The phenotype switching model also relies on human melanoma high plasticity and ability to be reprogrammed. As mentioned before, an example of the high plasticity of melanoma cells is vasculogenic mimicry (Seftor *et al.*, 2002). Some aggressive melanoma cells have been found to express molecular markers typical of other cell types, such as endothelial cells, which presumably allow melanoma cells to form vasculogenic networks that mimic blood vessels, and that could serve as an alternative route of escape for melanoma cells. This phenomenon has also been reported to occur *in vivo* in aggressive human melanoma

(Bittner *et al.*, 2000; Haqq *et al.*, 2005; Hoek, 2007; Pavey *et al.*, 2004).

2006; Goodall *et al.*, 2008; Hoek *et al.*, 2008).

demonstrated that under stress conditions like serum withdrawal or lack of extracellular matrix attachment, PEDF has a certain capacity to induce melanoma cell apoptosis although with a weaker potency than in endothelial cells (Garcia *et al.*, 2004). Reduced survival of melanoma cells caused by the absence of attachment in the presence of high PEDF circulating levels has important consequences for the outcome of tumor cells in transit to the metastatic site.

There are still a number PEDF's putative actions on the various tumor vascularization mechanisms described so far that remain to be explored and that are of critical importance: (i) does PEDF affect haematopoyetic precursor cells recruitment?, (ii) does PEDF block vasculogenesis (formation of new vessels from endothelial progenitor cells)?, (iii) does PEDF induce lymphatic vessels regression? There is no report linking PEDF to vasculogenesis, but it has recently been described that PEDF decreases lymph node metastasis of prostate cancer, while paradoxically increases extratumoral lymphangiogenesis by unknown mechanisms (Halin *et al.*, 2010).

In the context of spread of the disease an attractive benefit of antiangiogenic strategies is their potential to keep micrometastases in a dormant state (Gao *et al.*, 2008; Goss and Chambers, 2010). Thrombospondin-1 has been proven to be effective in preventing the growth of dormant pulmonary micrometastases in human melanoma xenografts after surgical resection or curative radiation of the primary tumor (Rofstad *et al.*, 2004; Rofstad *et al.*, 2003). PEDF's capability to maintain dormant micrometastases in check, preventing their growth to macrometastases remains to be evaluated, but it could be of relevant therapeutic interest.

Moreover, it has been shown that PEDF is also produced by a wide variety of epithelial cell types in different tissues and its role in controlling primary tumor growth, angiogenesis and metastatic spread has been explored in a wide range of tumors using diverse mouse models, and analysis of human biopsies. Levels of angiostatic PEDF decrease during the progression of a number of cancers, such as hepatocelular carcinoma (Matsumoto *et al.*, 2004), prostate cancer (Halin *et al.*, 2004; Qingyi *et al.*, 2009), breast adenocarcinoma (Cai *et al.*, 2006), glioblastoma (Guan *et al.*, 2003) and Wilm´s tumors (Abramson *et al.*, 2003). A number of recent excellent reviews cover the antitumor and antimetastatic action of PEDF on several tumor types (Broadhead *et al.*, 2009; Fernandez-Garcia *et al.*, 2007; Hoshina *et al.*, 2010).

#### **3. Halting melanoma progression. The role of pigment epithelium-derived factor**

#### **3.1 Cutaneous melanoma. Current models of melanoma progression**

Melanoma cells arise from melanocytes, normal cells specialized in the production of pigment melanin, which reside in the basal layer of the epidermis, among other locations, and whose homeostasis is tightly controlled by epidermal keratinocytes (Gray-Schopfer *et al.*, 2007).The classical model of melanoma progression describes melanoma development as a series of histopathological steps regarding the thickness and the grade of invasion of the lesion (Clark, 1991). Nevi are relatively benign lesions that rarely progress to melanoma, in part because most of melanocytes in nevocytic lesions are in a state of senescence-associated growth arrest (Michaloglou *et al.*, 2005). RGP, melanomas have high proliferative potential but null or very low invasive ability, being confined to the epidermis (Gray-Schopfer *et al.*, 2007). In contrast, VGP, melanomas are competent for metastasis, fully invade the upper part of the epidermis as well as the dermis and the subcutaneous tissue, being able to reach

demonstrated that under stress conditions like serum withdrawal or lack of extracellular matrix attachment, PEDF has a certain capacity to induce melanoma cell apoptosis although with a weaker potency than in endothelial cells (Garcia *et al.*, 2004). Reduced survival of melanoma cells caused by the absence of attachment in the presence of high PEDF circulating levels has important consequences for the outcome of tumor cells in transit to the

There are still a number PEDF's putative actions on the various tumor vascularization mechanisms described so far that remain to be explored and that are of critical importance: (i) does PEDF affect haematopoyetic precursor cells recruitment?, (ii) does PEDF block vasculogenesis (formation of new vessels from endothelial progenitor cells)?, (iii) does PEDF induce lymphatic vessels regression? There is no report linking PEDF to vasculogenesis, but it has recently been described that PEDF decreases lymph node metastasis of prostate cancer, while paradoxically increases extratumoral lymphangiogenesis by unknown

In the context of spread of the disease an attractive benefit of antiangiogenic strategies is their potential to keep micrometastases in a dormant state (Gao *et al.*, 2008; Goss and Chambers, 2010). Thrombospondin-1 has been proven to be effective in preventing the growth of dormant pulmonary micrometastases in human melanoma xenografts after surgical resection or curative radiation of the primary tumor (Rofstad *et al.*, 2004; Rofstad *et al.*, 2003). PEDF's capability to maintain dormant micrometastases in check, preventing their growth to macrometastases remains to be evaluated, but it could be of relevant therapeutic

Moreover, it has been shown that PEDF is also produced by a wide variety of epithelial cell types in different tissues and its role in controlling primary tumor growth, angiogenesis and metastatic spread has been explored in a wide range of tumors using diverse mouse models, and analysis of human biopsies. Levels of angiostatic PEDF decrease during the progression of a number of cancers, such as hepatocelular carcinoma (Matsumoto *et al.*, 2004), prostate cancer (Halin *et al.*, 2004; Qingyi *et al.*, 2009), breast adenocarcinoma (Cai *et al.*, 2006), glioblastoma (Guan *et al.*, 2003) and Wilm´s tumors (Abramson *et al.*, 2003). A number of recent excellent reviews cover the antitumor and antimetastatic action of PEDF on several tumor types (Broadhead *et al.*, 2009; Fernandez-Garcia *et al.*, 2007; Hoshina *et al.*, 2010).

**3. Halting melanoma progression. The role of pigment epithelium-derived**

Melanoma cells arise from melanocytes, normal cells specialized in the production of pigment melanin, which reside in the basal layer of the epidermis, among other locations, and whose homeostasis is tightly controlled by epidermal keratinocytes (Gray-Schopfer *et al.*, 2007).The classical model of melanoma progression describes melanoma development as a series of histopathological steps regarding the thickness and the grade of invasion of the lesion (Clark, 1991). Nevi are relatively benign lesions that rarely progress to melanoma, in part because most of melanocytes in nevocytic lesions are in a state of senescence-associated growth arrest (Michaloglou *et al.*, 2005). RGP, melanomas have high proliferative potential but null or very low invasive ability, being confined to the epidermis (Gray-Schopfer *et al.*, 2007). In contrast, VGP, melanomas are competent for metastasis, fully invade the upper part of the epidermis as well as the dermis and the subcutaneous tissue, being able to reach

**3.1 Cutaneous melanoma. Current models of melanoma progression**

metastatic site.

interest.

**factor**

mechanisms (Halin *et al.*, 2010).

blood or lymph circulation and eventually colonize and develop secondary tumors at distant organs (Chin *et al.*, 2006; Gray-Schopfer *et al.*, 2007). This model is in agreement with the tumor clonal evolution model (Nowell, 1976), which hypothesizes that cancer evolves from a poorly to a highly metastatic phase through accumulation of molecular alterations (mutations and/or epigenetic changes) that enhance proliferative and invasive potential of tumor cells, and that would promote lineal progression from RGP to VGP to metastasis (Miller and Mihm, 2006).

Even though the classification of melanomas according to the thickness of the lesion is one of the most widely used methods for the diagnosis and prognosis of melanoma (Fecher *et al.*, 2007), frequently there are lesions whose thickness does not correlate with their actual aggressiveness and metastatic outcome (Lomuto *et al.*, 2004; Slingluff *et al.*, 1988). Besides, this model does not offer either convincing explanations for the heterogeneity found in metastatic melanoma cells, nor for the persistent failure of anti-melanoma therapies. Consequently, in the last decade researchers have aimed to establish a molecular classification of melanoma utilizing gene expression profiling tools (Fecher *et al.*, 2007; Hoek, 2007; Hoek, 2009). A number of studies have been able to classify collections of melanoma cell lines or biopsies into groups differing in their aggressiveness and metastatic potential, confirming the large heterogeneity of melanoma and the importance of the microenvironment in determining gene expression programs of melanoma cells and progression to metastasis (Bittner *et al.*, 2000; Haqq *et al.*, 2005; Hoek, 2007; Pavey *et al.*, 2004).

One of these studies, by means of genome-wide gene expression analysis and functional assays, described that most melanoma cell lines could be categorized according to their gene expression profile into two extreme phenotypes, proliferative or invasive (Hoek *et al.*, 2006). The proliferative gene signature encompasses a number of melanocytic lineage genes such as microphthalmia-associated transcription factor (MITF) and some of its targets, while the invasive phenotype signature is defined by suppressed expression of proliferative genes in favor of others related to the modification of the tumor microenvironment (Hoek *et al.*, 2006). Based on this and other studies a new melanoma progression model was proposed, which takes into account the heterogeneous nature of melanoma and the key role of the tumor microenvironment (Carreira *et al.*, 2006; Goodall *et al.*, 2008; Hoek, 2009; Hoek *et al.*, 2008; Hoek *et al.*, 2006). This new model, referred to as phenotype switching, considers metastatic potential split into two mutually exclusive and reversible states, proliferative and invasive, and hypothesizes that melanoma progression is driven by the reversible switching between these two phenotypes (Hoek, 2009). A primary lesion would be initially composed of proliferative phenotype cells. Signals from the microenvironment, such as hypoxia or inflammation, would make some cells switch their gene signature to become invasive, which would allow them to escape from the primary tumor and eventually reach and colonize a foreign distant organ. There, signals from the new environment would reprogram cells back to the proliferative signature, ultimately developing metastases (Carreira *et al.*, 2006; Goodall *et al.*, 2008; Hoek *et al.*, 2008).

