Wnt Signaling in Stem Cells and Lung Cancer

Department of Surgery, University of California San Francisco Cancer Center, 1600 Divisadero Street, Box 1724, 94143-1724 San Francisco, USA email: [email protected]

1 Overview of Wnt Signaling 28

1.1 Wnt Ligands 28

1.2 Wnt Receptors and Antagonists 29

1.3 Canonical Wnt Signaling: Cytosolic and Nuclear Components ... 30

2 Stem Cells, Cancer Stem Cells, and Lung Cancer 31

3 Wnt Signaling in Stem Cell Maintenance and Regeneration 35

4 Wnt Signaling in Lung Cancer 37

4.1 Wnt Ligands 37

4.2 Wnt Antagonists 39

4.3 Dishevelled (Dvl) 40

4.4 APC 41

4.5 P-Catenin 42

4.6 Non-canonical Pathway 42

5 Potential Therapeutic Approaches Targeting the Wnt Pathway ... 44

6 Summary 46

References 47

Abstract. The Wnt signal transduction pathway plays important roles during embryo development, regulating cell proliferation and survival of immature cells. However, its improper function can lead to harmful consequences for humans, such as aberrant cell proliferation and, therefore, cancer. Increasing evidence suggests that stem cells may be the source of mutant cells that cause cancers to develop and proliferate. Wnt signaling has been shown to promote self-renewal in both gut epithelial and hematopoietic stem cells (HSCs) and to trigger critical pathways in carcinogenesis. Although the function of stem cells in solid tumor development is unclear, the Wnt pathway's role in determining the fate and self-renewal potential of cancer stem cells suggests a critical role in carcinogenesis. The development of new inhibitors, such as antibodies or small molecules, to inhibit this pathway may be of great therapeutic utility against cancer.

1 Overview of Wnt Signaling

The Wnt signal transduction pathway was named after the wingless gene, the Drosophila homologous gene of the first mammalian Wnt gene characterized, int-1 (Rijsewijk et al. 1987). Secreted Wnt ligands have been shown to activate signal transduction pathways and trigger changes in gene expression, cell behavior, adhesion, and polarity. In mammalian species, Wnt proteins comprise a family of 19 highly conserved signaling molecules. Wnt signaling has been described in at least three pathways (Widelitz 2005), with the best-understood canonical pathway, in which Wnt ligands bind to two distinct families of cell surface receptors, the Frizzled (Fz) receptor family and the LDL receptor-related protein (LRP) family, and activate target genes through stabilization of P-catenin in the nucleus (Akiyama 2000). Wnt proteins can also signal by activating calmodulin kinase II and protein kinase C (known as the Wnt/Ca2+ pathway), which involves an increase in intracellular Ca2+, or Jun N-terminal kinase (JNK) (known as the planar cell polarity pathway), which controls cytoskeletal rearrangements and cell polarity (Veeman MT et al. 2003).

1.1 Wnt Ligands

Wnt proteins are secreted glycoproteins of around 40 kDa, with a large number of conserved cysteine residues. They are produced by different cell types, and in humans 19 Wnt proteins currently have been identified (Miller 2002). It was found that cysteine palmitoylation is essential for the function of Wnt proteins (Willert et al. 2003). Hofmann (2000) reported that Porcupine (Porc), required in Wnt-secreting cells, shows homology to acyltransferases in the endoplasmic reticulum (ER). Taken together, it appears that Porc may be the enzyme responsible for cys-

teine palmitoylation of the Wnt proteins (Zhai et al. 2004). In addition, studies in Drosophila revealed that the seven-transmembrane proteins Wntless (Wls) and Evenness interrupted (Evi) are essential for Wnt secretion (Banziger et al. 2006; Bartscherer et al. 2006). In the absence of Wls/Evi, primarily residing in the Golgi apparatus, Wnts are retained inside the Wnt-producing cells. Furthermore, extracellular heparan sulfate proteoglycans (HSPGs) may also play a role in the transport or stabilization of Wnt proteins. (Lin 2004).

