**3.4 ECM and angiogenesis**

As previously mentioned, anti-angiogenic therapy represent a promising strategy for cancer therapy and offers the chance to avoid resistance to chemotherapeutic treatments. J. Folkman's long-standing vision of angiogenesis as a therapeutic target has been increasingly validated in both traditional transplant tumor models and genetically engineered mouse models of cancer (Szentirmai et al., 2008). Important targets in this context are ECM molecules (Fig. 5) which can exert both pro-angiogenic or anti-angiogenic effects (Tabruyn & Griffioen, 2007).

Fig. 5. Schematic representation of the therapeutic strategies targeting angiogenesis hinging on ECM molecules; TSPs: thrombospondins; MMPs: metalloproteinases; bFGF: basic Fibroblast Growth Factor; HSPGs: Heparan Sulfate Proteoglycans; VEGF and VEGFR2: Vascular Endothelial Growth Factor and one of its receptors; MMRN2: MULTIMERIN2; ADAMTS: A Disintegrin And Metalloproteinase with Thrombospondin Motifs; PEDF: Pigment Epithelial-Derived Factor; cdk-5: cyclin dependent kinase 5.

agents (VDAs) is a potentially useful alternative strategy. These compounds that predominantly affect the tumor periphery and small tumor masses have a different mechanism of action and tolerability profile compared with conventional antiangiogenic drugs and thus may provide an additional clinical benefit for a wide patient population. VDAs act to disrupt the established tumor vasculature in order to create an extensive necrosis in the tumor core. On the basis of their mechanism of action, VDAs are divided into 2 groups. The first includes the tubulin-binding agents that selectively target tumor ECs by disrupting their cytoskeleton. The second VDAs group includes compounds related to flavone acetic acid and they act in two ways; they induce ECs apoptosis and indirectly increase the intra-tumoral concentrations of TNFα and other cytokines, as well as nitric oxide, leading to an inhibition of tumor flow (Boehm et al., 2010; McKeage & Baguley, 2010).

As previously mentioned, anti-angiogenic therapy represent a promising strategy for cancer therapy and offers the chance to avoid resistance to chemotherapeutic treatments. J. Folkman's long-standing vision of angiogenesis as a therapeutic target has been increasingly validated in both traditional transplant tumor models and genetically engineered mouse models of cancer (Szentirmai et al., 2008). Important targets in this context are ECM molecules (Fig. 5) which can

Fig. 5. Schematic representation of the therapeutic strategies targeting angiogenesis hinging on ECM molecules; TSPs: thrombospondins; MMPs: metalloproteinases; bFGF: basic Fibroblast Growth Factor; HSPGs: Heparan Sulfate Proteoglycans; VEGF and VEGFR2: Vascular Endothelial Growth Factor and one of its receptors; MMRN2: MULTIMERIN2; ADAMTS: A Disintegrin And Metalloproteinase with Thrombospondin Motifs; PEDF:

Pigment Epithelial-Derived Factor; cdk-5: cyclin dependent kinase 5.

exert both pro-angiogenic or anti-angiogenic effects (Tabruyn & Griffioen, 2007).

**3.4 ECM and angiogenesis** 

*Pro-angiogenic ECM proteins -* Many ECM proteins surrounding the vasculature including collagens, laminins, and fibronectins are pro-angiogenic promoting EC survival, proliferation, migration and tube formation. Pro-angiogenic factors such as VEGF, bFGF and TGF-β are bound and sequestered by the ECM via the heparan-like glycosaminoglycans. Fibronectin, a fairly ubiquitous and abundant ECM protein that becomes assembled into fibrils at the cell surface, is necessary for vasculogenesis, in fact if this gene is knocked out mice die before birth due among others, to vascular bed defects. An alternatively-spliced form of fibronectin that contains an extra B domain has been found in fetal and neoplastic tissues but not in normal adult tissues; this isoform was found to be synthesized by the vascular cells in malignant astrocytoma (Castellani et al., 2002) and its expression appears to be a precise diagnostic marker of the highest grade of glioma or glioblastoma. Also other studies demonstrate that this isoform may be a good marker of angiogenesis (Santimaria et al., 2003) and could also be a therapeutic target.

Osteopontin, a secreted cell attachment protein, seems to be over-expressed in association with increased tumor angiogenesis (Hirama et al., 2003). The proangiogenic function of osteopontin can be attributed to its ability to promote VEGF-directed dermal microvascular EC migration (Senger et al., 1996) and to increase MMP-2 levels in an RGD-dependent manner (Teti et al., 1998). Additionally, osteopontin-bound integrin αvβ3 inhibits NF-kBdependent EC apoptosis (Cooper et al., 2002).

Tenascin-C is a glycoprotein composed of six subunits covalently associated by disulfide bonds. Different human Tenascin-C isoforms are generated by alternative splicing with aberrantly regulation in neoplastic tissues (Jones & Jones, 2000). It is expressed around angiogenic vessels in many tumors and there is evidence that it promotes and regulates angiogenesis *in vitro* and *in vivo*. Indeed the antibodies directed against Tenascin-C in glioma patients induced a significant inhibition of tumor angiogenesis.

HSPGs are necessary for stable binding of the proangiogenic growth factor bFGF to its receptor (Ornitz et al., 1992) and alterations in HS glycosamminoglycan in breast carcinoma have been shown to result in an increase in bFGF binding and receptor complex assembly.

Perlecan deposited along blood vessels basement membrane is thought to mediate structural and functional interactions with different molecules (Iozzo, 2005; Mongiat et al., 2003a). Intact perlecan is thought to be a pro-angiogenic HSPG as: *i)* its expression is altered during embryonic vasculogenesis and in neoplasia (Tapanadechopone et al., 2001; Zhou et al., 2004) perlecan-null mice show severe and sometimes fatal vascular and chondrogenic defects (Costell et al., 1999); *iii)* antisense targeting of perlecan blocks tumor growth and angiogenesis *in vivo* (Sharma et al., 1998); and *iv)* increased perlecan expression stimulates angiogenesis (Jiang & Couchman, 2003)*.* Tumor cell secreted perlecan is thought to promote EC sprouting and proliferation, thereby promoting angiogenesis (Jiang et al., 2004). Interestingly a degradation product of perlecan (endorepellin) exerts an opposite effect (Mongiat et al., 2003b).

Transmembrane chondroitin sulphate proteoglycan NG2 is another protein involved in tumor angiogenesis and has been shown to promote EC spreading perycite function (Ozerdem & Stallcup, 2003). An additional proangiogenic mechanism by which NG2 promotes angiogenesis includes its ability to bind and sequester angiostatin, thus blocking its antiangiogenic function (Chekenya et al., 2002). All these molecules may represent important tools for the development of drugs able to counteract blood vessel formation.

*Anti-angiogenic ECM proteins -* Among the molecules that counteract blood vessel formation thrombospondin-1 (TSP-1) and thrombospondin-2 (TSP-2) are the best studied and their

Therapeutical Cues from the Tumor Microenvironment 251

The targeting of ECM components and ECM-remodeling enzymes that would prevent changes of the stroma homeostasis promoting cancer progression has received much attention and is becoming an increasingly attractive therapeutic approach for preventing

Fig. 6. Schematic representation of the therapeutic strategies targeting the metastatic processes. E-cdh: E-cadherin; EMT: Epithelial-Mesenchymal Transition; BM: Basement Membrane; WK-UT1: uPA (urokinase-type Plasminogen Activator) inhibitor; TNC:

Tenascin-c; LOX: lysil oxidase activity; BAPN: β-aminoproprionitrile LOX inhibitor; Ab0023 inhibitory monoclonal antibody specific for the LOX family member LOXL2; N-cdh: Ncadherin; hOS: hyaluronan oligosaccharides; HA: Hyaluronan; MU: HA syntetase inhibitor 4-methylumbelliferone; MMPs: metalloproteinases; SB-3CT: MMP-2 and -9 inhibitor; FN: fibronectin; FN-EDB: oncofetal form of fibronectin; TNFα: Tumor Necrosis Factor α.

*ECM components* - The peculiar expression of tumor-associated ECM components may offer the opportunity to develop new strategies for cancer treatment, either targeting their function associated to tumor progression or by exploiting their specific localization to delivery bioactive molecules to the tumors. The ECM components are highly abundant in tumors and are often more stable than cell surface antigens. One example of this type of approach is offered by tenascin-C (TNC), which exerts different effects both on tumor cells and also on the many cell types within the tumor, thus affecting cancer progression. The effects on tumor and stromal cells are exerted very early in tumorigenesis and persist during tumor progression. Currently, the most promising approach is to target TNC with a specific mAb (81C6) coupled to radioactive molecules. This strategy has been successful in patients with recurrent primary and metastatic brain tumors, and is currently in clinical trials (Xing et al., 2010). A similar approach targeting the oncofetal form of fibronectin (FN-EDB) containing an extra-domain, and often up-regulated in tumors was particularly successful. Anti-FN-EDB antibodies show specific localization to a range of tumors, including brain, lung and colorectal cancers. This antibody has been used as a vehicle for TNFα and has been

shown to induce necrosis in tumors (Allen & Louise, 2011).

**3.5 ECM molecules involved in invasion and metastasis.** 

cancer progression and metastasis (Fig. 6).

expression in tumor tissues is often inversely correlated with angiogenesis (Adams, 2001; Adams & Lawler, 2004; Armstrong & Bornstein, 2003)*.* The mechanisms by which they can inhibit angiogenesis include: *i)* the induction of EC apoptosis by the binding of TSP-1, and potentially TSP-2, to CD36, thereby inducing Fas ligand expression (Jimenez et al., 2000; Nor et al., 2000); *ii)* the clearance of MMP-2 following lysosomal degradation of the TSP-2/MMP-2 complex (Armstrong et al., 2002; Rodriguez-Manzaneque et al., 2001) and *iii)* inhibition of EC proliferation (Armstrong et al., 2002).