The phenotype switching model also relies on human melanoma high plasticity and ability to be reprogrammed. As mentioned before, an example of the high plasticity of melanoma cells is vasculogenic mimicry (Seftor *et al.*, 2002). Some aggressive melanoma cells have been found to express molecular markers typical of other cell types, such as endothelial cells, which presumably allow melanoma cells to form vasculogenic networks that mimic blood vessels, and that could serve as an alternative route of escape for melanoma cells. This phenomenon has also been reported to occur *in vivo* in aggressive human melanoma

Pigment Epithelium-Derived Factor –

An Angiostatic Factor with a Broader Function in Melanoma 151

which are overexpressed in cancer (Sahai, 2005). Rac promotes a more elongated shape that requires matrix metalloproteinases in order to invade, while RhoA favors a rounded morphology and a movement less dependent on proteases (Sanz-Moreno *et al.*, 2008). On the other hand, MMPs are upregulated in invasive melanoma and they are thought to promote melanoma dissemination, probably through different mechanisms since different MMPs are

As previously mentioned, the antiangiogenic activity of PEDF prompted the study of its potential antitumor effects. We (Fernandez-Garcia *et al.*, 2007; Garcia *et al.*, 2004) and others (Abe et al., 2004; Doll et al., 2003; Ek et al., 2006b) described a complex mechanism underlying the potent inhibition of melanoma metastasis by PEDF. PEDF's antitumor activity in melanoma and other tumors is based on its dual action on the tumor microenvironment and on the tumor cells themselves (Fernandez-Garcia *et al.*, 2007). PEDF inhibits tumor angiogenesis by means of induction of apoptosis on endothelial cells and modulation of the angiogenic profile of melanoma cells, eventually destroying the main source of nutrients to the primary tumor as well as one of the main routes of dissemination to distant organs. Additionally, PEDF exerts a potent inhibitory action on melanoma cells, inducing apoptosis under stress conditions (such as absence of growth factors or detachment from the extracellular matrix) and abrogating migration and invasion. As a whole, PEDF overexpression in melanoma cells leads to a decrease in primary tumor growth and an inhibition of lung metastasis formation (Fernandez-Garcia *et al.*, 2007; Filleur *et al.*, 2009). In the previous section we described that PEDF is produced by low aggressive melanomas. Given that cutaneous melanoma develops from skin melanocytes, two questions soon arise: i) are normal melanocytes expressing this factor endogenously?, ii) if so, which could be the role of PEDF in normal melanocytic cells? By means of assessing a large collection of primary cultures of cutaneous melanocytes and other cell types of the skin we found that melanocytes secrete very high levels of PEDF comparable to other cell types known to express this factor (Orgaz *et al.*, 2009). In agreement with previous reports, dermal fibroblasts also express high levels of PEDF, which has been described to be involved in the control of their proliferative potential (Francis *et al.*, 2004; Tresini *et al.*, 1999); additionally, PEDF secreted by fibroblasts could also play a role in maintaining a correct vascularization of the skin. In contrast, PEDF is expressed at very low levels by epidermal keratinocytes or

In melanocytes, PEDF is one of the players involved in the regulation of their proliferation and migratory ability (Fig. 1). PEDF silencing in primary melanocytes leads to an increase in their migration and invasion, as well as a moderate augment in their growth rate (Orgaz *et al.*, 2009). Melanocytes arise from highly migratory embryonic neural crest progenitors, and therefore display an enormous migratory potential (Gupta *et al.*, 2005; Zbytek *et al.*, 2008) that must be tightly controlled in the skin (Hsu *et al.*, 2002). In addition to the regulatory signals from adjacent keratinocytes, an additional brake to their uncontrolled dissemination could be self-imposed within melanocytes by expressing high levels of PEDF themselves. As suggested for the fibroblasts, melanocytes could be also participating in the maintenance of appropriate angiogenesis in the skin by secreting this potent antiangiogenic factor, taking into account previous studies reporting an excessive vascularization of multiple organs upon PEDF knockdown (Doll *et al.*, 2003). Additional paracrine actions on other cell types,

thought to have pro- or anti-invasive effects (Overall and Lopez-Otin, 2002).

**3.3 PEDF as a brake for melanoma progression**

microvascular endothelial cells (Orgaz *et al.*, 2009).

keratinocytes for instance, remain to be investigated.

samples, and correlates with poor outcome (Folberg *et al.*, 2000; Hendrix *et al.*, 2003; Maniotis *et al.*, 1999). Furthermore, poorly aggressive melanoma cells can be reprogrammed to highly aggressive and invasive by exposing them to matrices preconditioned by more aggressive melanoma cells, highlighting the importance of the tumor microenvironment in driving melanoma progression (Postovit *et al.*, 2006; Seftor *et al.*, 2006).

#### **3.2 Melanoma invasion**

Primary melanoma patients can be usually cured by surgical removal of the tumor when detected early, but most of the times melanoma rapidly metastasizes with a poor outcome (Gray-Schopfer *et al.*, 2007). Therefore it is essential to understand the mechanisms by which melanoma cells escape from the primary tumor and spread and invade other organs, in order to develop improved therapies that increase patient survival rates. Following there is a brief summary detailing some key molecules involved in melanoma migration/invasion and metastasis; interested readers are suggested to read topic reviews elsewhere (Gaggioli and Sahai, 2007; Uong and Zon, 2010).

MITF plays a pivotal role in melanocyte and melanoma biology, as it is involved not only in the control of migration and invasion, but also in proliferation, survival, and differentiation of melanocytic cells (Levy *et al.*, 2006). The fine tuning of MITF activity enables melanoma cells to switch between a proliferative (high MITF) or invasive (low MITF) state, while very high levels promote differentiation and complete absence of MITF is incompatible with survival (Carreira *et al.*, 2006; Gray-Schopfer *et al.*, 2007).

Additionally, a number of growth factors and cytokines are upregulated as melanoma becomes more invasive, such as hepatocyte growth factor (HGF), TGF-β, IL-8, Nodal and several members of fibroblast growth factor (FGF) family (al-Alousi *et al.*, 1996; Albino *et al.*, 1991; Topczewska *et al.*, 2006). In addition to the autocrine effect on melanoma cells themselves upregulating cell motility genes, these factors are thought to have also paracrine pro-invasive effects, since they can signal to other cell types of the microenvironment, such as fibroblasts, to produce more pro-invasive molecules, like tenascin C and HGF (De Wever *et al.*, 2004). On the other hand, HGF receptor c-Met has also been shown to have proinvasive effects in melanocytes and melanoma cells (McGill *et al.*, 2006).

Melanoma inhibitory activity (MIA) protein is highly expressed in malignant melanomas but not in melanocytes (Bosserhoff, 2005). Several studies have shown that MIA enhances melanocyte and melanoma migration and invasion (Tatzel *et al.*, 2005), and additionally have suggested a central role for MIA in early melanoma development by regulating important melanoma-related pathways (Bosserhoff, 2005).

The Snail family transcription factors Snail and Slug have also increased activity in melanoma, since they downregulate the expression of keratinocyte-interacting surface molecules, such as E-cadherin and occluding, while upregulate N-cadherin, favoring interaction with stromal cells and not keratinocytes (Gaggioli and Sahai, 2007; Kajita *et al.*, 2004). Furthermore, Snail co-ordinately upregulates the expression of cell motility genes including matrix metalloproteinase (MMP)- 2, secreted protein acidic and rich in cysteine (SPARC), tissue inhibitor of metalloproteinases 1 (TIMP-1) and RhoA (Kuphal *et al.*, 2005).

Recently several studies have highlighted that melanoma cells also display a high plasticity regarding their cell motility. Melanoma cells can migrate and invade with different modes of movement that presumably allow them to adapt to varying microenvironments (Sahai and Marshall, 2003; Sanz-Moreno *et al.*, 2008). The main switches of these melanoma cell motility programs are Rho-GTPases family members Rac and Rho (Sanz-Moreno et al., 2008),

samples, and correlates with poor outcome (Folberg *et al.*, 2000; Hendrix *et al.*, 2003; Maniotis *et al.*, 1999). Furthermore, poorly aggressive melanoma cells can be reprogrammed to highly aggressive and invasive by exposing them to matrices preconditioned by more aggressive melanoma cells, highlighting the importance of the tumor microenvironment in

Primary melanoma patients can be usually cured by surgical removal of the tumor when detected early, but most of the times melanoma rapidly metastasizes with a poor outcome (Gray-Schopfer *et al.*, 2007). Therefore it is essential to understand the mechanisms by which melanoma cells escape from the primary tumor and spread and invade other organs, in order to develop improved therapies that increase patient survival rates. Following there is a brief summary detailing some key molecules involved in melanoma migration/invasion and metastasis; interested readers are suggested to read topic reviews elsewhere (Gaggioli

MITF plays a pivotal role in melanocyte and melanoma biology, as it is involved not only in the control of migration and invasion, but also in proliferation, survival, and differentiation of melanocytic cells (Levy *et al.*, 2006). The fine tuning of MITF activity enables melanoma cells to switch between a proliferative (high MITF) or invasive (low MITF) state, while very high levels promote differentiation and complete absence of MITF is incompatible with

Additionally, a number of growth factors and cytokines are upregulated as melanoma becomes more invasive, such as hepatocyte growth factor (HGF), TGF-β, IL-8, Nodal and several members of fibroblast growth factor (FGF) family (al-Alousi *et al.*, 1996; Albino *et al.*, 1991; Topczewska *et al.*, 2006). In addition to the autocrine effect on melanoma cells themselves upregulating cell motility genes, these factors are thought to have also paracrine pro-invasive effects, since they can signal to other cell types of the microenvironment, such as fibroblasts, to produce more pro-invasive molecules, like tenascin C and HGF (De Wever *et al.*, 2004). On the other hand, HGF receptor c-Met has also been shown to have pro-

Melanoma inhibitory activity (MIA) protein is highly expressed in malignant melanomas but not in melanocytes (Bosserhoff, 2005). Several studies have shown that MIA enhances melanocyte and melanoma migration and invasion (Tatzel *et al.*, 2005), and additionally have suggested a central role for MIA in early melanoma development by regulating

The Snail family transcription factors Snail and Slug have also increased activity in melanoma, since they downregulate the expression of keratinocyte-interacting surface molecules, such as E-cadherin and occluding, while upregulate N-cadherin, favoring interaction with stromal cells and not keratinocytes (Gaggioli and Sahai, 2007; Kajita *et al.*, 2004). Furthermore, Snail co-ordinately upregulates the expression of cell motility genes including matrix metalloproteinase (MMP)- 2, secreted protein acidic and rich in cysteine (SPARC), tissue inhibitor of metalloproteinases 1 (TIMP-1) and RhoA (Kuphal *et al.*, 2005). Recently several studies have highlighted that melanoma cells also display a high plasticity regarding their cell motility. Melanoma cells can migrate and invade with different modes of movement that presumably allow them to adapt to varying microenvironments (Sahai and Marshall, 2003; Sanz-Moreno *et al.*, 2008). The main switches of these melanoma cell motility programs are Rho-GTPases family members Rac and Rho (Sanz-Moreno et al., 2008),

driving melanoma progression (Postovit *et al.*, 2006; Seftor *et al.*, 2006).

**3.2 Melanoma invasion**

and Sahai, 2007; Uong and Zon, 2010).

survival (Carreira *et al.*, 2006; Gray-Schopfer *et al.*, 2007).

important melanoma-related pathways (Bosserhoff, 2005).

invasive effects in melanocytes and melanoma cells (McGill *et al.*, 2006).

which are overexpressed in cancer (Sahai, 2005). Rac promotes a more elongated shape that requires matrix metalloproteinases in order to invade, while RhoA favors a rounded morphology and a movement less dependent on proteases (Sanz-Moreno *et al.*, 2008). On the other hand, MMPs are upregulated in invasive melanoma and they are thought to promote melanoma dissemination, probably through different mechanisms since different MMPs are thought to have pro- or anti-invasive effects (Overall and Lopez-Otin, 2002).