1.2 Wnt Receptors and Antagonists

Reception and transduction of Wnt signals involve interaction of Wnt proteins with members of two distinct families of cell surface receptors, the Frizzled (Fz) gene family and the LDL receptor-related protein (LRP) family. Fz proteins bind Wnts through an extracellular N-terminal cysteine-rich domain (CRD), and most Wnt proteins can bind to multiple Fz receptors and vice versa. (Bhanot et al. 1996). Ten human Fz proteins have been identified so far, and their general structure is similar to that of seven-transmembrane G protein-coupled receptors, suggesting that Fz proteins may use heterotrimeric G proteins to transduce Wnt signals (Liu et al. 2001). A single-pass transmembrane molecule of the LRP family, identified as LRP5 or 6, is also required for the signaling (Tamai et al. 2000). It appears that surface expression of both receptor families is required to initiate the Wnt signal, although formation of trimeric complexes involving Wnt molecules with Fz and LRP5/6 has yet to be validated. In addition, two tyrosine kinase receptors, Derailed and Ror2, have been shown to bind Wnts. Derailed binds Wnts through its extracellular WIF (Wnt inhibitory factor) domain, and Ror2 binds Wnts through a Wnt binding CRD motif. Signaling events downstream of these alternative Wnt receptors remain largely unclear (Lu et al. 2004; Mikels and Nusse 2006).

Secreted inhibitory proteins can sequester Wnt ligands from their receptors. Among these are the secreted Frizzled-related proteins (SFRPs) and the Wnt inhibitory factor-1 (WIF-1) (Hsieh et al. 1999; Jones and Jomary 2002). The human SFRP family consists of five members, each containing a CRD domain. The biology of SFRPs is, however, complex, and in some cases, they may act as Wnt agonists (Uren et al.

2000). WIF-1 does not have any sequence homology with SFRPs but contains a unique evolutionarily conserved WIF domain and five epidermal growth factor (EGF)-like repeats. A third class of extracellular Wnt inhibitor is represented by the Dickkopf (Dkk) family, which antagonizes Wnt signaling pathway through inactivation of the surface receptors LRP5/6 (Fedi et al. 1999).

1.3 Canonical Wnt Signaling:

Cytosolic and Nuclear Components

When Wnt signaling is in the "off state" (Fig. 1), cytosolic P-catenin is phosphorylated by the serine/threonine kinases casein kinase I (CKI) and GSK3P at four N-terminal residues (Amit et al. 2002). The scaffolding proteins Axin and APC mediate the interaction between the kinases and P-catenin (Hart et al. 1998). These proteins form a P-catenin degradation complex that allows phosphorylated P-catenin to be recognized by P-TrCP and subsequently targeted for ubiquitination and proteasome degradation (Aberle et al. 1997). In the nucleus, the TCF/DNA-binding proteins form a complex with Groucho and act as repressors of Wnt target genes when the Wnt signal is absent (Cavallo et al. 1998). Groucho can interact with histone deacetylases, which makes the DNA refractive to transcriptional activation (Chen et al. 1999). Upon interaction of the Wnt ligands with their receptors, the Fz/LRP coreceptor complex activates the canonical signaling pathway (the "on state" of Wnt signaling) (Fig. 2). Fz can physically interact with Dishevelled (Dvl), a cytosolic protein that functions upstream of P-catenin and the kinase GSK3P. Then the scaffold protein axin translocates to the membrane, where it interacts with either the intracellular tail of LRP or with Fz-bound Dvl (Cliffe et al. 2003). Removing axin from the destruction complex promotes P-catenin stabilization. The "on" and "off" states of Wnt signaling control phosphorylation status of Dvl protein (Wallingford and Habas 2005). It remains unclear, however, whether the binding of Wnt to Fz regulates a direct Fz-Dvl interaction and how phospho-rylated Dvl functions during Wnt signal transduction. With the help of BCL9 (Kramps et al. 2002; Krieghoff 2005; Sampietro 2006), stabilized P-catenin enters the nucleus and competes with Groucho for binding to TCF/LEF, recruits Pygopus, and converts the TCF repres-

sor complex into a transcriptional activator complex. A large number of Wnt signaling target genes, including c-Myc, cyclin D1, MMP-7, and WISP, have been identified (a list of the Wnt target genes can be found at http://www.stanford.edu/~rnusse/wntwindow.html). The Wnt signaling pathway plays an important role in cell differentiation and proliferation, and when aberrantly activated, it contributes to most of the features that characterize malignant tumors, including evasion of apoptosis, tissue invasion and metastasis, self-sufficiency of growth signals, insen-sitivity to growth inhibitors, and sustained angiogenesis (Ilyas 2005).