Another extensively studied molecule is angiostatin, the cleavage product of plasminogen, the only not ECM antiangiogenic protein among the molecules described above. Angiostatin was shown to inhibit EC proliferation and motility and to down-regulate the protein level of cyclin-dependent kinase 5 (cdk5), a cdk absent in quiescent EC and induced by bFGF (Sharma et al., 2004). Angiostatin also acts by upregulating the mRNA levels of FasL and reducing the level of c-Flip which activates the extrinsic apoptotic pathway.

Other endogenous angiogenesis inhibitors include cryptic fragments from ECM molecules such as endostatin, a specific and potent anti-angiogenic factor generated from the proteolytic cleavage of collagen XVIII by matrix metalloproteinases, elastases or cathepsins (Kim et al., 2009). Endostatin inhibits EC proliferation and migration and causes EC G1 arrest and apoptotic cell death (Dhanabal et al., 1999). These properties have provided clues for development of an anti-tumor strategy with proven therapeutic effectiveness in numerous models of neoplasia.

Pigment epithelial-derived factor (PEDF) was initially identified as an antiangiogenic protein. Its high expression has been linked to decreased microvessel density and suppression of tumor growth in a number of tumors including glioma as well as prostate carcinoma and melanoma (Abe et al., 2004). In support of a role for PEDF in inhibiting angiogenesis, increased microvessel density is found in PEDF deficient mouse tissues (Doll et al., 2003), in addition PEDF inhibits cornea neovascularisation. The mechanism whereby PEDF acts to inhibit angiogenesis likely resides on its ability to bind collagen and to potentially interfere with the adhesion of cells to the collagen (Meyer et al., 2002) as well as triggering Fas up-regulation and down-regulation of the VEGF mRNA levels (Takenaka et al., 2005).

MULTIMERIN2 (MMRN2), also known as EndoGlyx-1, is an ECM glycoprotein exclusively expressed in the blood vessel in close proximity with the endothelium. In neoplastic tissues, MMRN2 is consistently found to be deposited along tumor capillaries and, in certain tumors, in the "hot spots" of neoangiogenesis (Belien et al., 1999). This protein is characterized by a short cluster of charged amino acids (10 out of 27 residues) located between the coiled-coil region and the C1q-like domain. The basic amino acids are arranged in a sequence similar to that of the consensus motifs responsible for the ionic interactions with glucosaminoglycans, such as heparin and heparan sulfate (Hileman et al., 1998) and are also found in heparin binding proteins such as the von Willebrand factor (Sobel et al., 1992). Given its deposition in tight contact with the EC surface, we have hypothesized the involvement of this protein in the regulation of angiogenesis and demonstrated that MMRN2 functions as a homeostatic molecule for ECs preventing their sprouting to form new vessels (unpublished results). This strong anti-angiogenic effect is reflected in a potent *in vivo* anti-tumor activity effect following treatment with MMRN2. For these reasons we are confident that this molecule represents a promising tool for the development of novel approaches to impair angiogenesis and tumor growth.

expression in tumor tissues is often inversely correlated with angiogenesis (Adams, 2001; Adams & Lawler, 2004; Armstrong & Bornstein, 2003)*.* The mechanisms by which they can inhibit angiogenesis include: *i)* the induction of EC apoptosis by the binding of TSP-1, and potentially TSP-2, to CD36, thereby inducing Fas ligand expression (Jimenez et al., 2000; Nor et al., 2000); *ii)* the clearance of MMP-2 following lysosomal degradation of the TSP-2/MMP-2 complex (Armstrong et al., 2002; Rodriguez-Manzaneque et al., 2001) and *iii)* inhibition of

Another extensively studied molecule is angiostatin, the cleavage product of plasminogen, the only not ECM antiangiogenic protein among the molecules described above. Angiostatin was shown to inhibit EC proliferation and motility and to down-regulate the protein level of cyclin-dependent kinase 5 (cdk5), a cdk absent in quiescent EC and induced by bFGF (Sharma et al., 2004). Angiostatin also acts by upregulating the mRNA levels of FasL and

Other endogenous angiogenesis inhibitors include cryptic fragments from ECM molecules such as endostatin, a specific and potent anti-angiogenic factor generated from the proteolytic cleavage of collagen XVIII by matrix metalloproteinases, elastases or cathepsins (Kim et al., 2009). Endostatin inhibits EC proliferation and migration and causes EC G1 arrest and apoptotic cell death (Dhanabal et al., 1999). These properties have provided clues for development of an anti-tumor strategy with proven therapeutic effectiveness in

Pigment epithelial-derived factor (PEDF) was initially identified as an antiangiogenic protein. Its high expression has been linked to decreased microvessel density and suppression of tumor growth in a number of tumors including glioma as well as prostate carcinoma and melanoma (Abe et al., 2004). In support of a role for PEDF in inhibiting angiogenesis, increased microvessel density is found in PEDF deficient mouse tissues (Doll et al., 2003), in addition PEDF inhibits cornea neovascularisation. The mechanism whereby PEDF acts to inhibit angiogenesis likely resides on its ability to bind collagen and to potentially interfere with the adhesion of cells to the collagen (Meyer et al., 2002) as well as triggering Fas up-regulation and down-regulation of the VEGF mRNA levels (Takenaka et

MULTIMERIN2 (MMRN2), also known as EndoGlyx-1, is an ECM glycoprotein exclusively expressed in the blood vessel in close proximity with the endothelium. In neoplastic tissues, MMRN2 is consistently found to be deposited along tumor capillaries and, in certain tumors, in the "hot spots" of neoangiogenesis (Belien et al., 1999). This protein is characterized by a short cluster of charged amino acids (10 out of 27 residues) located between the coiled-coil region and the C1q-like domain. The basic amino acids are arranged in a sequence similar to that of the consensus motifs responsible for the ionic interactions with glucosaminoglycans, such as heparin and heparan sulfate (Hileman et al., 1998) and are also found in heparin binding proteins such as the von Willebrand factor (Sobel et al., 1992). Given its deposition in tight contact with the EC surface, we have hypothesized the involvement of this protein in the regulation of angiogenesis and demonstrated that MMRN2 functions as a homeostatic molecule for ECs preventing their sprouting to form new vessels (unpublished results). This strong anti-angiogenic effect is reflected in a potent *in vivo* anti-tumor activity effect following treatment with MMRN2. For these reasons we are confident that this molecule represents a promising tool for the development of novel

reducing the level of c-Flip which activates the extrinsic apoptotic pathway.

EC proliferation (Armstrong et al., 2002).

numerous models of neoplasia.

approaches to impair angiogenesis and tumor growth.

al., 2005).

#### **3.5 ECM molecules involved in invasion and metastasis.**

The targeting of ECM components and ECM-remodeling enzymes that would prevent changes of the stroma homeostasis promoting cancer progression has received much attention and is becoming an increasingly attractive therapeutic approach for preventing cancer progression and metastasis (Fig. 6).

Fig. 6. Schematic representation of the therapeutic strategies targeting the metastatic processes. E-cdh: E-cadherin; EMT: Epithelial-Mesenchymal Transition; BM: Basement Membrane; WK-UT1: uPA (urokinase-type Plasminogen Activator) inhibitor; TNC: Tenascin-c; LOX: lysil oxidase activity; BAPN: β-aminoproprionitrile LOX inhibitor; Ab0023 inhibitory monoclonal antibody specific for the LOX family member LOXL2; N-cdh: Ncadherin; hOS: hyaluronan oligosaccharides; HA: Hyaluronan; MU: HA syntetase inhibitor 4-methylumbelliferone; MMPs: metalloproteinases; SB-3CT: MMP-2 and -9 inhibitor; FN: fibronectin; FN-EDB: oncofetal form of fibronectin; TNFα: Tumor Necrosis Factor α.

*ECM components* - The peculiar expression of tumor-associated ECM components may offer the opportunity to develop new strategies for cancer treatment, either targeting their function associated to tumor progression or by exploiting their specific localization to delivery bioactive molecules to the tumors. The ECM components are highly abundant in tumors and are often more stable than cell surface antigens. One example of this type of approach is offered by tenascin-C (TNC), which exerts different effects both on tumor cells and also on the many cell types within the tumor, thus affecting cancer progression. The effects on tumor and stromal cells are exerted very early in tumorigenesis and persist during tumor progression. Currently, the most promising approach is to target TNC with a specific mAb (81C6) coupled to radioactive molecules. This strategy has been successful in patients with recurrent primary and metastatic brain tumors, and is currently in clinical trials (Xing et al., 2010). A similar approach targeting the oncofetal form of fibronectin (FN-EDB) containing an extra-domain, and often up-regulated in tumors was particularly successful. Anti-FN-EDB antibodies show specific localization to a range of tumors, including brain, lung and colorectal cancers. This antibody has been used as a vehicle for TNFα and has been shown to induce necrosis in tumors (Allen & Louise, 2011).