#### **3.3 PEDF as a brake for melanoma progression**

As previously mentioned, the antiangiogenic activity of PEDF prompted the study of its potential antitumor effects. We (Fernandez-Garcia *et al.*, 2007; Garcia *et al.*, 2004) and others (Abe et al., 2004; Doll et al., 2003; Ek et al., 2006b) described a complex mechanism underlying the potent inhibition of melanoma metastasis by PEDF. PEDF's antitumor activity in melanoma and other tumors is based on its dual action on the tumor microenvironment and on the tumor cells themselves (Fernandez-Garcia *et al.*, 2007). PEDF inhibits tumor angiogenesis by means of induction of apoptosis on endothelial cells and modulation of the angiogenic profile of melanoma cells, eventually destroying the main source of nutrients to the primary tumor as well as one of the main routes of dissemination to distant organs. Additionally, PEDF exerts a potent inhibitory action on melanoma cells, inducing apoptosis under stress conditions (such as absence of growth factors or detachment from the extracellular matrix) and abrogating migration and invasion. As a whole, PEDF overexpression in melanoma cells leads to a decrease in primary tumor growth and an inhibition of lung metastasis formation (Fernandez-Garcia *et al.*, 2007; Filleur *et al.*, 2009).

In the previous section we described that PEDF is produced by low aggressive melanomas. Given that cutaneous melanoma develops from skin melanocytes, two questions soon arise: i) are normal melanocytes expressing this factor endogenously?, ii) if so, which could be the role of PEDF in normal melanocytic cells? By means of assessing a large collection of primary cultures of cutaneous melanocytes and other cell types of the skin we found that melanocytes secrete very high levels of PEDF comparable to other cell types known to express this factor (Orgaz *et al.*, 2009). In agreement with previous reports, dermal fibroblasts also express high levels of PEDF, which has been described to be involved in the control of their proliferative potential (Francis *et al.*, 2004; Tresini *et al.*, 1999); additionally, PEDF secreted by fibroblasts could also play a role in maintaining a correct vascularization of the skin. In contrast, PEDF is expressed at very low levels by epidermal keratinocytes or microvascular endothelial cells (Orgaz *et al.*, 2009).

In melanocytes, PEDF is one of the players involved in the regulation of their proliferation and migratory ability (Fig. 1). PEDF silencing in primary melanocytes leads to an increase in their migration and invasion, as well as a moderate augment in their growth rate (Orgaz *et al.*, 2009). Melanocytes arise from highly migratory embryonic neural crest progenitors, and therefore display an enormous migratory potential (Gupta *et al.*, 2005; Zbytek *et al.*, 2008) that must be tightly controlled in the skin (Hsu *et al.*, 2002). In addition to the regulatory signals from adjacent keratinocytes, an additional brake to their uncontrolled dissemination could be self-imposed within melanocytes by expressing high levels of PEDF themselves. As suggested for the fibroblasts, melanocytes could be also participating in the maintenance of appropriate angiogenesis in the skin by secreting this potent antiangiogenic factor, taking into account previous studies reporting an excessive vascularization of multiple organs upon PEDF knockdown (Doll *et al.*, 2003). Additional paracrine actions on other cell types, keratinocytes for instance, remain to be investigated.

Pigment Epithelium-Derived Factor –

and unpublished data) (Fig. 1).

PEDF (Orgaz *et al.*, 2009).

An Angiostatic Factor with a Broader Function in Melanoma 153

progression is a complex task. Therefore, we utilized different approaches in order to interrogate levels of PEDF during melanoma malignization (Orgaz *et al.*, 2009). When taking into account histopathological criteria (classical model of melanoma progression), PEDF expression is greatly diminished or lost in metastasis-derived melanoma cell lines compared to cell lines established from RGP or VGP tumors, even though we found a significant variability in PEDF levels across melanoma cell lines. Accordingly, PEDF expression is lower in metastatic melanoma biopsies compared to primary melanomas (Orgaz *et al.,* 2009;

This first analysis was expanded taking advantage of the vast collection of publicly available gene expression data from large series of melanoma cell lines and primary cultures of melanocytes. Firstly, we confirmed the downregulation of PEDF expression in melanoma compared to primary melanocytes (Fig. 1) (Hoek, 2007; Orgaz *et al.*, 2009). When classifying a number of series of melanoma cell lines regarding their gene expression profile into proliferative or invasive phenotype, we found that there is a significant decrease of PEDF expression in invasive phenotype melanoma cell lines. This suggests again an inverse correlation between melanoma aggressiveness and invasiveness, and expression levels of

Furthermore, expression of PEDF is subject to certain plasticity and can be reprogrammed during the metastatic progression of melanoma. We utilized paired cell lines isolated from the same metastasis of a cutaneous melanoma patient, and that display extreme phenotypes, poorly or highly aggressive (Seftor *et al.*, 2005). Only the poorly aggressive melanoma cell line expresses PEDF at high levels comparable to those of melanocytes, while it is undetectable in the highly aggressive cell line. The heterogeneity of PEDF expression is also found *in vivo* when analyzed in biopsies from dermal or lymph node melanoma metastases. Most of the biopsies from metastases are negative for PEDF, but when positive they display a heterogeneous staining pattern of PEDF expression (Orgaz *et al.*, 2009). Both observations strongly suggest that PEDF expression could be reprogrammed during the metastatic process in melanoma, in the context of the phenotype switching model, being expressed only by the poorly aggressive subpopulation of tumor cells (Fig. 1). Regulatory signals from the tumor microenvironment could be responsible, at least in part, for this switching in PEDF expression. As an example, primary melanocytes grown on collagen matrix preconditioned by a highly aggressive melanoma cell line display decreased PEDF expression levels compared to melanocytes grown on untreated matrix (Seftor *et al.*, 2005). The inverse correlation between melanoma aggressiveness and PEDF expression raises additional important questions. Firstly, what is the functional significance of PEDF modulation during melanoma progression? We addressed this by silencing endogenous PEDF expression in several melanoma cell lines utilizing short hairpin RNAs specific to PEDF delivered by lentiviral transduction. As in melanocytes, endogenous PEDF restricts the migratory and invasive ability of melanoma cells, which greatly translates *in vivo*: we found that PEDF inhibits spontaneous lung colonization by melanoma cells as well as formation of spontaneous lung metastases from a primary tumor (Orgaz *et al.*, 2009). Inhibition of spontaneous metastases formation by PEDF is particularly noteworthy, since it highlights that this factor also restricts the initial steps of the metastatic cascade, local invasiveness from the primary tumor and intravasation. Additionally, PEDF impinges on vasculogenic mimicry ability of melanoma cells (Fig. 1), diminishing the formation of vasculogenic networks by melanoma cells both *in vitro* on collagen matrices and *in vivo* in the lung parenchyma after tail vein injection into immunocompromised mice (Orgaz *et al.*, 2009).

Importantly, recent evidence points to a possible role of PEDF in melanocytic lineagespecific functions, such as pigment production. Melanocytes utilize specialized membrane vesicles called melanosomes to synthesize, store and deliver melanin to the cell membrane, eventually being transferred to surrounding keratinocytes (Barral and Seabra, 2004; Lin and Fisher, 2007). In a recent study we have found that PEDF overexpression in melanoma cell lines modulates the expression of Rab27A and melanophilin, two regulators of melanosome trafficking and melanin transfer (Orgaz *et al.*, 2011). Accordingly, previous reports had proposed the involvement of PEDF in pigment production, as PEDF was found in immature melanosomes of melanoma cells (Chi *et al.*, 2006) and it was also shown to induce tyrosinase expression (Abul-Hassan *et al.*, 2000) and melanosome maturation in RPE cells (Malchiodi-Albedi *et al.*, 1998). However, further studies are warranted in order to elucidate the functional role of PEDF in melanin production and/or secretion.

Fig. 1. Regulation of expression of PEDF during melanoma progression and functional effects in melanocytes and melanoma cells. Skin melanocytes express high levels of PEDF, which contribute to restrict their migratory and proliferative ability, and to regulate skin vascularization. In melanoma, PEDF expression is regulated by yet unidentified mechanisms that determine high levels in proliferative phenotype cells, and low levels in invasive phenotype cells. Melanoma progression is driven by switching between proliferative and invasive phenotypes, which involves reprogramming of PEDF and heterogeneous expression of this factor in primary and metastatic melanomas

The high expression of PEDF in skin melanocytes and its antitumor effects in melanoma raise the question whether PEDF expression could be modulated during melanoma development as it takes place in other types of cancer. However, defining melanoma

Importantly, recent evidence points to a possible role of PEDF in melanocytic lineagespecific functions, such as pigment production. Melanocytes utilize specialized membrane vesicles called melanosomes to synthesize, store and deliver melanin to the cell membrane, eventually being transferred to surrounding keratinocytes (Barral and Seabra, 2004; Lin and Fisher, 2007). In a recent study we have found that PEDF overexpression in melanoma cell lines modulates the expression of Rab27A and melanophilin, two regulators of melanosome trafficking and melanin transfer (Orgaz *et al.*, 2011). Accordingly, previous reports had proposed the involvement of PEDF in pigment production, as PEDF was found in immature melanosomes of melanoma cells (Chi *et al.*, 2006) and it was also shown to induce tyrosinase expression (Abul-Hassan *et al.*, 2000) and melanosome maturation in RPE cells (Malchiodi-Albedi *et al.*, 1998). However, further studies are warranted in order to elucidate the

Fig. 1. Regulation of expression of PEDF during melanoma progression and functional effects in melanocytes and melanoma cells. Skin melanocytes express high levels of PEDF, which contribute to restrict their migratory and proliferative ability, and to regulate skin

mechanisms that determine high levels in proliferative phenotype cells, and low levels in

The high expression of PEDF in skin melanocytes and its antitumor effects in melanoma raise the question whether PEDF expression could be modulated during melanoma development as it takes place in other types of cancer. However, defining melanoma

vascularization. In melanoma, PEDF expression is regulated by yet unidentified

invasive phenotype cells. Melanoma progression is driven by switching between proliferative and invasive phenotypes, which involves reprogramming of PEDF and heterogeneous expression of this factor in primary and metastatic melanomas

functional role of PEDF in melanin production and/or secretion.

progression is a complex task. Therefore, we utilized different approaches in order to interrogate levels of PEDF during melanoma malignization (Orgaz *et al.*, 2009). When taking into account histopathological criteria (classical model of melanoma progression), PEDF expression is greatly diminished or lost in metastasis-derived melanoma cell lines compared to cell lines established from RGP or VGP tumors, even though we found a significant variability in PEDF levels across melanoma cell lines. Accordingly, PEDF expression is lower in metastatic melanoma biopsies compared to primary melanomas (Orgaz *et al.,* 2009; and unpublished data) (Fig. 1).

This first analysis was expanded taking advantage of the vast collection of publicly available gene expression data from large series of melanoma cell lines and primary cultures of melanocytes. Firstly, we confirmed the downregulation of PEDF expression in melanoma compared to primary melanocytes (Fig. 1) (Hoek, 2007; Orgaz *et al.*, 2009). When classifying a number of series of melanoma cell lines regarding their gene expression profile into proliferative or invasive phenotype, we found that there is a significant decrease of PEDF expression in invasive phenotype melanoma cell lines. This suggests again an inverse correlation between melanoma aggressiveness and invasiveness, and expression levels of PEDF (Orgaz *et al.*, 2009).