2 Stem Cells, Cancer Stem Cells, and Lung Cancer

Stem cells are distinguished by their ability to self-renew and differentiate into other cell types and are found in embryonic and adult tissues (Preston et al. 2003). Embryonic stem cells (ES) have the ability, known as pluripotency, to develop into any type of cell required for mammalian development. While it has been proposed that all adult tissues derive from tissue-specific stem cells, that link has been demonstrated in only a limited number of cell types. Examples include hematopoietic stem cells (HSCs) from which all red and white blood cells develop (Morrison and Weissman 1994; Baum et al. 1992; Spangrude et al. 1988) and mesenchymal stem cells (MSCs), present in the bone marrow for later differentiation into bone, cartilage, adipose tissue, and muscle (Zimmermann et al. 2003; Simonsen et al. 2002). In addition, it has been demonstrated that "local" stem cell populations in brain and muscle can repopulate the bone marrow of a radiation-ablated or immune-deficient mouse (Otto 2002).

Although the role of stem cells in cancinogenesis is not well characterized, emerging evidence is providing new insight into this process (Preston et al. 2003; Marx 2003). Although stem cells are rare within any given cell population, several factors make them a likely culprit in the development of cancers. First is their capacity for self-renewal and replication. Second is their long-lived potential as undifferentiated cells, creating a larger window of opportunity for molecular alterations that accumulate over time. Thus, it is not surprising that the Hedgehog (Hh) and Wnt pathways, regulatory mechanisms for stem cell renewal,

Wnt Lung Cancer

Fig. 1. The inactive canonical Wnt signaling pathway. In the absence of factors that activate Wnt signaling, the complex APC-Axin and GSK-3 binds to P-catenin with subsequent P-catenin phosphorylation, ubiquination, and degradation by proteasomes. TCF/LEF proteins repress target genes through a direct association with co-repressors

Fig. 1. The inactive canonical Wnt signaling pathway. In the absence of factors that activate Wnt signaling, the complex APC-Axin and GSK-3 binds to P-catenin with subsequent P-catenin phosphorylation, ubiquination, and degradation by proteasomes. TCF/LEF proteins repress target genes through a direct association with co-repressors tissue repair, and tissue regeneration, are implicated in the development of cancers when aberrantly activated.

Cancer has long been thought to originate and develop from cancer stem cells. Although constituting only a fraction of the cells within the tumor, cancer stem cells are nonetheless critical for its propagation (Park et al. 1971). The concept of cancer stem cells originated from findings in the hematopoietic system (Till 1961) and acute myelogenous leukemia (AML) (Till 1961; Hope et al. 2004; Bonnet and Dick 1997).

Lung Cell Development Wnt SignalingBonnet Dick Aml

vate Wnt signaling, Dvl binds to Axin and inhibits the degradation complex APC-Axin-GSK-3. Therefore, P-catenin cannot be phosphorylated and its level increases in the cytoplasm, allowing its translocation to the nucleus, where it converts TCF/LEF factors into transcriptional activators of Wnt target genes, such as c-myc, and cyclin D1, etc.

vate Wnt signaling, Dvl binds to Axin and inhibits the degradation complex APC-Axin-GSK-3. Therefore, P-catenin cannot be phosphorylated and its level increases in the cytoplasm, allowing its translocation to the nucleus, where it converts TCF/LEF factors into transcriptional activators of Wnt target genes, such as c-myc, and cyclin D1, etc.

Cancer stem cells in tumorigenesis have been demonstrated in several cancer types. An important criterion for cancer stem cells is that they enable serial propagation of tumors that retain the often diverse marker profile of the primary tumor (Singh et al. 2004). Cancer stem cells and normal stem cells share numerous properties, including the expression of common cell surface markers, the capacity to self-renew, and unlimited replication potential and the quality of being long-lived, allowing the accrual of multiple mutations over time, increasing the rate of cell proliferation and producing clinically significant cancer.

Lung cancer is the leading cause of cancer mortality in the USA. Approximately 173,770 new cases of lung cancer were diagnosed in 2004,13% of all new cancer cases, and an estimated 160,440 Americans died from the disease, accounting for 28% of all cancer deaths.