Therapeutical Cues from the Tumor Microenvironment 253

Likewise, increasing the cadherins expression could inhibit cell invasion by strengthening cell-cell adhesions, thus preventing the cell from escaping the primary tumor. Indeed, forcing the overexpression of cadherins has been the focus of some anti-invasion therapy

*Modification enzymes* – The inhibition of LOX activity reduces primary tumor growth and mechanotransduction in the mammary epithelium. LOX inhibition prevents the invasion of tumor cells *in vivo* and abrogates bone marrow derived cells recruitment and the establishment of metastasis. It also destabilizes already-formed metastases reducing their growth and improves patients' survival (Cox & Erler, 2011). LOX inhibitors are currently in development for clinical use. For example, a small-molecule lysyl oxidase inhibitor called βaminoproprionitrile (BAPN) is available for clinical studies and it prevents the invasion of metastatic breast cancer and melanoma cell lines *in vitro* (Baker et al., 2011). More recently the efficacy of BAPN was outperformed by the use of an inhibitory mAb (AB0023) specific for LOXL2, a member of the LOX family, that was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models (Barry-Hamilton et

*Proteases* - On the basis of the pivotal roles that MMPs play in several steps of cancer progression, many efforts have been spent with the aim of developing safe and effective agents targeting MMPs. Several generations of MMPs inhibitors have been tested in phase II clinical trials, including peptidomimetics, non-peptidomimetics and tetracycline derivates (Gialeli et al., 2011). Unfortunately so far the results have been disappointing; all the clinical trials involving the use of MMPs inhibitors have failed to show significant efficacy and to increase the rates of patients' survival (Cox & Erler, 2011). The poor success of this approach largely depended on the toxicity of the compounds for normal tissues, to the conflicting roles of MMPs in both promoting and reducing metastatic dissemination, and to the functional redundancy between the family members. Nevertheless, the ongoing challenge is the development of a new generation of highly specific MMPs inhibitors, as for example SB-3CT that binds to the active site of MMP-2 and -9 and re-establishes the pro-enzyme structure. These new compounds aim at targeting the enzymes involved in ECM remodeling avoiding unwanted side effects. To increase the specificity of MMPs inhibitors, the future of drug development comprises the use of molecules targeting specific exo-sites, specific binding sites outside the active domain of the metalloprotease involved in substrate

Finally, also the uPA system (uPAS), due its involvement in many steps of cancer progression, including angiogenesis, cell proliferation, invasion and growth at the metastatic site, represents a suitable source for the development of new anti-cancer drugs. Several therapeutical approaches aimed at inhibiting the uPA/uPAR functions lead to antitumor effects in xenograft models, including selective inhibitors of uPA activity such as WK-UK1, antagonist peptides, mAbs able to prevent uPA/uPAR binding and gene therapy techniques aimed at silencing uPA/uPAR expression. However, although promising, all these strategies need definitive confirmation in humans as, up to now, only few uPA

The cells and molecules of the tumor microenvironment hold the potential for the development of innovative therapeutic approaches. Many of the molecules and approaches

inhibitors have been used in clinical trial (Ulisse et al., 2009).

studies (Junxia et al., 2010).

selection (Gialeli et al., 2011).

**4. Conclusions** 

al., 2010).

*Cell-cell and cell-ECM interactions* - Integrin-targeted therapeutics have recently been proved beneficial in delivering chemotherapeutics, oncolytic viruses, proapoptotic peptides and redionucleotides to both tumor cells and the supporting vasculature. Recent studies showed that delivery of targeted nanoparticles loaded with doxorubicin to integrin αVβ3-positive tumor vasculature inhibited metastases while eliminating the toxicity and patients' weight loss associated with the systemic administration of this drug (Desgrosellier & Cheresh, 2010).

HA contributes to cancer progression in many types of carcinomas and it is up-regulated in tumor stroma; its activated CD44 receptor is over-expressed in solid tumors unlike the nontumorigenic counterparts and this characteristic may also be exploited to develop new therapies. The membrane receptor CD44 can internalize HA, thus, HA-carrying drugs have the potential to be used as targeted drugs. HA–drug conjugates are internalized via CD44, and the drug is released and activated mainly by intracellular enzymatic hydrolysis. Several preclinical studies have shown that HA chemically conjugated to cytotoxic agents improves the anticancer properties of the agent in vitro (Sironen et al., 2011). Manipulation of HA synthesis alters the tumorigenicity of malignant tumors, including breast cancer. Recently, the HA syntetase inhibitor 4-methylumbelliferone (MU), which has been reported to inhibit HA synthesis dose-dependently in several cell types, was shown to exert anti-tumor effects inhibiting *in vitro* cell proliferation, migration and invasion (Urakawa et al., 2011). MU suppresses intra-osseous tumor growth, suggesting its potential as a therapeutic candidate for established breast cancer-derived bone metastasis. Additive effects of MU in combination with trastuzumab for the treatment of trastuzumab-resistant breast cancers have also been reported both in *in vitro* and *in vivo* experiments.

The expression of integrins by various cell types involved in tumor progression and their ability to crosstalk with growth factor receptors has made them appealing therapeutic targets. Integrin inhibitors have been extensively studied as anti-cancer agents because integrins play a dual function: they are involved in invasion of tumor cells out of the primary tumor site to the metastatic sites, but they also regulate ECs migration into the tumor mass during angiogenesis (Veiseh et al., 2011). Velociximab, a chimeric mAb that inhibits integrin α5β1, has been used in clinical phase II trials for renal cell carcinoma, metastatic melanoma and pancreatic cancer. The rationale of using velociximab in cancer therapy is based on the fact that this specific integrin is expressed by endothelial cells and up-regulated in tumor vasculature (Jarvelainen et al., 2009). The use of etaracizumab, a blocking antibody for αVβ3 integrin, directly affects not only cancer cell growth and angiogenesis, but also osteoclast attachment, suggesting a possible efficacy in reducing bone metastasis (Desgrosellier & Cheresh, 2010).

Another strategy is based on the development of small-molecules compounds or peptide mimetics in order to block the integrin signaling pathways activated by ECM remodeling. A successful example of this strategy is cilengitide, a cyclic RGD peptide that inhibits integrins αVβ3 and αVβ5, that has been proven promising in lung and prostate cancer patients and for the treatment of glioblastoma (Allen & Louise, 2011). ATN-61 is a non-RGD-based peptide inhibitor of integrin α5β1 blocking breast cancer growth and metastasis and, in combination with fluorouracil, significantly reduces liver metastasis in mouse models of colon cancer (Desgrosellier & Cheresh, 2010). Targeting other types of ECM receptors has also been attempted. For example the administration of hyaluronan oligosaccharides (hOS), which interfere with the CD44-hyaluronan interaction are effective inhibitors of the progression of several tumor types, including mammary and lung carcinoma.

*Cell-cell and cell-ECM interactions* - Integrin-targeted therapeutics have recently been proved beneficial in delivering chemotherapeutics, oncolytic viruses, proapoptotic peptides and redionucleotides to both tumor cells and the supporting vasculature. Recent studies showed that delivery of targeted nanoparticles loaded with doxorubicin to integrin αVβ3-positive tumor vasculature inhibited metastases while eliminating the toxicity and patients' weight loss associated with the systemic administration of this drug (Desgrosellier & Cheresh,

HA contributes to cancer progression in many types of carcinomas and it is up-regulated in tumor stroma; its activated CD44 receptor is over-expressed in solid tumors unlike the nontumorigenic counterparts and this characteristic may also be exploited to develop new therapies. The membrane receptor CD44 can internalize HA, thus, HA-carrying drugs have the potential to be used as targeted drugs. HA–drug conjugates are internalized via CD44, and the drug is released and activated mainly by intracellular enzymatic hydrolysis. Several preclinical studies have shown that HA chemically conjugated to cytotoxic agents improves the anticancer properties of the agent in vitro (Sironen et al., 2011). Manipulation of HA synthesis alters the tumorigenicity of malignant tumors, including breast cancer. Recently, the HA syntetase inhibitor 4-methylumbelliferone (MU), which has been reported to inhibit HA synthesis dose-dependently in several cell types, was shown to exert anti-tumor effects inhibiting *in vitro* cell proliferation, migration and invasion (Urakawa et al., 2011). MU suppresses intra-osseous tumor growth, suggesting its potential as a therapeutic candidate for established breast cancer-derived bone metastasis. Additive effects of MU in combination with trastuzumab for the treatment of trastuzumab-resistant breast cancers

The expression of integrins by various cell types involved in tumor progression and their ability to crosstalk with growth factor receptors has made them appealing therapeutic targets. Integrin inhibitors have been extensively studied as anti-cancer agents because integrins play a dual function: they are involved in invasion of tumor cells out of the primary tumor site to the metastatic sites, but they also regulate ECs migration into the tumor mass during angiogenesis (Veiseh et al., 2011). Velociximab, a chimeric mAb that inhibits integrin α5β1, has been used in clinical phase II trials for renal cell carcinoma, metastatic melanoma and pancreatic cancer. The rationale of using velociximab in cancer therapy is based on the fact that this specific integrin is expressed by endothelial cells and up-regulated in tumor vasculature (Jarvelainen et al., 2009). The use of etaracizumab, a blocking antibody for αVβ3 integrin, directly affects not only cancer cell growth and angiogenesis, but also osteoclast attachment, suggesting a possible efficacy in reducing bone

Another strategy is based on the development of small-molecules compounds or peptide mimetics in order to block the integrin signaling pathways activated by ECM remodeling. A successful example of this strategy is cilengitide, a cyclic RGD peptide that inhibits integrins αVβ3 and αVβ5, that has been proven promising in lung and prostate cancer patients and for the treatment of glioblastoma (Allen & Louise, 2011). ATN-61 is a non-RGD-based peptide inhibitor of integrin α5β1 blocking breast cancer growth and metastasis and, in combination with fluorouracil, significantly reduces liver metastasis in mouse models of colon cancer (Desgrosellier & Cheresh, 2010). Targeting other types of ECM receptors has also been attempted. For example the administration of hyaluronan oligosaccharides (hOS), which interfere with the CD44-hyaluronan interaction are effective inhibitors of the progression of

have also been reported both in *in vitro* and *in vivo* experiments.

several tumor types, including mammary and lung carcinoma.

metastasis (Desgrosellier & Cheresh, 2010).