Furthermore, expression of PEDF is subject to certain plasticity and can be reprogrammed during the metastatic progression of melanoma. We utilized paired cell lines isolated from the same metastasis of a cutaneous melanoma patient, and that display extreme phenotypes, poorly or highly aggressive (Seftor *et al.*, 2005). Only the poorly aggressive melanoma cell line expresses PEDF at high levels comparable to those of melanocytes, while it is undetectable in the highly aggressive cell line. The heterogeneity of PEDF expression is also found *in vivo* when analyzed in biopsies from dermal or lymph node melanoma metastases. Most of the biopsies from metastases are negative for PEDF, but when positive they display a heterogeneous staining pattern of PEDF expression (Orgaz *et al.*, 2009). Both observations strongly suggest that PEDF expression could be reprogrammed during the metastatic process in melanoma, in the context of the phenotype switching model, being expressed only by the poorly aggressive subpopulation of tumor cells (Fig. 1). Regulatory signals from the tumor microenvironment could be responsible, at least in part, for this switching in PEDF expression. As an example, primary melanocytes grown on collagen matrix preconditioned by a highly aggressive melanoma cell line display decreased PEDF expression levels compared to melanocytes grown on untreated matrix (Seftor *et al.*, 2005).

The inverse correlation between melanoma aggressiveness and PEDF expression raises additional important questions. Firstly, what is the functional significance of PEDF modulation during melanoma progression? We addressed this by silencing endogenous PEDF expression in several melanoma cell lines utilizing short hairpin RNAs specific to PEDF delivered by lentiviral transduction. As in melanocytes, endogenous PEDF restricts the migratory and invasive ability of melanoma cells, which greatly translates *in vivo*: we found that PEDF inhibits spontaneous lung colonization by melanoma cells as well as formation of spontaneous lung metastases from a primary tumor (Orgaz *et al.*, 2009). Inhibition of spontaneous metastases formation by PEDF is particularly noteworthy, since it highlights that this factor also restricts the initial steps of the metastatic cascade, local invasiveness from the primary tumor and intravasation. Additionally, PEDF impinges on vasculogenic mimicry ability of melanoma cells (Fig. 1), diminishing the formation of vasculogenic networks by melanoma cells both *in vitro* on collagen matrices and *in vivo* in the lung parenchyma after tail vein injection into immunocompromised mice (Orgaz *et al.*, 2009).

Pigment Epithelium-Derived Factor –

*al.*, 2007).

metastasis.

*et al.*, 1999; Utikal *et al.*, 2007).

**4.1 Biochemical features of PEDF**

secreted serpin family member.

An Angiostatic Factor with a Broader Function in Melanoma 155

interesting to assess whether p73 is implicated in regulating PEDF expression during melanoma progression. Human melanomas display either oncogenic NRAS (20% melanomas) or BRAF (50-70%), thought to be responsible of the initial transformation of melanocytes, uncoupling cell growth from external mitogenic stimuli (Chin, 2003; Chin *et al.*, 2006). However, by means of an analysis of publicly available microarray data from melanoma cell lines, we did not observe a significant correlation between PEDF expression levels and BRAF or NRAS mutation status (unpublished data). Finally, loss of expression of PEDF could occur upon epigenetic silencing of its promoter. Maspin, another member of Serpin family with anti-invasive and anti-tumor activities, is expressed at high levels in melanocytes but it is silenced in naevi and melanomas by epigenetic mechanisms (Denk *et*

Although molecular aspects of the mechanism of action of PEDF on endothelial cells or neural derived cells have been described little is known about the molecular mechanisms underlying PEDF´s actions in melanoma, particularly in the inhibition of invasion and

Interestingly, a recent study by us utilizing a gene expression analysis upon PEDF overexpression in melanoma cell lines has started to reveal some factors and pathways that could be mediating PEDF antimetastatic effects (Orgaz *et al.*, 2011). PEDF downregulates several key promigratory and proangiogenic factors such as IL-8, TGF-α and TGF-β, as well as a number of proteases and extracellular matrix proteins, like collagen IV, that could account for the lesser invasive ability and angiogenic potential of melanoma cells expressing PEDF. Additionally, PEDF modulates genes previously involved in melanoma progression toward a trend in agreement with decreased aggressiveness and invasiveness, such as factors from the Notch (Pinnix and Herlyn, 2007) or Wnt (Weeraratna, 2005) pathways, as well as FGF13 (Hoek *et al.*, 2004), insuling-like growth factor binding protein 3 (IGFBP3) (Xi *et al.*, 2006) or inhibin beta A (INHBA) (Hoek *et al.*, 2006), among many others. Interestingly, a number of melanoma markers with increased levels upon melanoma malignization are predominantly downregulated by PEDF overexpression, such as MIA or S100-β (Deichmann

**4. Therapeutic applications of pigment epithelium derived-factor**

several proline residues preceding the cleavage site (Simonovic *et al.*, 2001).

Therapeutic applications of PEDF are closely related to the cellular niches where this factor is produced and to the multiplicity of cellular functions and activities ascribed to this

SERPINs are a large superfamily of genes that codes for serine protease inhibitors in mammals (Tombran-Tink *et al.*, 2005). These proteins are able to control several processes such as blood coagulation, complement activation and extracellular matrix remodelling (Filleur *et al.*, 2009; Tombran-Tink *et al.*, 2005). However, there is a small number of serpin family members with non-inhibitory protease activity, among which is included PEDF (*SERPINF1*) (Becerra *et al.*, 1995; Lawrence *et al.*, 1990; Steele *et al.*, 1993). The inhibitory activity against proteinases resides in a domain called reactive centre loop (RCL). The reason why PEDF lacks protease inhibitory capability is due to the presence in the RCL of

Amino acid analyses indicate that PEDF shares considerable sequence homology with other members of the serpin family (Steele *et al.*, 1993); however, residues 40-67, at the N-terminal

Therefore, in the model that we propose reprogramming of PEDF expression is important for the switching between proliferative and invasive phenotypes that are thought to drive metastatic progression of melanoma. Unknown mechanisms so far determine that PEDF expression is high in proliferative phenotype primary tumor cells, where it restricts migratory and invasive abilities, angiogenic potential and vasculogenic mimicry, as a whole leading to a diminished metastatic potential. However, loss of PEDF helps melanoma cells to acquire an invasive phenotype essential to disseminate and colonize distant organs. There, some metastatic cells would be reprogrammed by signals from the new microenvironment toward proliferative phenotype cells, switching back to express PEDF again. Eventually these cells will develop micrometastases and macrometastases, where additional reprogramming events will take place leading to a metastatic lesion heterogeneous for PEDF expression. Additionally, mechanisms yet to be described determine a loss of PEDF expression in fully developed melanoma metastases (Fig. 1).

Despite the knowledge we have gathered regarding PEDF modulation in melanoma, important questions still remain to be resolved. First of all, which are the regulators of PEDF during melanoma progression? Elucidating the mechanisms that promote or repress PEDF expression would help better understand how PEDF changes in the course of transformation and malignant progression of melanoma. Our results suggest that PEDF expression could be modulated by two general types of mechanisms, reprogramming events and loss of expression.

Assessing PEDF expression during maturation and migration of neural crest precursors toward epidermis could allow investigating the relationship between migratory potential and differentiation state and PEDF expression in a physiological context. Eventually this could also help identify possible factors involved in reprogramming PEDF in the different environments that neural crest precursors encounter in their way toward epidermis. Some of these factors are likely to be responsible for modulating PEDF expression in melanoma cells, given that tumor cells frequently activate signaling pathways and utilize regulatory mechanisms typical of progenitor cells.

Signals from the microenvironment such as hypoxia and inflammation are thought to reprogram and switch melanoma cells toward an invasive phenotype, and therefore, could be responsible for the downregulation of PEDF during melanoma progression. As a matter of a fact, earlier reports described that PEDF expression is decreased by hypoxia in retinoblastoma (Dawson *et al.*, 1999) and RPE (Notari *et al.*, 2005). Additionally, transcription factors that drive and are at the core of each phenotype gene signature could be direct regulators of PEDF expression. MITF is an appealing candidate, since it is a key factor in melanoma and melanocyte biology (Levy *et al.*, 2006) with anti-invasive effects and whose expression is tightly associated with the proliferative phenotype (Carreira *et al.*, 2006; Hoek *et al.*, 2008). Similar to PEDF, some studies have reported a trend toward loss of MITF expression in metastases compared to primary melanomas (Carreira *et al.*, 2006; Goodall *et al.*, 2008).

Additionally, genetic and epigenetic mechanisms could lead to a loss of PEDF expression as melanoma evolves to metastatic. The expression of a number of angiogenesis regulators in cancer is controlled by gain of expression of oncogenes and loss of tumor suppressors (Bouck, 1990). PEDF is a direct target of p53-related p63 and p73 proteins in colorectal carcinoma (Sasaki *et al.*, 2005). In melanoma, TA-p73 isoform has been recently described to inhibit anchor independent growth through KCNK1 protein, whose expression decreases in melanoma compared to normal skin (Beitzinger *et al.*, 2008). Therefore it would be

Therefore, in the model that we propose reprogramming of PEDF expression is important for the switching between proliferative and invasive phenotypes that are thought to drive metastatic progression of melanoma. Unknown mechanisms so far determine that PEDF expression is high in proliferative phenotype primary tumor cells, where it restricts migratory and invasive abilities, angiogenic potential and vasculogenic mimicry, as a whole leading to a diminished metastatic potential. However, loss of PEDF helps melanoma cells to acquire an invasive phenotype essential to disseminate and colonize distant organs. There, some metastatic cells would be reprogrammed by signals from the new microenvironment toward proliferative phenotype cells, switching back to express PEDF again. Eventually these cells will develop micrometastases and macrometastases, where additional reprogramming events will take place leading to a metastatic lesion heterogeneous for PEDF expression. Additionally, mechanisms yet to be described determine a loss of PEDF expression in fully

Despite the knowledge we have gathered regarding PEDF modulation in melanoma, important questions still remain to be resolved. First of all, which are the regulators of PEDF during melanoma progression? Elucidating the mechanisms that promote or repress PEDF expression would help better understand how PEDF changes in the course of transformation and malignant progression of melanoma. Our results suggest that PEDF expression could be modulated by two general types of mechanisms, reprogramming events and loss of

Assessing PEDF expression during maturation and migration of neural crest precursors toward epidermis could allow investigating the relationship between migratory potential and differentiation state and PEDF expression in a physiological context. Eventually this could also help identify possible factors involved in reprogramming PEDF in the different environments that neural crest precursors encounter in their way toward epidermis. Some of these factors are likely to be responsible for modulating PEDF expression in melanoma cells, given that tumor cells frequently activate signaling pathways and utilize regulatory

Signals from the microenvironment such as hypoxia and inflammation are thought to reprogram and switch melanoma cells toward an invasive phenotype, and therefore, could be responsible for the downregulation of PEDF during melanoma progression. As a matter of a fact, earlier reports described that PEDF expression is decreased by hypoxia in retinoblastoma (Dawson *et al.*, 1999) and RPE (Notari *et al.*, 2005). Additionally, transcription factors that drive and are at the core of each phenotype gene signature could be direct regulators of PEDF expression. MITF is an appealing candidate, since it is a key factor in melanoma and melanocyte biology (Levy *et al.*, 2006) with anti-invasive effects and whose expression is tightly associated with the proliferative phenotype (Carreira *et al.*, 2006; Hoek *et al.*, 2008). Similar to PEDF, some studies have reported a trend toward loss of MITF expression in metastases compared to primary melanomas (Carreira *et al.*, 2006; Goodall *et*

Additionally, genetic and epigenetic mechanisms could lead to a loss of PEDF expression as melanoma evolves to metastatic. The expression of a number of angiogenesis regulators in cancer is controlled by gain of expression of oncogenes and loss of tumor suppressors (Bouck, 1990). PEDF is a direct target of p53-related p63 and p73 proteins in colorectal carcinoma (Sasaki *et al.*, 2005). In melanoma, TA-p73 isoform has been recently described to inhibit anchor independent growth through KCNK1 protein, whose expression decreases in melanoma compared to normal skin (Beitzinger *et al.*, 2008). Therefore it would be

developed melanoma metastases (Fig. 1).

mechanisms typical of progenitor cells.

expression.