There are two major pathological groups in lung cancer: non-small cell lung cancer (NSCLC), comprising 80% of the total, and small cell lung cancer (SCLC) comprising the remaining 20%. Increasing evidence shows that lung cancer occurs through a multistep oncogenic process. Bronchioalveolar carcinoma (BAC) and atypical adenomatous hyperplasia (AAH), a premalignant lesion believed to be a precursor to BAC, are often present near areas of invasive adenocarcinoma (Yoshida et al. 2005; Saad et al. 2004; Kitamura et al. 1999; Okubo et al. 1999). Both tumor suppressor genes and oncogenes play important roles in the development of lung cancer (Bishop 1991; Weinberg 1991). For example, single allele mutation in a proto-oncogene is often sufficient for aberrant transformation with crucial effects on signal transduction and transcription processes. Amplification, translocation, rearrangement, and point mutations in dominant oncogenes lead to aberrant transformation. Homozygous loss of function in tumor suppressor genes by genetic, epigenetic, or both events leads to abnormal regulation of transcription. Conventional treatments, including chemotherapy and radiation and, in earlier-stage cases, surgery, have succeeded only in slowing the inexorable march of the disease, and overall five-year survival has remained more or less constant at 15% for over a decade. If cancer stem cells are the driving force for cancer formation, then traditional therapeutic interventions that target the main tumor mass, but not the cancer stem cells, are not likely to succeed.

Recently agents targeting the underlying molecular signaling pathways in lung cancer have entered clinical trials with more encouraging results (Smith and Khuri 2004). Therefore better understanding the molecular mechanisms of lung cancer development should improve the diagnosis and treatment of this deadly disease. Identification and characterization of cancer stem cells should accelerate the development of therapeutic drugs that target them. In fact, a recent study in a mouse model by Kim et al. (2005) discovered that some cells at the bronchioalveolar duct junction exhibit features of stem cells. They discovered that cells at this junction carrying Clara cell and alveolar cell mark ers ("bronchioalveolar stem cells") appear refractory to naphthalene treatment and start to divide after naphthalene-induced damage, resulting in repair of damaged lung epithelial tissue. Furthermore, their data suggest that these double-positive cells with their stem cell-like features may play an important role in the transformation of normal lung epithelia into adenocarcinomas (Kim et al. 2005). However, there are questions that still need to be answered, such as whether these bron-chioalveolar stem cells give rise to lung cancer stem cells, and if so, what events are required to achieve this transformation.

The activation of Hh and Wnt pathways in promoting stem cell renewal, transiently in normal tissues and aberrantly in carcinogenesis, has attracted great scientific interest. Hh pathway activity was initially linked to cancer through identification of mutations in a negative regulator of the Hh receptor, Patched (Ptch) (Wechsler-Reya and Scott 2001; Taipale and Beachy 2001). Recent studies employing Hh-blocking antibodies and a specific Smoothened (Smo) inhibitor, cyclopamine, have demonstrated that Hh pathway activity, which requires ligand activation, is important in the growth of many lethal cancers including SCLC (Thayer et al. 2003; Berman et al. 2003; Watkins et al. 2003). Here we discuss the role the Wnt pathway plays in the maintenance of normal stem cells and the development of lung cancer.

3 Wnt Signaling in Stem Cell Maintenance and Regeneration

Wnt signaling has many functions in animal development including a crucial role in the morphogenesis of the gastrointestinal tract (Ko-rinek et al. 1998), mammary glands (Brennan and Brown 2004), cardiovascular system (Pandur et al. 2002), and bone marrow (Reya et al. 2003). Recent study data also highlight its developmental role in embryogenesis and in the adult lung. For example, Wnt signaling regulates important aspects of epithelial and mesenchymal development during gestation (Morrisey 2003).

More specifically, studies of knockout mice demonstrated the importance of Wnt-2, Wnt-5a, and Wnt-7b in lung maturation (Li et al. 2002; Shu et al. 2002; Weidenfeld et al. 2002; Yamaguchi et al. 1999). Using oligonucleotide arrays, Bonner et al. demonstrated the contribution of the Wnt pathway to various stages of murine lung development (Bonner et al. 2003). Finally, the Wnt/ß-catenin pathway has been shown to be activated in lung inflammatory processes, including idiopathic pulmonary fibrosis (Chilosi et al. 2003).

In addition to its traditional role in embryogenesis, the Wnt pathway has recently been implicated in the maintenance of stem and progenitor cells in adult tissues of the skin, blood, gut, prostate, muscle, and nervous system (Bhardwaj et al. 2001; Karhadkar et al. 2004; Ko-rinek et al. 1998; Lai et al. 2003; Machold et al. 2003; Owens and Watt 2003; Pinto et al. 2003; Perez-Losada and Balmain 2003; Polesskaya et al. 2003; Ramalho-Santos et al. 2000; Reya et al. 2003; Zhang and Kalderon 2001). For example, studies of transgenic mice suggest that activation of the Wnt pathway in epidermal stem cells may lead to epithelial cancers (Gat et al. 1998). It was found consistently that gene expression patterns in colon cancer cells and colon stem cells resembled one another (van de Wetering et al. 2002).