2010).

Likewise, increasing the cadherins expression could inhibit cell invasion by strengthening cell-cell adhesions, thus preventing the cell from escaping the primary tumor. Indeed, forcing the overexpression of cadherins has been the focus of some anti-invasion therapy studies (Junxia et al., 2010).

*Modification enzymes* – The inhibition of LOX activity reduces primary tumor growth and mechanotransduction in the mammary epithelium. LOX inhibition prevents the invasion of tumor cells *in vivo* and abrogates bone marrow derived cells recruitment and the establishment of metastasis. It also destabilizes already-formed metastases reducing their growth and improves patients' survival (Cox & Erler, 2011). LOX inhibitors are currently in development for clinical use. For example, a small-molecule lysyl oxidase inhibitor called βaminoproprionitrile (BAPN) is available for clinical studies and it prevents the invasion of metastatic breast cancer and melanoma cell lines *in vitro* (Baker et al., 2011). More recently the efficacy of BAPN was outperformed by the use of an inhibitory mAb (AB0023) specific for LOXL2, a member of the LOX family, that was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models (Barry-Hamilton et al., 2010).

*Proteases* - On the basis of the pivotal roles that MMPs play in several steps of cancer progression, many efforts have been spent with the aim of developing safe and effective agents targeting MMPs. Several generations of MMPs inhibitors have been tested in phase II clinical trials, including peptidomimetics, non-peptidomimetics and tetracycline derivates (Gialeli et al., 2011). Unfortunately so far the results have been disappointing; all the clinical trials involving the use of MMPs inhibitors have failed to show significant efficacy and to increase the rates of patients' survival (Cox & Erler, 2011). The poor success of this approach largely depended on the toxicity of the compounds for normal tissues, to the conflicting roles of MMPs in both promoting and reducing metastatic dissemination, and to the functional redundancy between the family members. Nevertheless, the ongoing challenge is the development of a new generation of highly specific MMPs inhibitors, as for example SB-3CT that binds to the active site of MMP-2 and -9 and re-establishes the pro-enzyme structure. These new compounds aim at targeting the enzymes involved in ECM remodeling avoiding unwanted side effects. To increase the specificity of MMPs inhibitors, the future of drug development comprises the use of molecules targeting specific exo-sites, specific binding sites outside the active domain of the metalloprotease involved in substrate selection (Gialeli et al., 2011).

Finally, also the uPA system (uPAS), due its involvement in many steps of cancer progression, including angiogenesis, cell proliferation, invasion and growth at the metastatic site, represents a suitable source for the development of new anti-cancer drugs. Several therapeutical approaches aimed at inhibiting the uPA/uPAR functions lead to antitumor effects in xenograft models, including selective inhibitors of uPA activity such as WK-UK1, antagonist peptides, mAbs able to prevent uPA/uPAR binding and gene therapy techniques aimed at silencing uPA/uPAR expression. However, although promising, all these strategies need definitive confirmation in humans as, up to now, only few uPA inhibitors have been used in clinical trial (Ulisse et al., 2009).

### **4. Conclusions**

The cells and molecules of the tumor microenvironment hold the potential for the development of innovative therapeutic approaches. Many of the molecules and approaches

Therapeutical Cues from the Tumor Microenvironment 255

Belien, J.A., Somi, S., de Jong, J.S., van Diest, P.J. & Baak, J.P. (1999). Fully automated

Bishop, J.R., Schuksz, M. & Esko, J.D. (2007). Heparan sulphate proteoglycans fine-tune

Boehm, S., Rothermundt, C., Hess, D. & Joerger, M. (2010). Antiangiogenic drugs in

Bouzin, C. & Feron, O. (2007). Targeting tumor stroma and exploiting mature tumor vasculature to improve anti-cancer drug delivery, *Drug Resist. Updat.* 10, 109-120 Brooks, S.A., Lomax-Browne, H.J., Carter, T.M., Kinch, C.E. & Hall, D.M. (2010). Molecular

Campbell, N.E., Kellenberger, L., Greenaway, J., Moorehead, R.A., Linnerth-Petrik, N.M. &

Carmeliet, P., Lampugnani, M.G., Moons, L., Breviario, F., Compernolle, V., Bono, F.,

Castellani, P., Borsi, L., Carnemolla, B., Biro, A., Dorcaratto, A., Viale, G.L., Neri, D. & Zardi, L.

Chekenya, M., Hjelstuen, M., Enger, P.O., Thorsen, F., Jacob, A.L., Probst, B., Haraldseth, O.,

Chetty, C., Lakka, S.S., Bhoopathi, P., Kunigal, S., Geiss, R. & Rao, J.S. (2008). Tissue

metalloproteinase 2-down-regulated lung cancer, *Cancer Res* 68, 4736-4745 Chlenski, A., Liu, S., Crawford, S.E., Volpert, O.V., DeVries, G.H., Evangelista, A., Yang, Q.,

Chua, C.C., Rahimi, N., Forsten-Williams, K. & Nugent, M.A. (2004). Heparan sulfate

Coghlin, C. & Murray, G.I. (2010). Current and emerging concepts in tumour metastasis, *J* 

Cooper, C.R., Chay, C.H. & Pienta, K.J. (2002). The role of alpha(v)beta(3) in prostate cancer

Costell, M., Gustafsson, E., Aszodi, A., Morgelin, M., Bloch, W., Hunziker, E., Addicks, K.,

Cox, T.R. & Erler, J.T. (2011). Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer, *Dis. Model. Mech.* 4, 165-178

extracellular signal-regulated kinases 1 and 2, *Circ Res* 94, 316-323

some basement membranes, *J Cell Biol* 147, 1109-1122

sections in invasive breast cancer, *J Clin Pathol* 52, 184-192

interactions in cancer cell metastasis, *Acta Histochem.* 112, 3-25

mediated endothelial survival and angiogenesis, *Cell* 98, 147-157

mammalian physiology, *Nature* 446, 1030-1037

303-309

2010, 586905

7357-7363

*Pathol* 222, 1-15

angiostatin, *FASEB J* 16, 586-588

progression, *Neoplasia.* 4, 191-194

microvessel counting and hot spot selection by image processing of whole tumour

oncology: a focus on drug safety and the elderly - a mini-review, *Gerontology* 56,

Petrik, J. (2010). Extracellular matrix proteins and tumor angiogenesis, *J Oncol.*

Balconi, G., Spagnuolo, R., Oosthuyse, B., Dewerchin, M., Zanetti, A., Angellilo, A., Mattot, V., Nuyens, D., Lutgens, E., Clotman, F., de Ruiter, M.C., Gittenberger-de, G.A., Poelmann, R., Lupu, F., Herbert, J.M., Collen, D. & Dejana, E. (1999). Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-

(2002). Differentiation between high- and low-grade astrocytoma using a human recombinant antibody to the extra domain-B of fibronectin, *Am J Pathol* 161, 1695-1700

Pilkington, G., Butt, A., Levine, J.M. & Bjerkvig, R. (2002). NG2 proteoglycan promotes angiogenesis-dependent tumor growth in CNS by sequestering

inhibitor of metalloproteinase 3 suppresses tumor angiogenesis in matrix

Salwen, H.R., Farrer, R., Bray, J. & Cohn, S.L. (2002). SPARC is a key Schwannianderived inhibitor controlling neuroblastoma tumor angiogenesis, *Cancer Res* 62,

proteoglycans function as receptors for fibroblast growth factor-2 activation of

Timpl, R. & Fassler, R. (1999). Perlecan maintains the integrity of cartilage and

that we have taken into account in this chapter are currently in clinical trials, while some have only been suggested based on preliminary results and require further investigations. To avoid problems of toxicity and resistance to the treatments the future of cancer therapy is likely to involve the use of combinatorial treatments that will include conventional chemotherapeutic agents at lower doses and novel molecules able to halt tumor development with little or no toxicity for the patient.

#### **5. References**


that we have taken into account in this chapter are currently in clinical trials, while some have only been suggested based on preliminary results and require further investigations. To avoid problems of toxicity and resistance to the treatments the future of cancer therapy is likely to involve the use of combinatorial treatments that will include conventional chemotherapeutic agents at lower doses and novel molecules able to halt tumor

Abe, R., Shimizu, T., Yamagishi, S., Shibaki, A., Amano, S., Inagaki, Y., Watanabe, H.,

Abrams, T.J., Lee, L.B., Murray, L.J., Pryer, N.K. & Cherrington, J.M. (2003). SU11248 inhibits

Adams, J.C. (2001). Thrombospondins: multifunctional regulators of cell interactions, *Annu.* 

Allavena, P., Signorelli, M., Chieppa, M., Erba, E., Bianchi, G., Marchesi, F., Olimpio, C.O.,

Allen, M. & Louise, J.J. (2011). Jekyll and Hyde: the role of the microenvironment on the

Armstrong, L.C., Bjorkblom, B., Hankenson, K.D., Siadak, A.W., Stiles, C.E. & Bornstein, P.

Armstrong, L.C. & Bornstein, P. (2003). Thrombospondins 1 and 2 function as inhibitors of

Augsten, M., Hagglof, C., Pena, C. & Ostman, A. (2010). A digest on the role of the tumor microenvironment in gastrointestinal cancers, *Cancer Microenviron.* 3, 167-176 Baker, A.M., Cox, T.R., Bird, D., Lang, G., Murray, G.I., Sun, X.F., Southall, S.M., Wilson, J.R.