*al.*, 2008).

interesting to assess whether p73 is implicated in regulating PEDF expression during melanoma progression. Human melanomas display either oncogenic NRAS (20% melanomas) or BRAF (50-70%), thought to be responsible of the initial transformation of melanocytes, uncoupling cell growth from external mitogenic stimuli (Chin, 2003; Chin *et al.*, 2006). However, by means of an analysis of publicly available microarray data from melanoma cell lines, we did not observe a significant correlation between PEDF expression levels and BRAF or NRAS mutation status (unpublished data). Finally, loss of expression of PEDF could occur upon epigenetic silencing of its promoter. Maspin, another member of Serpin family with anti-invasive and anti-tumor activities, is expressed at high levels in melanocytes but it is silenced in naevi and melanomas by epigenetic mechanisms (Denk *et al.*, 2007).

Although molecular aspects of the mechanism of action of PEDF on endothelial cells or neural derived cells have been described little is known about the molecular mechanisms underlying PEDF´s actions in melanoma, particularly in the inhibition of invasion and metastasis.

Interestingly, a recent study by us utilizing a gene expression analysis upon PEDF overexpression in melanoma cell lines has started to reveal some factors and pathways that could be mediating PEDF antimetastatic effects (Orgaz *et al.*, 2011). PEDF downregulates several key promigratory and proangiogenic factors such as IL-8, TGF-α and TGF-β, as well as a number of proteases and extracellular matrix proteins, like collagen IV, that could account for the lesser invasive ability and angiogenic potential of melanoma cells expressing PEDF. Additionally, PEDF modulates genes previously involved in melanoma progression toward a trend in agreement with decreased aggressiveness and invasiveness, such as factors from the Notch (Pinnix and Herlyn, 2007) or Wnt (Weeraratna, 2005) pathways, as well as FGF13 (Hoek *et al.*, 2004), insuling-like growth factor binding protein 3 (IGFBP3) (Xi *et al.*, 2006) or inhibin beta A (INHBA) (Hoek *et al.*, 2006), among many others. Interestingly, a number of melanoma markers with increased levels upon melanoma malignization are predominantly downregulated by PEDF overexpression, such as MIA or S100-β (Deichmann *et al.*, 1999; Utikal *et al.*, 2007).

## **4. Therapeutic applications of pigment epithelium derived-factor**

#### **4.1 Biochemical features of PEDF**

Therapeutic applications of PEDF are closely related to the cellular niches where this factor is produced and to the multiplicity of cellular functions and activities ascribed to this secreted serpin family member.

SERPINs are a large superfamily of genes that codes for serine protease inhibitors in mammals (Tombran-Tink *et al.*, 2005). These proteins are able to control several processes such as blood coagulation, complement activation and extracellular matrix remodelling (Filleur *et al.*, 2009; Tombran-Tink *et al.*, 2005). However, there is a small number of serpin family members with non-inhibitory protease activity, among which is included PEDF (*SERPINF1*) (Becerra *et al.*, 1995; Lawrence *et al.*, 1990; Steele *et al.*, 1993). The inhibitory activity against proteinases resides in a domain called reactive centre loop (RCL). The reason why PEDF lacks protease inhibitory capability is due to the presence in the RCL of several proline residues preceding the cleavage site (Simonovic *et al.*, 2001).

Amino acid analyses indicate that PEDF shares considerable sequence homology with other members of the serpin family (Steele *et al.*, 1993); however, residues 40-67, at the N-terminal

Pigment Epithelium-Derived Factor –

2007).

**4.2 PEDF receptors**

An Angiostatic Factor with a Broader Function in Melanoma 157

the TGA epitope, and an internal fragment of 44-mer, referred to as ERT. Filleur and collaborators showed *in vivo* that the epitopes TGA and the complete 34-mer inhibit tumor angiogenesis in prostate adenocarcinoma by inducing apoptosis of endothelial cells and blocking endothelial cell migration. Curiously, ERT despite of being located inside the neurotrophic 44-mer peptide, also presents antiangiogenic activity in prostate adenocarcinoma. Complete 44-mer is able to induce neurite outgrowth in Y-79 retinoblastoma cell line and causes apoptosis of endothelial cells, blocking migration and angiogenesis (Filleur *et al.*, 2005). In a more recent study, smaller regions of 34-mer epitope (named P14, P18 and P23, according to their respective length) were tested for angioinhibitory activity *in vitro* and *in vivo*. P14 and P23 display antiangiogenic activity *in vitro* (both blocking endothelial chemotaxis, and inducing apoptosis in the case of P23), but not *in vivo*; while P18 is a more potent antiangiogenic peptide than 34-mer in prostate cancer, being able to block bFGF and VEGF-dependent angiogenesis in the *in vivo* cornea neovascularisation assay (Mirochnik *et al.*, 2009). Another study by Ek and colleagues identified other small peptides with antitumoral activity in an orthotopic osteosarcoma model. They generated six 25-mer peptides along the functionally distinct regions of PEDF characterized so far. Residues 78-102 inhibit proliferation, whereas residues 90-114 stimulate adhesion of PEDF to type I collagen. Furthermore, residues 387 to 411 inhibit invasion of osteosarcoma cells *in vitro* and residues 40-64 promote osteogenic differentiation (Ek *et al.*,

The multifunctional character of PEDF and the evidence of the different roles displayed depending on the cell type, suggest that PEDF could be acting through distinct domains recognized by several specific receptors. The identification of these putative receptors and the characterization of the binding affinity of each functionally identified peptide toward them could make a breakthrough in the understanding of PEDF's mechanism of action and,

Several studies have characterized the affinity of PEDF for the surface of human retinoblastoma cells, cerebellar granular neurons (Alberdi *et al.*, 1999), motor neurons (Bilak *et al.*, 2002), neural retina (Aymerich *et al.*, 2001) and endothelial cells (Yamagishi *et al.*, 2004). PEDF could be sequestered in the extracellular matrix based on ionic interactions with sulphated (heparin, heparin sulfate and condroitin sulfate) (Alberdi *et al.*, 1998), and nonsulfated (hyaluronan) (Becerra *et al.*, 2008) glycosaminglycans and type I collagen (Meyer *et al.*, 2002). In order to identify the potential receptor(s) of PEDF, Simonovic and collaborators carried out a three-dimensional study of PEDF, and identified an asymmetric charge distribution, with a high acidic region located at C-ter and basic amino acids at opposite region of PEDF protein (Simonovic *et al.*, 2001). This basic region is involved in the binding of PEDF to heparin through three clustered basic amino acid residues, Lys146, Lys147 and Arg149. Moreover, Asp256, Asp258 and Asp300 residues present in the acidic region of PEDF are

Using the 34-mer and 44-mer epitopes it was possible to propose the existence of two distinct putative receptors for PEDF. These epitopes are able to bind the surface of endothelial and prostate cells, but they do not compete for receptor binding (Filleur *et al.*, 2005). This result suggests the existence of two PEDF receptors with different functions: PEDF-RN, that interacts with 44-mer epitope and regulates the neurotrophic and neuroprotective activities of PEDF; and PEDF-RA, which is involved in blocking

therefore, the possibility of its therapeutic use in multiple pathological contexts.

crucial to type I collagen binding (Meyer *et al.*, 2002; Yasui *et al.*, 2003).

(N-ter) region, and residues 277-301 at C-terminal (C-ter) are specific to PEDF. This feature suggests that these residues could be involved in maintaining the distinct structure of PEDF in these regions or in determining specific functions of this non-inhibitory serpin (Tombran-Tink *et al.*, 2005; Xu *et al.*, 2006). Tombran-Tink and collaborators also compared sequence homology of PEDF protein among different species and analyzed structural homology. They found a high degree of conservation in the leader sequence, responsible for protein secretion, a C-ter glycosilation site, and four specific regions: two domains present at N-ter region, corresponding to residues 40-67 and 78-95; and other two regions, 277-301 and 384- 415, located at C-ter of the protein (Tombran-Tink *et al.*, 2005).

PEDF was initially isolated from conditioned medium of human fetal RPE cells and identified as a neurotrophic factor (Tombran-Tink and Johnson, 1989), although further studies showed that it is widely expressed throughout fetal and adult tissues (Ek *et al.*, 2006b). This broad expression is suggestive of a general and relevant function of PEDF in mammals. PEDF is also known as EPC-1 (early population doubling cDNA-1), and has been shown to participate in cell cycle regulation, initially in fibroblasts (Pignolo *et al.*, 1993; Tombran-Tink and Johnson, 1989; Tombran-Tink *et al.*, 1995) and later in other cell types like endothelial cells (Duh *et al.*, 2002; Hutchings *et al.*, 2002). PEDF's cell cycle regulatory function requires the presence of a putative nuclear localization sequence (residues 141-151) that is highly conserved in *SERPINF1* among different species, and could mediate PEDF translocation to the nucleus (Tombran-Tink *et al.*, 2005). One of the most relevant functional characteristics of PEDF is its antiangiogenic activity, being considered as the most potent natural inhibitor of physiological and pathological angiogenesis (Dawson *et al.*, 1999). PEDF is also a very effective neurotrophic factor that induces cell differentiation, cell survival, and protection from cell death in many cell types of the nervous system. PEDF prevents degeneration of retinal neurons that are exposed to transient ischemic reperfusion (Ogata *et al.*, 2001), and also protects other regions of the brain and spinal cord from the damaging effects caused by oxidative stress and glutamate toxicity (Bilak *et al.*, 1999; Cao *et al.*, 1999; DeCoster *et al.*, 1999; Taniwaki *et al.*, 1995). It has been shown the implication of NFkB in all these processes, which induces the expression of neurotrophic factors and anti-apoptotic genes that participate in the control of cell survival, proliferation and death (Barnstable and Tombran-Tink, 2004; Yabe *et al.*, 2001). Neurotrophic activity has been mapped to the N-ter region of PEDF, encompassing residues Val58-Thr101 (Simonovic *et al.*, 2001). This region is involved in binding to a putative plasma membrane receptor in cerebellar granule neurons and retinoblastoma cells (Alberdi *et al.*, 1999). PEDF is also secreted by ependymal and endothelial cells from the subventricular zone of the brain, where it promotes the selfrenewal of adult neural stem cells (Andreu-Agullo *et al.*, 2009; Ramirez-Castillejo *et al.*, 2006). Furthermore, PEDF presents three phosphorylation sites located in Ser24, Ser114 and Ser227, and the regulation of their phosphorylation state modulates the switch of PEDF's function from neurotrophic (Ser24 and Ser114 phosphorylated) to angiangiogenic (Ser227 phosphorylated) (Becerra, 2006).

