor microenvironment into a milieu that enhances anticancer activity (DE SILVA et al., 2010). Angiogenesis is significantly involved in tumor progression and VEGF is one of the dominant drivers of angiogenesis in neoplasms.

Therefore, inhibition of VEGF signaling represents an attractive cancer treatment (ROSKOSKI, 2007). Some viruses possess inherent antivascular properties. These viruses have the ability to infect both developing and established tumor vasculature without destroying normal vessels (ANGARITA et al., 2013). The mechanism behind this tropism for tumor vasculature is still not clear, but there are three suggested mechanisms by which oncolytic viruses affect tumor vasculature: first through direct infection of endothelial cells of tumor vasculature; second through the induction of virus-mediated immune responses which affect tumor perfusion and third, the expression of viral proteins which have antiangiogenic properties (ANGARITA et al., 2013). Pancreatic cancer cell lines infected with an adenovirus which retained the normal early region 1A (E1A) protein showed an oncolytic effect and inhibited tumor angiogenesis significantly (SAITO et al., 2006). Similarly, intravenous administration of vesicular stomatitis virus (VSV) initiates an inflammatory reaction including a neutrophil-dependent production of microclots within the tumor blood vessels and ultimately tumor vascular collapse (BREITBACH et al., 2011). Another interesting oncolytic

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virus, the replication-restricted herpes simplex virus HSV-1716, directly infects tumor endothelium and thereby exerts a direct antiangiogenic effect following intratumoral administration in ovarian carcinoma (BENENCIA et al., 2005). In addition, oncolytic viruses have been genetically engineered to carry antiangiogenic genes that either deliver antiangiogenic factors or target the expression of angiogenic proteins. Interesting examples of this principle are the oncolytic Lister vaccine strain of vaccinia virus which is armed with an endostatin-angiostatin fusion gene, the recombinant human endostatin adenovirus (Ad-hEndo) and the oncolytic herpes simplex virus expressing endostatin (YANG et al., 2010; TYSOME et al., 2011; GOODWIN et al., 2012). Other oncolytic viruses have also been developed to target the VEGF signaling pathway (KANG et al., 2008). Ad-DeltaE1-KOX is an oncolytic adenovirus expressing a VEGF promoter targeted artificial zinc-finger protein that elicited a pronounced antitumor effect against a human glioblastoma xenograft model (KANG et al., 2008).

Another promising therapeutic approach is the use of oncolytic viruses for targeting MMPs, which play an important role in tumor progression as described above (OVERALL and LOPEZ-OTIN, 2002). Infection of glioma cell lines with an oncolytic adenovirus expressing TIMP-3 called AdDelta24TIMP-3, showed enhanced oncolytic activity and significantly reduced tumor growth (LAMFERS et al., 2005). Oncolytic viruses are also able to target the tumors by activating and redirecting functional innate and adaptive immune responses against the transformed cells (CHIOCCA and RABKIN, 2014). Initially, the immune system was seen as a negative factor in oncolytic virus therapy because the immune system limits infection and replication of the virus within the neoplastic tissue (MELCHER et al., 2011).

However, a number of recent studies demonstrate that the immune system plays an important role in the efficacy of intratumoral oncolytic viral therapy, which is sometimes even more important than the direct oncolytic effect of the virus itself (PRESTWICH et al., 2008). This has been shown by using vesicular stomatitis virus (VSV), reovirus and herpes simplex virus (HSV; DIAZ et al., 2007; PRESTWICH et al., 2009; SOBOL et al., 2011; WORKENHE et al., 2014).

2.4.3. Canine distemper virus (CDV)

CDV is an enveloped, non-segmented, single-stranded RNA virus and is a member of the Morbilliviruses, thus closely related to measles virus (LAMB and KOLAKOFSKY, 2001;

VANDEVELDE and ZURBRIGGEN, 2005). CDV possesses a nucleocapsid containing the viral genome, which comprises of the nucleoprotein (N) and the polymerase complex