Barry-Hamilton, V., Spangler, R., Marshall, D., McCauley, S., Rodriguez, H.M., Oyasu, M.,

caspase-independent mechanism, *Mol. Biol Cell* 13, 1893-1905

metastasis of colorectal cancer, *J Natl. Cancer Inst.* 103, 407-424

Adams, J.C. & Lawler, J. (2004). The thrombospondins, *Int. J Biochem. Cell Biol* 36, 961-968 Adderley, S.R. & Fitzgerald, D.J. (2000). Glycoprotein IIb/IIIa antagonists induce apoptosis in rat cardiomyocytes by caspase-3 activation, *J Biol Chem.* 275, 5760-5766 Allavena, P., Sica, A., Garlanda, C. & Mantovani, A. (2008). The Yin-Yang of tumor-

human small cell lung cancer, *Mol. Cancer Ther.* 2, 471-478

Sugawara, H., Nakamura, H., Takeuchi, M., Imaizumi, T. & Shimizu, H. (2004). Overexpression of pigment epithelium-derived factor decreases angiogenesis and inhibits the growth of human malignant melanoma cells in vivo, *Am J Pathol* 164,

KIT and platelet-derived growth factor receptor beta in preclinical models of

associated macrophages in neoplastic progression and immune surveillance,

Bonardi, C., Garbi, A., Lissoni, A., de, B.F., Jimeno, J. & D'Incalci, M. (2005). Antiinflammatory properties of the novel antitumor agent yondelis (trabectedin): inhibition of macrophage differentiation and cytokine production, *Cancer Res* 65,

(2002). Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a

& Erler, J.T. (2011). The role of lysyl oxidase in SRC-dependent proliferation and

Mikels, A., Vaysberg, M., Ghermazien, H., Wai, C., Garcia, C.A., Velayo, A.C., Jorgensen, B., Biermann, D., Tsai, D., Green, J., Zaffryar-Eilot, S., Holzer, A., Ogg, S., Thai, D., Neufeld, G., Van, V.P. & Smith, V. (2010). Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment, *Nat* 

development with little or no toxicity for the patient.

**5. References** 

1225-1232

2964-2971

*Rev Cell Dev. Biol* 17, 25-51

*Immunol. Rev* 222, 155-161

progression of cancer, *J Pathol* 223, 162-176

angiogenesis, *Matrix Biol* 22, 63-71

*Med.* 16, 1009-1017


Therapeutical Cues from the Tumor Microenvironment 257

Geho, D.H., Bandle, R.W., Clair, T. & Liotta, L.A. (2005). Physiological mechanisms of tumor-cell invasion and migration, *Physiology (Bethesda. )* 20, 194-200 Gialeli, C., Theocharis, A.D. & Karamanos, N.K. (2011). Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting, *FEBS J* 278, 16-27 Giatromanolaki, A., Sivridis, E. & Koukourakis, M.I. (2006). Angiogenesis in colorectal cancer: prognostic and therapeutic implications, *Am J Clin Oncol.* 29, 408-417

Ginath, S., Menczer, J., Friedmann, Y., Aingorn, H., Aviv, A., Tajima, K., Dantes, A.,

Giralt, J., Navalpotro, B., Hermosilla, E., de, T., I, Espin, E., Reyes, V., Cerezo, L., de las,

Gorringe, K.L., Jacobs, S., Thompson, E.R., Sridhar, A., Qiu, W., Choong, D.Y. & Campbell,

Gregory, A.D. & Houghton, A.M. (2011). Tumor-Associated Neutrophils: New Targets for

Guiducci, C., Vicari, A.P., Sangaletti, S., Trinchieri, G. & Colombo, M.P. (2005). Redirecting

Guruvayoorappan, C. (2008). Tumor versus tumor-associated macrophages: how hot is the

Guttery, D.S., Shaw, J.A., Lloyd, K., Pringle, J.H. & Walker, R.A. (2010). Expression of tenascin-C and its isoforms in the breast, *Cancer Metastasis Rev* 29, 595-606 Han, S., Sidell, N., Roser-Page, S. & Roman, J. (2004). Fibronectin stimulates human lung

Haubeiss, S., Schmid, J.O., Murdter, T.E., Sonnenberg, M., Friedel, G., van der Kuip, H. &

Hawinkels, L.J., Zuidwijk, K., Verspaget, H.W., de Jonge-Muller, E.S., van, D.W., Ferreira,

Hazan, R.B., Qiao, R., Keren, R., Badano, I. & Suyama, K. (2004). Cadherin switch in tumor

Herbst, R.S. (2004). Review of epidermal growth factor receptor biology, *Int. J Radiat. Oncol.* 

Hermiston, M.L. & Gordon, J.I. (1995). In vivo analysis of cadherin function in the mouse

and regulation of programmed cell death, *J Cell Biol* 129, 489-506

Glezerman, M., Vlodavsky, I. & Amsterdam, A. (2001). Expression of heparanase,

H.M., Cajal, S., Armengol, M. & Benavente, S. (2006). Prognostic significance of vascular endothelial growth factor and cyclooxygenase-2 in patients with rectal

I.G. (2007). High-resolution single nucleotide polymorphism array analysis of epithelial ovarian cancer reveals numerous microdeletions and amplifications, *Clin* 

in vivo elicited tumor infiltrating macrophages and dendritic cells towards tumor

carcinoma cell growth by inducing cyclooxygenase-2 (COX-2) expression, *Int. J.* 

Aulitzky, W.E. (2010). Dasatinib reverses cancer-associated fibroblasts (CAFs) from primary lung carcinomas to a phenotype comparable to that of normal fibroblasts,

V., Fontijn, R.D., David, G., Hommes, D.W., Lamers, C.B. & Sier, C.F. (2008). VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer

intestinal epithelium: essential roles in adhesion, maintenance of differentiation,

Gilmore, A.P. (2005). Anoikis, *Cell Death. Differ.* 12 Suppl 2, 1473-1477

Mdm2, and erbB2 in ovarian cancer, *Int. J Oncol.* 18, 1133-1144

summary: panitumumab (Vectibix), *Oncologist.* 12, 577-583 Goldberg, R.M. (2005). Cetuximab, *Nat Rev Drug Discov.* Suppl, S10-S11

*Cancer Res* 13, 4731-4739

*Cancer* 111, 322-331

*Mol. Cancer* 9, 168

*Biol Phys.* 59, 21-26

Cancer Therapy, *Cancer Res* 

rejection, *Cancer Res* 65, 3437-3446

link?, *Integr. Cancer Ther.* 7, 90-95

angiogenesis, *Eur. J Cancer* 44, 1904-1913

progression, *Ann. N. Y. Acad. Sci.* 1014, 155-163

cancer treated with preoperative radiotherapy, *Oncology* 71, 312-319 Giusti, R.M., Shastri, K.A., Cohen, M.H., Keegan, P. & Pazdur, R. (2007). FDA drug approval


Dancey, J. & Sausville, E.A. (2003). Issues and progress with protein kinase inhibitors for

Deckers, M.M., van Bezooijen, R.L., van der Horst, G., Hoogendam, J., van Der, B.C.,

Desgrosellier, J.S. & Cheresh, D.A. (2010). Integrins in cancer: biological implications and

Dhanabal, M., Volk, R., Ramchandran, R., Simons, M. & Sukhatme, V.P. (1999). Cloning,

Doliana, R., Bot, S., Bonaldo, P. & Colombatti, A. (2000). EMI, a novel cysteine-rich domain

Doll, J.A., Stellmach, V.M., Bouck, N.P., Bergh, A.R., Lee, C., Abramson, L.P., Cornwell, M.L.,

Eble, J.A. & Haier, J. (2006). Integrins in cancer treatment, *Curr. Cancer Drug Targets.* 6, 89-

Elbauomy, E.S., Green, A.R., Lambros, M.B., Turner, N.C., Grainge, M.J., Powe, D., Ellis, I.O.

Erez, N., Truitt, M., Olson, P., Arron, S.T. & Hanahan, D. (2010). Cancer-Associated

Inflammation in an NF-kappaB-Dependent Manner, *Cancer Cell* 17, 135-147 Erler, J.T., Bennewith, K.L., Cox, T.R., Lang, G., Bird, D., Koong, A., Le, Q.T. & Giaccia, A.J.

Erler, J.T. & Giaccia, A.J. (2006). Lysyl oxidase mediates hypoxic control of metastasis, *Cancer* 

Ferrara, N., Hillan, K.J. & Novotny, W. (2005). Bevacizumab (Avastin), a humanized anti-

Folkman, J. (1971). Tumor angiogenesis: therapeutic implications, *N. Engl. J. Med.* 285, 1182-

Fridlender, Z.G., Sun, J., Kim, S., Kapoor, V., Cheng, G., Ling, L., Worthen, G.S. & Albelda,

Frisch, S.M. & Screaton, R.A. (2001). Anoikis mechanisms, *Curr. Opin. Cell Biol* 13, 555-562 Fukumoto, M., Takahashi, J.A., Murai, N., Ueba, T., Kono, K. & Nakatsu, S. (2000). Induction

fibroblast growth factor autocrine loop, *Anticancer Res* 20, 4059-4065

recruitment to form the premetastatic niche, *Cancer Cell* 15, 35-44

Folkman, J. & Klagsbrun, M. (1987). Angiogenic factors, *Science* 235, 442-447

Papapoulos, S.E. & Lowik, C.W. (2002). Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A,

expression, and in vitro activity of human endostatin, *Biochem. Biophys. Res* 

of EMILINs and other extracellular proteins, interacts with the gC1q domains and

Pins, M.R., Borensztajn, J. & Crawford, S.E. (2003). Pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas, *Nat Med.* 9, 774-780 Dunn, J.R., Reed, J.E., du Plessis, D.G., Shaw, E.J., Reeves, P., Gee, A.L., Warnke, P. &

Walker, C. (2006). Expression of ADAMTS-8, a secreted protease with antiangiogenic properties, is downregulated in brain tumours, *Br. J Cancer* 94, 1186-

& Reis-Filho, J.S. (2007). FGFR1 amplification in breast carcinomas: a chromogenic

Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting

(2009). Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell

VEGF monoclonal antibody for cancer therapy, *Biochem. Biophys. Res Commun.* 333,

S.M. (2009). Polarization of tumor-associated neutrophil phenotype by TGF-beta:

of apoptosis in glioma cells: an approach to control tumor growth by blocking basic

cancer treatment, *Nat Rev Drug Discov.* 2, 296-313

therapeutic opportunities, *Nat Rev Cancer* 10, 9-22

participates in multimerization, *FEBS Lett.* 484, 164-168

in situ hybridisation analysis, *Breast Cancer Res* 9, R23

*Endocrinology* 143, 1545-1553

*Commun.* 258, 345-352

*Res* 66, 10238-10241

Folkman, J. (2006). Angiogenesis, *Annu. Rev Med.* 57, 1-18

"N1" versus "N2" TAN, *Cancer Cell* 16, 183-194

328-335

1186

1193

105


Therapeutical Cues from the Tumor Microenvironment 259

Junxia, W., Ping, G., Yuan, H., Lijun, Z., Jihong, R., Fang, L., Min, L., Xi, W., Ting, H., Ke, D.