In order to better understand the mechanism of action of PEDF, it was necessary to identify the structural domains responsible for each of its described biological functions. Two major epitopes were identified at the N-ter region of PEDF: 34-mer peptide (residues 24-57) responsible of the antiangiogenic and pro-apoptotic actions of PEDF; and a second epitope, 44-mer peptide (residues 58-101), which induces neuronal differentiation of retinoblastoma cells (Filleur *et al.*, 2005) and plays a neurotrophic role in many neuronal cell types (Bilak *et al.*, 2002). There are another two highly conserved smaller epitopes, one upstream 34-mer,

(N-ter) region, and residues 277-301 at C-terminal (C-ter) are specific to PEDF. This feature suggests that these residues could be involved in maintaining the distinct structure of PEDF in these regions or in determining specific functions of this non-inhibitory serpin (Tombran-Tink *et al.*, 2005; Xu *et al.*, 2006). Tombran-Tink and collaborators also compared sequence homology of PEDF protein among different species and analyzed structural homology. They found a high degree of conservation in the leader sequence, responsible for protein secretion, a C-ter glycosilation site, and four specific regions: two domains present at N-ter region, corresponding to residues 40-67 and 78-95; and other two regions, 277-301 and 384-

PEDF was initially isolated from conditioned medium of human fetal RPE cells and identified as a neurotrophic factor (Tombran-Tink and Johnson, 1989), although further studies showed that it is widely expressed throughout fetal and adult tissues (Ek *et al.*, 2006b). This broad expression is suggestive of a general and relevant function of PEDF in mammals. PEDF is also known as EPC-1 (early population doubling cDNA-1), and has been shown to participate in cell cycle regulation, initially in fibroblasts (Pignolo *et al.*, 1993; Tombran-Tink and Johnson, 1989; Tombran-Tink *et al.*, 1995) and later in other cell types like endothelial cells (Duh *et al.*, 2002; Hutchings *et al.*, 2002). PEDF's cell cycle regulatory function requires the presence of a putative nuclear localization sequence (residues 141-151) that is highly conserved in *SERPINF1* among different species, and could mediate PEDF translocation to the nucleus (Tombran-Tink *et al.*, 2005). One of the most relevant functional characteristics of PEDF is its antiangiogenic activity, being considered as the most potent natural inhibitor of physiological and pathological angiogenesis (Dawson *et al.*, 1999). PEDF is also a very effective neurotrophic factor that induces cell differentiation, cell survival, and protection from cell death in many cell types of the nervous system. PEDF prevents degeneration of retinal neurons that are exposed to transient ischemic reperfusion (Ogata *et al.*, 2001), and also protects other regions of the brain and spinal cord from the damaging effects caused by oxidative stress and glutamate toxicity (Bilak *et al.*, 1999; Cao *et al.*, 1999; DeCoster *et al.*, 1999; Taniwaki *et al.*, 1995). It has been shown the implication of NFkB in all these processes, which induces the expression of neurotrophic factors and anti-apoptotic genes that participate in the control of cell survival, proliferation and death (Barnstable and Tombran-Tink, 2004; Yabe *et al.*, 2001). Neurotrophic activity has been mapped to the N-ter region of PEDF, encompassing residues Val58-Thr101 (Simonovic *et al.*, 2001). This region is involved in binding to a putative plasma membrane receptor in cerebellar granule neurons and retinoblastoma cells (Alberdi *et al.*, 1999). PEDF is also secreted by ependymal and endothelial cells from the subventricular zone of the brain, where it promotes the selfrenewal of adult neural stem cells (Andreu-Agullo *et al.*, 2009; Ramirez-Castillejo *et al.*, 2006). Furthermore, PEDF presents three phosphorylation sites located in Ser24, Ser114 and Ser227, and the regulation of their phosphorylation state modulates the switch of PEDF's function from neurotrophic (Ser24 and Ser114 phosphorylated) to angiangiogenic (Ser227 phosphorylated)

In order to better understand the mechanism of action of PEDF, it was necessary to identify the structural domains responsible for each of its described biological functions. Two major epitopes were identified at the N-ter region of PEDF: 34-mer peptide (residues 24-57) responsible of the antiangiogenic and pro-apoptotic actions of PEDF; and a second epitope, 44-mer peptide (residues 58-101), which induces neuronal differentiation of retinoblastoma cells (Filleur *et al.*, 2005) and plays a neurotrophic role in many neuronal cell types (Bilak *et al.*, 2002). There are another two highly conserved smaller epitopes, one upstream 34-mer,

415, located at C-ter of the protein (Tombran-Tink *et al.*, 2005).

(Becerra, 2006).

the TGA epitope, and an internal fragment of 44-mer, referred to as ERT. Filleur and collaborators showed *in vivo* that the epitopes TGA and the complete 34-mer inhibit tumor angiogenesis in prostate adenocarcinoma by inducing apoptosis of endothelial cells and blocking endothelial cell migration. Curiously, ERT despite of being located inside the neurotrophic 44-mer peptide, also presents antiangiogenic activity in prostate adenocarcinoma. Complete 44-mer is able to induce neurite outgrowth in Y-79 retinoblastoma cell line and causes apoptosis of endothelial cells, blocking migration and angiogenesis (Filleur *et al.*, 2005). In a more recent study, smaller regions of 34-mer epitope (named P14, P18 and P23, according to their respective length) were tested for angioinhibitory activity *in vitro* and *in vivo*. P14 and P23 display antiangiogenic activity *in vitro* (both blocking endothelial chemotaxis, and inducing apoptosis in the case of P23), but not *in vivo*; while P18 is a more potent antiangiogenic peptide than 34-mer in prostate cancer, being able to block bFGF and VEGF-dependent angiogenesis in the *in vivo* cornea neovascularisation assay (Mirochnik *et al.*, 2009). Another study by Ek and colleagues identified other small peptides with antitumoral activity in an orthotopic osteosarcoma model. They generated six 25-mer peptides along the functionally distinct regions of PEDF characterized so far. Residues 78-102 inhibit proliferation, whereas residues 90-114 stimulate adhesion of PEDF to type I collagen. Furthermore, residues 387 to 411 inhibit invasion of osteosarcoma cells *in vitro* and residues 40-64 promote osteogenic differentiation (Ek *et al.*, 2007).

The multifunctional character of PEDF and the evidence of the different roles displayed depending on the cell type, suggest that PEDF could be acting through distinct domains recognized by several specific receptors. The identification of these putative receptors and the characterization of the binding affinity of each functionally identified peptide toward them could make a breakthrough in the understanding of PEDF's mechanism of action and, therefore, the possibility of its therapeutic use in multiple pathological contexts.

#### **4.2 PEDF receptors**

Several studies have characterized the affinity of PEDF for the surface of human retinoblastoma cells, cerebellar granular neurons (Alberdi *et al.*, 1999), motor neurons (Bilak *et al.*, 2002), neural retina (Aymerich *et al.*, 2001) and endothelial cells (Yamagishi *et al.*, 2004). PEDF could be sequestered in the extracellular matrix based on ionic interactions with sulphated (heparin, heparin sulfate and condroitin sulfate) (Alberdi *et al.*, 1998), and nonsulfated (hyaluronan) (Becerra *et al.*, 2008) glycosaminglycans and type I collagen (Meyer *et al.*, 2002). In order to identify the potential receptor(s) of PEDF, Simonovic and collaborators carried out a three-dimensional study of PEDF, and identified an asymmetric charge distribution, with a high acidic region located at C-ter and basic amino acids at opposite region of PEDF protein (Simonovic *et al.*, 2001). This basic region is involved in the binding of PEDF to heparin through three clustered basic amino acid residues, Lys146, Lys147 and Arg149. Moreover, Asp256, Asp258 and Asp300 residues present in the acidic region of PEDF are crucial to type I collagen binding (Meyer *et al.*, 2002; Yasui *et al.*, 2003).

Using the 34-mer and 44-mer epitopes it was possible to propose the existence of two distinct putative receptors for PEDF. These epitopes are able to bind the surface of endothelial and prostate cells, but they do not compete for receptor binding (Filleur *et al.*, 2005). This result suggests the existence of two PEDF receptors with different functions: PEDF-RN, that interacts with 44-mer epitope and regulates the neurotrophic and neuroprotective activities of PEDF; and PEDF-RA, which is involved in blocking

Pigment Epithelium-Derived Factor –

previously described 60-kDa receptor.

**4.3 Therapeutic prospects for PEDF**

translating PEDF's effects, remains to be investigated.

unique candidate for a therapeutic agent in many diseases.

An Angiostatic Factor with a Broader Function in Melanoma 159

Further studies identified on the surface of the retina and human immortalized retinoblastoma cells (ARPE-19) the human Transport Secretion Protein 2-2 (TTS-2.2) /Independent phospholipase A2 (PLA2)ξ (also known in mice as adipose triglyceride lipase-ATGL, desnutrin, and patatin-like phospholipase domain containing protein-PNPLP2), an important lipase involved in triglyceride metabolism, as a specific PEDF receptor (Notari *et al.*, 2006). The phospholipase A2 domain of this receptor releases bioactive fatty acids that function as second messengers. Therefore, depending on the lipid released, this receptor could activate different signal transduction pathways. It is still not fully demonstrated if the

Recently, a new receptor for PEDF has been identified, the non-integrin 67-kDa laminin receptor (67LR) (Bernard *et al.*, 2009), which may be related to the 60-kDa receptor previously reported in endothelial cells (Yamagishi *et al.*, 2004). This hypothesis is supported by the observation of antiangiogenic effects (inhibition of endothelial cell migration and induction of endothelial cell apoptosis) when 34-mer epitope binds to 67LR and the

However, whether these characterized receptors are expressed on the surface of melanocytes and melanoma cells to mediate multiple PEDF's biological activities, and whether other unknown melanocytic lineage-specific receptors could play a role in

During the last two decades many groups have described PEDF as a multifunctional protein that plays effective neuroprotective and antiangiogenic activities. The wide diversity of PEDF functions, along with the fact that it is an endogenous molecule, makes PEDF a

Several studies have already shown the role of PEDF in various pathological conditions such as ocular and chronic inflammatory diseases, atherosclerosis and diabetes. Moreover, several groups have demonstrated, *in vivo* and *in vitro*, the antitumor and antimetastatic potential of PEDF. They described the capability of PEDF to induce tumor cell differentiation in neuroblastoma (Crawford *et al.*, 2001) and prostate cancer (Filleur *et al.*, 2005); a direct tumor suppression action in osteosarcoma (Takenaka *et al.*, 2005), melanoma (Abe *et al.*, 2004; Garcia *et al.*, 2004) and prostate cancer (Filleur *et al.*, 2005; Guan *et al.*, 2007); and its angiostatic action in a wide number of tumor types that include, among others, melanoma

The use of PEDF as a therapeutic agent in the clinic requires deeper knowledge of its biological effects and underlying molecular mediators. An important feature is the development of effective therapeutic agents based on the optimum combination of biological activities beneficial for each specific pathological context, together with the development of optimum delivery systems to allow proper targeting and stability during treatment. One of the possibilities is the use of recombinant full-length PEDF protein (rPEDF) produced in human embryonic kidney cells. Also, as PEDF is secreted at high levels by RPE cells, endogenous PEDF protein can be purified from the conditioned medium of RPEs. rPEDF has been successfully tested *in vitro* and *in vivo* in osteosarcoma (Takenaka *et al.*, 2005), prostate cancer (Doll *et al.*, 2003) and neuroblastoma (Crawford *et al.*, 2001). Due to its endogenous nature, short-term treatment would not lead to any immune response after systemic administration. However, the main disadvantage of this strategy is the susceptibility of rPEDF to cleavage by proteases, and therefore a limited bioavailability.