Kalluri, R. (2003). Basement membranes: structure, assembly and role in tumour

Kim, H.S., Lim, S.J. & Park, Y.K. (2009). Anti-angiogenic factor endostatin in osteosarcoma,

Kowanetz, M. & Ferrara, N. (2006). Vascular endothelial growth factor signaling pathways:

Kraman, M., Bambrough, P.J., Arnold, J.N., Roberts, E.W., Magiera, L., Jones, J.O., Gopinathan,

human breast tissues in mice, *Proc. Natl. Acad. Sci. U. S. A* 101, 4966-4971 Lee, S., Jilani, S.M., Nikolova, G.V., Carpizo, D. & Iruela-Arispe, M.L. (2005). Processing of

Lewen, S., Zhou, H., Hu, H.D., Cheng, T., Markowitz, D., Reisfeld, R.A., Xiang, R. & Luo, Y.

Li, H., Fan, X. & Houghton, J. (2007). Tumor microenvironment: the role of the tumor stroma

Liu, L., Cao, Y., Chen, C., Zhang, X., McNabola, A., Wilkie, D., Wilhelm, S., Lynch, M. &

Loeffler, M., Kruger, J.A., Niethammer, A.G. & Reisfeld, R.A. (2006). Targeting tumor-

Luo, Y., Zhou, H., Krueger, J., Kaplan, C., Lee, S.H., Dolman, C., Markowitz, D., Wu, W.,

Mantovani, A., Allavena, P. & Sica, A. (2004). Tumour-associated macrophages as a

Mantovani, A., Sozzani, S., Locati, M., Allavena, P. & Sica, A. (2002). Macrophage

mononuclear phagocytes, *Trends Immunol.* 23, 549-555

as a novel strategy against breast cancer, *J Clin Invest* 116, 2132-2141 Malinda, K.M., Nomizu, M., Chung, M., Delgado, M., Kuratomi, Y., Yamada, Y., Kleinman,

A., Tuveson, D.A. & Fearon, D.T. (2010). Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-alpha, *Science* 330, 827-830 Kuperwasser, C., Chavarria, T., Wu, M., Magrane, G., Gray, J.W., Carey, L., Richardson, A.

& Weinberg, R.A. (2004). Reconstruction of functionally normal and malignant

VEGF-A by matrix metalloproteinases regulates bioavailability and vascular

(2008). A Legumain-based minigene vaccine targets the tumor stroma and suppresses breast cancer growth and angiogenesis, *Cancer Immunol. Immunother.* 57, 507-515 Lewis, C.E. & Pollard, J.W. (2006). Distinct Role of Macrophages in Different Tumor

Carter, C. (2006). Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model

associated fibroblasts improves cancer chemotherapy by increasing intratumoral

Liu, C., Reisfeld, R.A. & Xiang, R. (2006). Targeting tumor-associated macrophages

H.K. & Ponce, M.L. (1999). Identification of laminin alpha1 and beta1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting,

prototypic type II polarised phagocyte population: role in tumour progression, *Eur.* 

polarization: tumor-associated macrophages as a paradigm for polarized M2

Kalluri, R. & Zeisberg, M. (2006). Fibroblasts in cancer, *Nat. Rev. Cancer* 6, 392-401

therapeutic perspective, *Clin Cancer Res* 12, 5018-5022

in vitro and in vivo, *Cancer Sci.* 101, 1790-1796

angiogenesis, *Nat. Rev. Cancer* 3, 422-433

patterning in tumors, *J Cell Biol* 169, 681-691

Microenvironments, *Cancer Res.* 66, 605-612

in cancer, *J. Cell Biochem.* 101, 805-815

PLC/PRF/5, *Cancer Res* 66, 11851-11858

drug uptake, *J Clin Invest* 116, 1955-1962

*FASEB J* 13, 53-62

*J Cancer* 40, 1660-1667

*APMIS* 117, 716-723

& Huizhong, Z. (2010). Double strand RNA-guided endogeneous E-cadherin upregulation induces the apoptosis and inhibits proliferation of breast carcinoma cells


Hileman, R.E., Fromm, J.R., Weiler, J.M. & Linhardt, R.J. (1998). Glycosaminoglycan-protein

Hinoda, Y., Sasaki, S., Ishida, T. & Imai, K. (2004). Monoclonal antibodies as effective

Hirama, M., Takahashi, F., Takahashi, K., Akutagawa, S., Shimizu, K., Soma, S., Shimanuki,

Houghton, A.M. (2010). The paradox of tumor-associated neutrophils: fueling tumor growth

Hu, M. & Polyak, K. (2008). Microenvironmental regulation of cancer development, *Curr.* 

Hubbard, S.R. & Miller, W.T. (2007). Receptor tyrosine kinases: mechanisms of activation

Hunter, T. (1995). Protein kinases and phosphatases: the yin and yang of protein

Ikeguchi, M., Fukuda, K., Yamaguchi, K., Kondo, A., Tsujitani, S. & Kaibara, N. (2003a).

Ikeguchi, M., Hirooka, Y. & Kaibara, N. (2003b). Heparanase gene expression and its

Imai, K. & Takaoka, A. (2006). Comparing antibody and small-molecule therapies for cancer,

Iozzo, R.V. (2005). Basement membrane proteoglycans: from cellar to ceiling, *Nat Rev Mol.* 

Iozzo, R.V. & San Antonio, J.D. (2001). Heparan sulfate proteoglycans: heavy hitters in the

Ishizuka, T., Tanabe, C., Sakamoto, H., Aoyagi, K., Maekawa, M., Matsukura, N., Tokunaga,

Jarvelainen, H., Sainio, A., Koulu, M., Wight, T.N. & Penttinen, R. (2009). Extracellular matrix molecules: potential targets in pharmacotherapy, *Pharmacol. Rev* 61, 198-223

Jiang, X. & Couchman, J.R. (2003). Perlecan and tumor angiogenesis, *J Histochem. Cytochem.*

Jiang, X., Multhaupt, H., Chan, E., Schaefer, L., Schaefer, R.M. & Couchman, J.R. (2004).

Jimenez, B., Volpert, O.V., Crawford, S.E., Febbraio, M., Silverstein, R.L. & Bouck, N. (2000).

Jin, Z. & El-Deiry, W.S. (2005). Overview of cell death signaling pathways, *Cancer Biol Ther.*

Jones, P.L. & Jones, F.S. (2000). Tenascin-C in development and disease: gene regulation and

A., Tajiri, T., Yoshida, T., Terada, M. & Sasaki, H. (2002). Gene amplification profiling of esophageal squamous cell carcinomas by DNA array CGH, *Biochem.* 

Essential contribution of tumor-derived perlecan to epidermal tumor growth and

Signals leading to apoptosis-dependent inhibition of neovascularization by

Quantitative analysis of heparanase gene expression in esophageal squamous cell

correlation with spontaneous apoptosis in hepatocytes of cirrhotic liver and

therapeutic agents for solid tumors, *Cancer Sci.* 95, 621-625

with cytotoxic substances, *Cell Cycle* 9, 1732-1737

and signaling, *Curr. Opin. Cell Biol* 19, 117-123

phosphorylation and signaling, *Cell* 80, 225-236

carcinoma, *Ann. Surg. Oncol.* 10, 297-304

angiogenesis arena, *J Clin Invest* 108, 349-355

angiogenesis, *J Histochem. Cytochem.* 52, 1575-1590

carcinoma, *Eur. J Cancer* 39, 86-90

*Biophys. Res Commun.* 296, 152-155

thrombospondin-1, *Nat. Med.* 6, 41-48

cell function, *Matrix Biol* 19, 581-596

*Nat Rev Cancer* 6, 714-727

*Cell Biol* 6, 646-656

51, 1393-1410

4, 139-163

*Bioessays* 20, 156-167

*Opin. Genet. Dev.* 18, 27-34

117

interactions: definition of consensus sites in glycosaminoglycan binding proteins,

Y., Nishio, K. & Fukuchi, Y. (2003). Osteopontin overproduced by tumor cells acts as a potent angiogenic factor contributing to tumor growth, *Cancer Lett.* 198, 107-


Therapeutical Cues from the Tumor Microenvironment 261

Nor, J.E., Mitra, R.S., Sutorik, M.M., Mooney, D.J., Castle, V.P. & Polverini, P.J. (2000).