(Abe *et al.*, 2004; Garcia *et al.*, 2004; Orgaz *et al.*, 2009; Yang and Grossniklaus, 2010).

80-kDa (PLA2)ξ receptor is the previously identified PEDF-RN receptor (Fig. 2).

angiogenesis by binding to 34-mer epitope (Fig. 2). The differential expression of these two putative PEDF receptors in the diverse cell types analyzed supports the idea of distinct functions for each receptor type. PEDF-RN is an 80-kDa receptor, which is located on the surface of human retinoblastoma cells, neural retina, cerebellar granular and motor neurons, whereas PEDF-RA is a 60-kDa receptor specifically present on endothelial cells (Filleur *et al.*, 2009).

Fig. 2. Three-dimensional structure of PEDF, small peptides derived from PEDF and receptors. Crystal structure of PEDF molecule showing the location of 34-mer and 44-mer peptides. PEDF could be sequestered in the extracellular matrix by ionic interactions with type I collagen and glycosaminglycans. 34-mer, TGA and ERT peptides display antiangiogenic action through their bind to 60-kDa receptor (PEDF-RA, proposed as the nonintegrin 67-kDa laminin receptor) in endothelial cells. 34-mer also has the capability to induce apoptosis in tumor cells. 44-mer and ERT peptides present a neurotrophic action through their binding to the TTS-2.2/ (PLA2) ξ receptor (putatively identified as the PEDF-RN) in several cell types. Abbreviations: 67LR: Non-Integrin 67-kDa laminin receptor; TTS-2.2/(PLA2)ξ: Transport secretion protein 2.2/Independent phospholipase A2; Y79: human retinoblastoma cell line; CGN: cerebellar granular neurons

angiogenesis by binding to 34-mer epitope (Fig. 2). The differential expression of these two putative PEDF receptors in the diverse cell types analyzed supports the idea of distinct functions for each receptor type. PEDF-RN is an 80-kDa receptor, which is located on the surface of human retinoblastoma cells, neural retina, cerebellar granular and motor neurons, whereas PEDF-RA is a 60-kDa receptor specifically present on endothelial cells (Filleur *et al.*,

Fig. 2. Three-dimensional structure of PEDF, small peptides derived from PEDF and receptors. Crystal structure of PEDF molecule showing the location of 34-mer and 44-mer peptides. PEDF could be sequestered in the extracellular matrix by ionic interactions with

antiangiogenic action through their bind to 60-kDa receptor (PEDF-RA, proposed as the nonintegrin 67-kDa laminin receptor) in endothelial cells. 34-mer also has the capability to induce apoptosis in tumor cells. 44-mer and ERT peptides present a neurotrophic action through their binding to the TTS-2.2/ (PLA2) ξ receptor (putatively identified as the PEDF-RN) in several cell types. Abbreviations: 67LR: Non-Integrin 67-kDa laminin receptor; TTS-2.2/(PLA2)ξ: Transport secretion protein 2.2/Independent phospholipase A2; Y79: human

type I collagen and glycosaminglycans. 34-mer, TGA and ERT peptides display

retinoblastoma cell line; CGN: cerebellar granular neurons

2009).

Further studies identified on the surface of the retina and human immortalized retinoblastoma cells (ARPE-19) the human Transport Secretion Protein 2-2 (TTS-2.2) /Independent phospholipase A2 (PLA2)ξ (also known in mice as adipose triglyceride lipase-ATGL, desnutrin, and patatin-like phospholipase domain containing protein-PNPLP2), an important lipase involved in triglyceride metabolism, as a specific PEDF receptor (Notari *et al.*, 2006). The phospholipase A2 domain of this receptor releases bioactive fatty acids that function as second messengers. Therefore, depending on the lipid released, this receptor could activate different signal transduction pathways. It is still not fully demonstrated if the 80-kDa (PLA2)ξ receptor is the previously identified PEDF-RN receptor (Fig. 2).

Recently, a new receptor for PEDF has been identified, the non-integrin 67-kDa laminin receptor (67LR) (Bernard *et al.*, 2009), which may be related to the 60-kDa receptor previously reported in endothelial cells (Yamagishi *et al.*, 2004). This hypothesis is supported by the observation of antiangiogenic effects (inhibition of endothelial cell migration and induction of endothelial cell apoptosis) when 34-mer epitope binds to 67LR and the previously described 60-kDa receptor.

However, whether these characterized receptors are expressed on the surface of melanocytes and melanoma cells to mediate multiple PEDF's biological activities, and whether other unknown melanocytic lineage-specific receptors could play a role in translating PEDF's effects, remains to be investigated.

#### **4.3 Therapeutic prospects for PEDF**

During the last two decades many groups have described PEDF as a multifunctional protein that plays effective neuroprotective and antiangiogenic activities. The wide diversity of PEDF functions, along with the fact that it is an endogenous molecule, makes PEDF a unique candidate for a therapeutic agent in many diseases.

Several studies have already shown the role of PEDF in various pathological conditions such as ocular and chronic inflammatory diseases, atherosclerosis and diabetes. Moreover, several groups have demonstrated, *in vivo* and *in vitro*, the antitumor and antimetastatic potential of PEDF. They described the capability of PEDF to induce tumor cell differentiation in neuroblastoma (Crawford *et al.*, 2001) and prostate cancer (Filleur *et al.*, 2005); a direct tumor suppression action in osteosarcoma (Takenaka *et al.*, 2005), melanoma (Abe *et al.*, 2004; Garcia *et al.*, 2004) and prostate cancer (Filleur *et al.*, 2005; Guan *et al.*, 2007); and its angiostatic action in a wide number of tumor types that include, among others, melanoma (Abe *et al.*, 2004; Garcia *et al.*, 2004; Orgaz *et al.*, 2009; Yang and Grossniklaus, 2010).

The use of PEDF as a therapeutic agent in the clinic requires deeper knowledge of its biological effects and underlying molecular mediators. An important feature is the development of effective therapeutic agents based on the optimum combination of biological activities beneficial for each specific pathological context, together with the development of optimum delivery systems to allow proper targeting and stability during treatment. One of the possibilities is the use of recombinant full-length PEDF protein (rPEDF) produced in human embryonic kidney cells. Also, as PEDF is secreted at high levels by RPE cells, endogenous PEDF protein can be purified from the conditioned medium of RPEs. rPEDF has been successfully tested *in vitro* and *in vivo* in osteosarcoma (Takenaka *et al.*, 2005), prostate cancer (Doll *et al.*, 2003) and neuroblastoma (Crawford *et al.*, 2001). Due to its endogenous nature, short-term treatment would not lead to any immune response after systemic administration. However, the main disadvantage of this strategy is the susceptibility of rPEDF to cleavage by proteases, and therefore a limited bioavailability.

Pigment Epithelium-Derived Factor –

**5. Conclusions**

**6. Acknowledgment**

contract.

**7. References**

An Angiostatic Factor with a Broader Function in Melanoma 161

promising therapy for cancer and other diseases, there are still some problems and patient risks that have to be solved, such as (i) obtaining clinically effective viral titres, (ii) stable transgene expression in individuals requiring long-term treatment, and (iii) the risk of *de*

The biological effect of small peptides derived from PEDF retaining different functional activities has not yet been explored in melanoma, although it would be very relevant for the therapeutic development of PEDF in the context of aggressive melanoma. Also, characterization of PEDF's receptors expressed in melanoma cells is very important in order to understand the molecular mechanism of action underpinning the multiple biological actions of PEDF on melanoma cells, as well as to develop new therapeutic strategies based

Collectively, our findings strongly support PEDF as a potent biological modifier that effectively halts the metastatic spread of human melanoma by combining distinct functional epitopes respectively impinging on the vascular component of the tumor microenvironment and on the set of capabilities that a melanoma cell must acquire in order to successfully

Supported by grants Ministerio de Educacion y Ciencia SAF2007-62292 and SAF2010-19256 to BJ. AFB has been supported by a CSIC-JAE fellowship and JLO by a SAF2007-62292

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

factor in a xenograft model. *J Pediatr Surg* 38: 336-42; discussion 336-42. Abul-Hassan K., Walmsley R., Tombran-Tink J., & Boulton M. (2000). Regulation of

al-Alousi S., Carlson JA., Blessing K., Cook M., Karaoli T., & Barnhill RL. (1996). Expression of basic fibroblast growth factor in desmoplastic melanoma. *J Cutan Pathol* 23: 118-25. Alberdi E., Hyde CC., & Becerra SP. (1998). Pigment epithelium-derived factor (PEDF) binds to glycosaminoglycans: analysis of the binding site. *Biochemistry* 37: 10643-52. Alberdi E., Aymerich MS., & Becerra SP. (1999). Binding of pigment epithelium-derived

Albino AP., Davis BM., & Nanus DM. (1991). Induction of growth factor RNA expression in human malignant melanoma: markers of transformation. *Cancer Res* 51: 4815-20.

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: 1225-32. Abramson LP., Stellmach V., Doll JA., Cornwell M., Arensman RM., & Crawford SE. (2003).

Wilms' tumor growth is suppressed by antiangiogenic pigment epithelium-derived

tyrosinase expression and activity in cultured human retinal pigment epithelial

factor (PEDF) to retinoblastoma cells and cerebellar granule neurons. Evidence for

on the receptors and pathways that mediate PEDF's actions in aggressive melanoma.

*novo* cancer initiation via recombination within the patient's cell genome.

leave its primary site to colonize distant foreign environments.

cells. *Pigment Cell Res* 13: 436-41.

a PEDF receptor. *J Biol Chem* 274: 31605-12.

PEDF is a protein that is 418 amino acids in length. Filleur and collaborators elucidated that two small fragments of PEDF, 34-mer and 44-mer, were able to display the antiangiogenic and neurotrophic roles (respectively) as the complete protein (Filleur *et al.*, 2005). Shorter peptides improve the stability and delivery, and reduce the possibility of being recognized by the immune system. However, the use of rPEDF or small peptides derived from functionally active regions requires a systemic distribution; and therefore due to the antiangiogenic activity of this factor or derived peptides, its presence in plasma could have unexplored side effects on physiologic vascularization during the menstrual cycle and wound healing (Ek *et al.*, 2006a).