Nozue, M., Isaka, N. & Fukao, K. (2001). Over-expression of vascular endothelial growth

Ogasawara, S., Yano, H., Iemura, A., Hisaka, T. & Kojiro, M. (1996). Expressions of basic

Orimo, A., Gupta, P.B., Sgroi, D.C., Arenzana-Seisdedos, F., Delaunay, T., Naeem, R., Carey,

Ornitz, D.M., Yayon, A., Flanagan, J.G., Svahn, C.M., Levi, E. & Leder, P. (1992). Heparin is

Ozerdem, U. & Stallcup, W.B. (2003). Early contribution of pericytes to angiogenic sprouting

Pang, R. & Poon, R.T. (2006). Angiogenesis and antiangiogenic therapy in hepatocellular

Paolillo, M., Russo, M.A., Serra, M., Colombo, L. & Schinelli, S. (2009). Small molecule integrin antagonists in cancer therapy, *Mini. Rev Med. Chem.* 9, 1439-1446 Park, J.W., Finn, R.S., Kim, J.S., Karwal, M., Li, R.K., Ismail, F., Thomas, M., Harris, R.,

Pepper, M.S. (2001). Role of the matrix metalloproteinase and plasminogen activatorplasmin systems in angiogenesis, *Arterioscler Thromb Vasc Biol* 21, 1104-1117 Pietras, K. & Ostman, A. (2010). Hallmarks of cancer: interactions with the tumor stroma,

Pietras, K., Ostman, A., Sjoquist, M., Buchdunger, E., Reed, R.K., Heldin, C.H. & Rubin, K.

Pollard, J.W. (2004). Tumour-educated macrophages promote tumour progression and

Rasanen, K. & Vaheri, A. (2010). Activation of fibroblasts in cancer stroma, *Exp. Cell Res* 316,

Reed, J.C. & Pellecchia, M. (2005). Apoptosis-based therapies for hematologic malignancies,

Reichert, J.M., Rosensweig, C.J., Faden, L.B. & Dewitz, M.C. (2005). Monoclonal antibody

Reid, A., Vidal, L., Shaw, H. & de, B.J. (2007). Dual inhibition of ErbB1 (EGFR/HER1) and

Rodriguez-Manzaneque, J.C., Lane, T.F., Ortega, M.A., Hynes, R.O., Lawler, J. & Iruela-

endothelial growth factor, *Proc. Natl. Acad. Sci. U. S. A* 98, 12485-12490

Arispe, M.L. (2001). Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular

activating the caspase death pathway, *J Vasc Res* 37, 209-218

human hepatocellular carcinoma cell lines, *Hepatology* 24, 198-205

through elevated SDF-1/CXCL12 secretion, *Cell* 121, 335-348

and for mitogenesis in whole cells, *Mol. Cell Biol* 12, 240-247

and tube formation, *Angiogenesis.* 6, 241-249

carcinoma, *Cancer Lett.* 242, 151-167

*Cancer Res* 17, 1973-1983

*Exp. Cell Res* 316, 1324-1331

metastasis, *Nat Rev Cancer* 4, 71-78

successes in the clinic, *Nat Biotechnol.* 23, 1073-1078

ErbB2 (HER2/neu), *Eur. J Cancer* 43, 481-489

2934

2713-2722

*Blood* 106, 408-418

Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by

factor after preoperative radiation therapy for rectal cancer, *Oncol. Rep.* 8, 1247-1249

fibroblast growth factor and its receptors and their relationship to proliferation of

V.J., Richardson, A.L. & Weinberg, R.A. (2005). Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis

required for cell-free binding of basic fibroblast growth factor to a soluble receptor

Baudelet, C., Walters, I. & Raoul, J.L. (2011). Phase II, Open-Label Study of Brivanib as First-Line Therapy in Patients with Advanced Hepatocellular Carcinoma, *Clin* 

(2001). Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors, *Cancer Res* 61, 2929-


Mantovani, A., Porta, C., Rubino, L., Allavena, P. & Sica, A. (2006). Tumor-associated

Marastoni, S., Ligresti, G., Lorenzon, E., Colombatti, A. & Mongiat, M. (2008). Extracellular

McKeage, M.J. & Baguley, B.C. (2010). Disrupting established tumor blood vessels: an

Mendel, D.B., Laird, A.D., Xin, X., Louie, S.G., Christensen, J.G., Li, G., Schreck, R.E.,

pharmacokinetic/pharmacodynamic relationship, *Clin Cancer Res* 9, 327-337 Meredith, J.E., Jr., Fazeli, B. & Schwartz, M.A. (1993). The extracellular matrix as a cell

Meyer, C., Notari, L. & Becerra, S.P. (2002). Mapping the type I collagen-binding site on

Misra, S., Heldin, P., Hascall, V.C., Karamanos, N.K., Skandalis, S.S., Markwald, R.R. &

Mongiat, M., Fu, J., Oldershaw, R., Greenhalgh, R., Gown, A.M. & Iozzo, R.V. (2003a).

Mongiat, M., Ligresti, G., Marastoni, S., Lorenzon, E., Doliana, R. & Colombatti, A. (2007).

Mongiat, M., Marastoni, S., Ligresti, G., Lorenzon, E., Schiappacassi, M., Perris, R., Frustaci,

Mongiat, M., Mungiguerra, G., Bot, S., Mucignat, M.T., Giacomello, E., Doliana, R. &

Mongiat, M., Sweeney, S.M., San Antonio, J.D., Fu, J. & Iozzo, R.V. (2003b). Endorepellin, a

Motzer, R.J. & Bukowski, R.M. (2006). Targeted therapy for metastatic renal cell carcinoma, *J* 

Motzer, R.J., Rini, B.I., Bukowski, R.M., Curti, B.D., George, D.J., Hudes, G.R., Redman, B.G.,

Murdoch, C., Muthana, M., Coffelt, S.B. & Lewis, C.E. (2008). The role of myeloid cells in the

promotion of tumour angiogenesis, *Nat Rev Cancer* 8, 618-631

glycoprotein EMILIN2, *Mol. Cell Biol.* 27, 7176-7187

pigment epithelium-derived factor. Implications for its antiangiogenic activity, *J* 

Ghatak, S. (2011). Hyaluronan-CD44 interactions as potential targets for cancer

Perlecan protein core interacts with extracellular matrix protein 1 (ECM1), a glycoprotein involved in bone formation and angiogenesis, *J. Biol. Chem.* 278, 17491-

Regulation of the extrinsic apoptotic pathway by the extracellular matrix

S. & Colombatti, A. (2010). The extracellular matrix glycoprotein elastin microfibril interface located protein 2: a dual role in the tumor microenvironment, *Neoplasia.*

Colombatti, A. (2000). Self-assembly and supramolecular organization of EMILIN,

novel inhibitor of angiogenesis derived from the C terminus of perlecan, *J. Biol.* 

Margolin, K.A., Merchan, J.R., Wilding, G., Ginsberg, M.S., Bacik, J., Kim, S.T., Baum, C.M. & Michaelson, M.D. (2006). Sunitinib in patients with metastatic renal

matrix: a matter of life and death, *Connect. Tissue Res.* 49, 203-206

emerging therapeutic strategy for cancer, *Cancer* 116, 1859-1871

*Therapeutic Strategies* 3, 361-366

survival factor, *Mol. Biol Cell* 4, 953-961

*Biol Chem.* 277, 45400-45407

*J. Biol. Chem.* 275, 25471-25480

*Chem.* 278, 4238-4249

*Clin Oncol.* 24, 5601-5608

cell carcinoma, *JAMA* 295, 2516-2524

therapy, *FEBS J* 

17499

12, 294-304

macrophages (TAMs) as new target in anticancer therapy, *Drug Discovery Today:* 

Abrams, T.J., Ngai, T.J., Lee, L.B., Murray, L.J., Carver, J., Chan, E., Moss, K.G., Haznedar, J.O., Sukbuntherng, J., Blake, R.A., Sun, L., Tang, C., Miller, T., Shirazian, S., McMahon, G. & Cherrington, J.M. (2003). In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a


Therapeutical Cues from the Tumor Microenvironment 263

Sharma, M.R., Tuszynski, G.P. & Sharma, M.C. (2004). Angiostatin-induced inhibition of

Simon, R., Richter, J., Wagner, U., Fijan, A., Bruderer, J., Schmid, U., Ackermann, D., Maurer,

Sironen, R.K., Tammi, M., Tammi, R., Auvinen, P.K., Anttila, M. & Kosma, V.M. (2011).

Sobel, M., Soler, D.F., Kermode, J.C. & Harris, R.B. (1992). Localization and characterization

Sottile, J. (2004). Regulation of angiogenesis by extracellular matrix, *Biochim. Biophys. Acta*

Steeg, P.S. (2006). Tumor metastasis: mechanistic insights and clinical challenges, *Nat Med.*

Szentirmai, O., Baker, C.H., Bullain, S.S., Lin, N., Takahashi, M., Folkman, J., Mulligan, R.C.

Tabruyn, S.P. & Griffioen, A.W. (2007). Molecular pathways of angiogenesis inhibition,

Tai, I.T., Dai, M., Owen, D.A. & Chen, L.B. (2005). Genome-wide expression analysis of

Takenaka, K., Yamagishi, S., Jinnouchi, Y., Nakamura, K., Matsui, T. & Imaizumi, T. (2005).

Tang, M.J. & Tai, I.T. (2007). A novel interaction between procaspase 8 and SPARC enhances

Tapanadechopone, P., Tumova, S., Jiang, X. & Couchman, J.R. (2001). Epidermal

Teti, A., Farina, A.R., Villanova, I., Tiberio, A., Tacconelli, A., Sciortino, G., Chambers, A.F.,

Theocharis, A.D., Skandalis, S.S., Tzanakakis, G.N. & Karamanos, N.K. (2010). Proteoglycans

Tilghman, R.W., Cowan, C.R., Mih, J.D., Koryakina, Y., Gioeli, D., Slack-Davis, J.K.,

RGD and cell shape change dependent, *Int. J Cancer* 77, 82-93

cancer cell growth and cellular phenotype, *PLoS ONE* 5, e12905

pharmacological targeting, *FEBS J* 277, 3904-3923

of a heparin binding domain peptide of human von Willebrand factor, *J Biol Chem.*

& Carter, B.S. (2008). Successful inhibition of intracranial human glioblastoma multiforme xenograft growth via systemic adenoviral delivery of soluble endostatin and soluble vascular endothelial growth factor receptor-2: laboratory

therapy-resistant tumors reveals SPARC as a novel target for cancer therapy, *J Clin* 

Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human

apoptosis and potentiates chemotherapy sensitivity in colorectal cancers, *J. Biol.* 

transformation leads to increased perlecan synthesis with heparin-binding-growth-

Gulino, A. & Mackay, A.R. (1998). Activation of MMP-2 by human GCT23 giant cell tumour cells induced by osteopontin, bone sialoprotein and GRGDSP peptides is

in health and disease: novel roles for proteoglycans in malignancy and their

Blackman, B.R., Tschumperlin, D.J. & Parsons, J.T. (2010). Matrix rigidity regulates

cell cycle regulatory protein cdk5, *J Cell Biochem.* 91, 398-409

Hyaluronan in human malignancies, *Exp. Cell Res* 317, 383-391

urinary bladder cancer, *Cancer Res* 61, 4514-4519

investigation, *J Neurosurg.* 108, 979-988

*Biochem. Biophys. Res Commun.* 355, 1-5

osteosarcoma cells, *Life Sci.* 77, 3231-3241

factor affinity, *Biochem. J* 355, 517-527

267, 8857-8862

1654, 13-22

12, 895-904

*Invest* 115, 1492-1502

*Chem.* 282, 34457-34467

endothelial cell proliferation/apoptosis is associated with the down-regulation of

R., Alund, G., Knonagel, H., Rist, M., Wilber, K., Anabitarte, M., Hering, F., Hardmeier, T., Schonenberger, A., Flury, R., Jager, P., Fehr, J.L., Schraml, P., Moch, H., Mihatsch, M.J., Gasser, T. & Sauter, G. (2001). High-throughput tissue microarray analysis of 3p25 (RAF1) and 8p12 (FGFR1) copy number alterations in


Roskoski, R., Jr. (2007). Vascular endothelial growth factor (VEGF) signaling in tumor

Rothhammer, T., Bataille, F., Spruss, T., Eissner, G. & Bosserhoff, A.K. (2007). Functional

Rouet, V., Hamma-Kourbali, Y., Petit, E., Panagopoulou, P., Katsoris, P., Barritault, D.,

Sahadevan, K., Darby, S., Leung, H.Y., Mathers, M.E., Robson, C.N. & Gnanapragasam, V.J.

Santimaria, M., Moscatelli, G., Viale, G.L., Giovannoni, L., Neri, G., Viti, F., Leprini, A.,

Sasisekharan, R., Shriver, Z., Venkataraman, G. & Narayanasami, U. (2002). Roles of heparan-sulphate glycosaminoglycans in cancer, *Nat Rev Cancer* 2, 521-528 Sato, S., Kigawa, J., Minagawa, Y., Okada, M., Shimada, M., Takahashi, M., Kamazawa, S. &

Sattler, M. & Salgia, R. (2004). Targeting c-Kit mutations: basic science to novel therapies,

Scatena, M., Almeida, M., Chaisson, M.L., Fausto, N., Nicosia, R.F. & Giachelli, C.M. (1998).

Schiller, J.H. (2003). New directions for ZD1839 in the treatment of solid tumors, *Semin.* 

Seidler, D.G., Goldoni, S., Agnew, C., Cardi, C., Thakur, M.L., Owens, R.T., McQuillan, D.J.

Senger, D.R., Ledbetter, S.R., Claffey, K.P., Papadopoulos-Sergiou, A., Peruzzi, C.A. &

Seo, D.W., Li, H., Guedez, L., Wingfield, P.T., Diaz, T., Salloum, R., Wei, B.Y. & Stetler-

Sessa, C., de, B.F., Perotti, A., Bauer, J., Curigliano, G., Noberasco, C., Zanaboni, F., Gianni,

Sharma, B., Handler, M., Eichstetter, I., Whitelock, J.M., Nugent, M.A. & Iozzo, R.V. (1998).

apoptosis via caspase-3 activation, *J. Biol. Chem.* 281, 26408-26418

implication of BMP4 expression on angiogenesis in malignant melanoma, *Oncogene*

Caruelle, J.P. & Courty, J. (2005). A synthetic glycosaminoglycan mimetic binds vascular endothelial growth factor and modulates angiogenesis, *J Biol Chem.* 280,

(2007). Selective over-expression of fibroblast growth factor receptors 1 and 4 in

Borsi, L., Castellani, P., Zardi, L., Neri, D. & Riva, P. (2003). Immunoscintigraphic detection of the ED-B domain of fibronectin, a marker of angiogenesis, in patients

Terakawa, N. (1999). Chemosensitivity and p53-dependent apoptosis in epithelial

NF-kappaB mediates alphavbeta3 integrin-induced endothelial cell survival, *J Cell* 

& Iozzo, R.V. (2006). Decorin protein core inhibits in vivo cancer growth and metabolism by hindering epidermal growth factor receptor function and triggering

Detmar, M. (1996). Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin, *Am J* 

Stevenson, W.G. (2003). TIMP-2 mediated inhibition of angiogenesis: an MMP-

L., Marsoni, S., Jimeno, J., D'Incalci, M., Dall'o, E. & Colombo, N. (2005). Trabectedin for women with ovarian carcinoma after treatment with platinum and

Antisense targeting of perlecan blocks tumor growth and angiogenesis in vivo, *J* 

progression, *Crit Rev Oncol. Hematol.* 62, 179-213

clinical prostate cancer, *J Pathol* 213, 82-90

with cancer, *Clin Cancer Res* 9, 571-579

ovarian carcinoma, *Cancer* 86, 1307-1313

independent mechanism, *Cell* 114, 171-180

taxanes fails, *J Clin Oncol.* 23, 1867-1874

*Clin Invest* 102, 1599-1608

*Leuk. Res* 28 Suppl 1, S11-S20

*Biol* 141, 1083-1093

*Pathol* 149, 293-305

*Oncol.* 30, 49-55

26, 4158-4170

32792-32800


**12**

*1Germany 2Canada* 

**Cyclin-Dependent Kinases (Cdk) as Targets** 

Aberration in proliferation and consequently in cell cycle control is a common aspect in carcinogenesis. As master cell cycle regulating proteins in all eukaryotic cells the Cyclindependent kinases (Cdk) were identified by Leland Hartwell, Paul Nurse, and Timothy Hunt in the 1970s and 1980s. Chronological activation of respective Cdk according to respective cell cycle phase G1, S, G2 or M is mediated through association with a regulatory Cyclin subunit, phosphorylation of Cdk and binding of endogenous activators and

In human cells four Cdk are essential components of the cell cycle machinery with key functions also in human cancer cells: Cdk1, Cdk2, Cdk4, and Cdk6 (Fig. 1) (Malumbres & Barbacid, 2009). First, Cyclin D-dependent kinases Cdk4 and Cdk6 are activated in human cell cycle in response to mitogenic signals to initiate G1 phase progression and prepare DNA duplication in S phase (Malumbres & Barbacid, 2005). Cdk4-Cyclin D or Cdk6-Cyclin D and later also Cdk2-Cyclin E complexes sequentially phosphorylate retinoblastoma proteins (Rb) on different serine and threonine residues. Resulting Rb protein inactivation is required for the transcriptional activation of genes in G1/S phase (Harbour & Dean, 2000). In G1 phase endogenous inhibitors of monomeric Cdk4 and Cdk6 like INK4 and inhibitors of Cdk2/Cdk4/Cdk6-Cyclin complexes like Cip and Kip proteins exert important influence on Cdk catalytic activity (Blain, 2008; Sherr & Roberts, 1999). Once the cell irreversibly passed restriction point R at the end of G1 phase, Cdk2-Cyclin A complex is formed, facilitating orderly execution of S phase events like DNA replication and centrosome cycle through phosphorylation of various proteins (Malumbres & Barbacid, 2005). Activation of Cdk1 by Cyclin A is required for DNA damage checkpoint control, later Cdk1-Cyclin B for G2/ M phase transition and initiation of mitosis, especially chromosome condensation and microtubule dynamics (Malumbres & Barbacid, 2009). Therefore, active Cdk1-Cyclin complexes mediate phosphorylation of about 70 substrates, e. g., minichromosome

Initiation of cell re-entrance from G0 to G1 phase and early inactivation of Rb is assigned to Cdk3-Cyclin C (Ren & Rollins, 2004). Another Cyclin-dependent kinase, Cdk5, is involved in

inhibitors, as well as subcellular localization (Shapiro, 2006).

maintenance (MCM), p53, lamins, and dyneins.

the regulation of neuronal function (Cruz & Tsai, 2004).

**1. Introduction** 

**for Cancer Therapy and Imaging** 

Franziska Graf1, Frank Wuest2 and Jens Pietzsch*<sup>1</sup>*

*2Department of Oncology, University of Alberta* 

*1Institute of Radiopharmacy, Helmholtz-Zentrum Dresden-Rossendorf* 