An alternative strategy that has been widely tested by a number of groups in mouse models of cancer is the use of viral vectors for the delivery of either full length PEDF or small peptides derived from the diverse functional regions identified. Gene transfer using viral or plasmid vectors is an attractive tool for human cancer gene therapy. Several studies have used this strategy in different types of cancer, such as pancreatic cancer (Hase *et al.*, 2005), neuroblastoma (Streck *et al.*, 2005), prostate cancer (Guan *et al.*, 2007) and melanoma (Abe *et al.*, 2004; Garcia *et al.*, 2004; Orgaz *et al.*, 2009; Yang and Grossniklaus, 2010), with a reduction of primary tumor size and number of metastases after PEDF delivery.

In melanoma, Abe's group and us were the first to describe the antitumor effect of PEDF *in vitro* and *in vivo* in malignant melanoma cell lines (Abe *et al.*, 2004; Garcia *et al.*, 2004). To this aim, Abe and collaborators overexpressed PEDF by transfection of G361 melanoma cell line and observed in nude mice a reduction of tumor angiogenesis and an almost complete inhibition of G361 growth in melanoma xenografts. These effects are the result of suppression of tumor angiogenesis and induction of Fas ligand-dependent apoptosis in tumor cells (Abe *et al.*, 2004). We used a retroviral strategy to transduce the human melanoma cell line UCD-Mel-N, which does not express significant levels of endogenous PEDF. We observed a considerable inhibition of primary tumor growth in subcutaneous xenotransplants in immunocompromised mice and a complete abrogation of lung metastases formation in the tail vein injection model. We demonstrated that the inhibition of primary melanoma tumor growth by PEDF is based on selective destruction of immature vessels, together with a significant direct induction of apoptosis in melanoma cells. Although it was first demonstrated that PEDF inhibits endothelial cell migration and induces apoptosis in remodelling endothelium, we showed that PEDF also has direct effects on melanoma cells, inhibiting melanoma cell migration and inducing apoptosis under stress conditions like absence of growth factors or detachment from the extracellular matrix (Garcia *et al.*, 2004). In a recent work we also used a lentiviral transduction strategy to silence PEDF in poorly aggressive melanoma cell lines with high expression of endogenous PEDF. PEDF knockdown in these melanoma cell lines enables the acquisition of an invasive phenotype, showing the critical importance of PEDF for the malignant progression of human melanoma (Orgaz *et al.*, 2009).

Retroviral and lentiviral vectors are attractive tools for human cancer gene therapy and, based on their ability to integrate into the genome, they have the potential to achieve longterm stable expression and maintain therapeutic levels of secreted peptides (Hase *et al.*, 2005). Both types of virus are able to transduce proliferating cells, although only lentiviruses present the advantage of transducing non-dividing cells. This feature is of great advantage for gene transfer as a complementary treatment in cancer, due to the fact that chemotherapy is only effective in actively proliferative cells, allowing non-dividing cells to be resistant to treatment and enabling the development of metastasis. Although viral systems seem to be a promising therapy for cancer and other diseases, there are still some problems and patient risks that have to be solved, such as (i) obtaining clinically effective viral titres, (ii) stable transgene expression in individuals requiring long-term treatment, and (iii) the risk of *de novo* cancer initiation via recombination within the patient's cell genome.

The biological effect of small peptides derived from PEDF retaining different functional activities has not yet been explored in melanoma, although it would be very relevant for the therapeutic development of PEDF in the context of aggressive melanoma. Also, characterization of PEDF's receptors expressed in melanoma cells is very important in order to understand the molecular mechanism of action underpinning the multiple biological actions of PEDF on melanoma cells, as well as to develop new therapeutic strategies based on the receptors and pathways that mediate PEDF's actions in aggressive melanoma.

## **5. Conclusions**

160 Breakthroughs in Melanoma Research

PEDF is a protein that is 418 amino acids in length. Filleur and collaborators elucidated that two small fragments of PEDF, 34-mer and 44-mer, were able to display the antiangiogenic and neurotrophic roles (respectively) as the complete protein (Filleur *et al.*, 2005). Shorter peptides improve the stability and delivery, and reduce the possibility of being recognized by the immune system. However, the use of rPEDF or small peptides derived from functionally active regions requires a systemic distribution; and therefore due to the antiangiogenic activity of this factor or derived peptides, its presence in plasma could have unexplored side effects on physiologic vascularization during the menstrual cycle and

An alternative strategy that has been widely tested by a number of groups in mouse models of cancer is the use of viral vectors for the delivery of either full length PEDF or small peptides derived from the diverse functional regions identified. Gene transfer using viral or plasmid vectors is an attractive tool for human cancer gene therapy. Several studies have used this strategy in different types of cancer, such as pancreatic cancer (Hase *et al.*, 2005), neuroblastoma (Streck *et al.*, 2005), prostate cancer (Guan *et al.*, 2007) and melanoma (Abe *et al.*, 2004; Garcia *et al.*, 2004; Orgaz *et al.*, 2009; Yang and Grossniklaus, 2010), with a reduction

In melanoma, Abe's group and us were the first to describe the antitumor effect of PEDF *in vitro* and *in vivo* in malignant melanoma cell lines (Abe *et al.*, 2004; Garcia *et al.*, 2004). To this aim, Abe and collaborators overexpressed PEDF by transfection of G361 melanoma cell line and observed in nude mice a reduction of tumor angiogenesis and an almost complete inhibition of G361 growth in melanoma xenografts. These effects are the result of suppression of tumor angiogenesis and induction of Fas ligand-dependent apoptosis in tumor cells (Abe *et al.*, 2004). We used a retroviral strategy to transduce the human melanoma cell line UCD-Mel-N, which does not express significant levels of endogenous PEDF. We observed a considerable inhibition of primary tumor growth in subcutaneous xenotransplants in immunocompromised mice and a complete abrogation of lung metastases formation in the tail vein injection model. We demonstrated that the inhibition of primary melanoma tumor growth by PEDF is based on selective destruction of immature vessels, together with a significant direct induction of apoptosis in melanoma cells. Although it was first demonstrated that PEDF inhibits endothelial cell migration and induces apoptosis in remodelling endothelium, we showed that PEDF also has direct effects on melanoma cells, inhibiting melanoma cell migration and inducing apoptosis under stress conditions like absence of growth factors or detachment from the extracellular matrix (Garcia *et al.*, 2004). In a recent work we also used a lentiviral transduction strategy to silence PEDF in poorly aggressive melanoma cell lines with high expression of endogenous PEDF. PEDF knockdown in these melanoma cell lines enables the acquisition of an invasive phenotype, showing the critical importance of PEDF for the malignant progression of

Retroviral and lentiviral vectors are attractive tools for human cancer gene therapy and, based on their ability to integrate into the genome, they have the potential to achieve longterm stable expression and maintain therapeutic levels of secreted peptides (Hase *et al.*, 2005). Both types of virus are able to transduce proliferating cells, although only lentiviruses present the advantage of transducing non-dividing cells. This feature is of great advantage for gene transfer as a complementary treatment in cancer, due to the fact that chemotherapy is only effective in actively proliferative cells, allowing non-dividing cells to be resistant to treatment and enabling the development of metastasis. Although viral systems seem to be a

of primary tumor size and number of metastases after PEDF delivery.

wound healing (Ek *et al.*, 2006a).

human melanoma (Orgaz *et al.*, 2009).

Collectively, our findings strongly support PEDF as a potent biological modifier that effectively halts the metastatic spread of human melanoma by combining distinct functional epitopes respectively impinging on the vascular component of the tumor microenvironment and on the set of capabilities that a melanoma cell must acquire in order to successfully leave its primary site to colonize distant foreign environments.

## **6. Acknowledgment**

Supported by grants Ministerio de Educacion y Ciencia SAF2007-62292 and SAF2010-19256 to BJ. AFB has been supported by a CSIC-JAE fellowship and JLO by a SAF2007-62292 contract.

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**9**

*Japan*

**The Influence of the Skin Microenvironment**

Shuji Toda1, Shigehisa Aoki1, Kazuyoshi Uchihashi1, Aki Matsunobu1, Mai Yakushiji1, Akifumi Ootani2, Eisuke Koike3 and Nobuhisa Yonemitsu4

*Faculty of Medicine & Graduate School of Medicine, Saga University, Saga 849-8501,*

*Faculty of Medicine & Graduate School of Medicine, Saga University, Saga 849-8501,*

The skin that is exposed to atmospheric air is situated at an air–liquid interface (ALI) through its contact with gas phase. Thus, ALI is an essential microenvironment of the skin. In fact, ALI promotes the growth and differentiation of normal keratinocytes (Sugihara et al., 1991). In cutaneous squamous cell carcinoma cells, ALI also accelerates their invasive growth more greatly than submerged condition without ALI (Inoue et al., 2001). ALI is thus

The critical skin neoplasm melanoma is still at present responsible for about 80% of all skin cancer-related deaths (Miller et al., 2006). This tumor is considered to originate from epidermal melanocytes or their precursor cells (Bennett, 1993). Thus, the epidermal microenvironments seem crucial for the initiation and progression of melanoma cells. Furthermore, the prognosis of melanoma with ulcer formation is worse than that of melanoma without ulcer formation (Grande et al., 2006). Given that skin ulcer causes cutaneous cell types under the epidermis to situate at ALI by the ulcer-induced exposure of them to atmospheric air, ALI may be related to the growth and invasion of melanoma cells. However, it has been unclear whether the skin microenvironment ALI directly affects the biological behavior of

On the basis of the background above, we have recently demonstrated that ALI promotes the growth of melanoma cells, but not their apoptosis and invasion, through mitogen-activated protein kinase (MAPK) activation (Chong et al., 2010). In this article, we introduce ALI culture system and describe the effects of ALI on the biological behavior of melanoma cells.

Air-liquid interface (ALI) is a common microenvironment of the skin. However, the influences of ALI on the biological behavior of melanoma cells have been poorly

considered to be a critical factor for both normal and neoplastic subjects of the skin.

**1. Introduction**

melanoma cells.

**2. Effects of ALI on melanoma cell behavior**

**Air-Liquid Interface on Melanoma**

*3Department of Surgery, Koike Hospital, Saga 840-0861,*

*4Department of Pathology, Sasebo Chuo Hospital, Nagasaki 857-1195*

*1Department of Pathology & Microbiology,*

*2Department of Internal Medicine*

## **The Influence of the Skin Microenvironment Air-Liquid Interface on Melanoma**

Shuji Toda1, Shigehisa Aoki1, Kazuyoshi Uchihashi1, Aki Matsunobu1, Mai Yakushiji1, Akifumi Ootani2, Eisuke Koike3 and Nobuhisa Yonemitsu4 *1Department of Pathology & Microbiology,*

*Faculty of Medicine & Graduate School of Medicine, Saga University, Saga 849-8501, 2Department of Internal Medicine*

*Faculty of Medicine & Graduate School of Medicine, Saga University, Saga 849-8501, 3Department of Surgery, Koike Hospital, Saga 840-0861, 4Department of Pathology, Sasebo Chuo Hospital, Nagasaki 857-1195 Japan*

## **1. Introduction**

170 Breakthroughs in Melanoma Research

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