Other evidence points as well to Wnt pathway involvement in stem cell development. Treatment of isolated HSCs with Wnt proteins in vitro increases their proliferative capacity and improves their ability to form colonies, both in vitro and in vivo (Reya et al. 2003). While inhibiting Wnt pathway activation in the intestine fails to prevent the development, initially, of normal epithelial architecture, its inactivation eventually results in a progressive degradation of epithelial structure. This effect is associated with a loss of proliferative activity in the crypts, where stem cells reside (Korinek et al. 1998; Pinto et al. 2003).

Reports have also suggested a role for the Wnt pathway in regenerative responses. For example, its activation has been closely associated with regeneration of muscle (Polesskaya et al. 2003), bile duct (Shackel et al. 2001), kidney (Surendran and Simon 2003), and liver (Monga et al. 2001) after injuries. In mice, it has been demonstrated that Wnt pathway activation enriches the population of mammary progenitors through increased levels of Wnt ligand or stabilized ß-catenin (Liu et al. 2004). Moreover, it has been shown that muscle regeneration is inhibited by Wnt antagonists, such as secreted Frizzled-related proteins (SFRPs) (Polesskaya et al. 2003).

Wnt pathway activity has been associated with both chronic tissue injury and carcinogenesis (Dvorak 1986; Coussens and Werb 2002). Indeed, both processes may be related. First, genetic and/or epigenetic events lead to aberrant Wnt pathway activity, preventing activated stem or progenitor cells, once tissue regeneration is complete, from returning to a normal quiescent state, in effect a condition of unregulated tissue repair. In this way, conversion of a normal stem cell into a cancerous one may lock the cell in an active state of renewal. For example, when the lung or skin is continuously exposed to environmental insults, they may shift into a constant renewal state and ultimately become the sites of new cancers.

4 Wnt Signaling in Lung Cancer

In addition to its role in stem cell self-renewal, tissue regeneration, and lung development, Wnt signaling is also intimately involved in tumori-genesis and cancer progression (Polakis 2000; Bienz and Clevers 2000). For example, the organs where Wnt signaling influences stem cell self-renewal are the same organs where those Wnt-pathway-dependent cancers originate. Numerous reports have demonstrated aberrant Wnt activation in many human cancers, including colorectal (Morin et al. 1997; Korinek et al. 1997), head and neck (Rhee et al. 2002), melanoma (Weer-aratna et al. 2002), and leukemia (Lu 2004). This activation can be caused by mutations and/or deregulation of many different Wnt signaling components. Mutations in Wnt pathway components are rarely found in lung cancer. Instead, nongenetic events appear to be the major cause of aberrant activation of Wnt signaling in lung cancer.

4.1 Wnt Ligands

Wnt-1 was first identified from retroviral integration that caused mammary tumors in mice (Nusse and Varmus 1982) and was found to be upregulated in a number of human cancers (Katoh 2003; Wong et al. 2002). Moreover, cancer cells expressing Wnt-1 are resistant to therapies that mediate apoptosis (Chen et al. 2001). Overexpression of Wnt-1 has been demonstrated in NSCLC cell lines and primary cancer tissues (He et al. 2004). Blockade of Wnt-1 signaling induces apoptosis in vitro and suppresses tumor growth in vivo (He et al. 2004). Similar results were observed in head and neck squamous cell carcinoma (Rhee et al. 2002), suggesting that Wnt-1 signaling is a key mediator of apoptosis in epithelial cancers.

The human Wnt-2 gene, located on chromosome 7q31.3, is highly expressed in fetal lung and weakly expressed in placenta (Katoh 2001a). The link between Wnt-2 and tumorigenesis was first proposed after data indicated that Wnt-2 was amplified in human cancers (Yoshida et al. 1988). Similarly, Wnt-2 has been implicated in mouse mammary tumorigenesis through gene amplification (Roelink et al. 1992). Wnt-2 was later shown to be upregulated in gastric cancers (Nessling et al. 1998; Katoh 2001b), colorectal cancers (Vider et al. 1996; Holcombe et al. 2002; Katoh 2001c), and melanoma (Pham et al. 2003).

Recently, we demonstrated overexpression of Wnt-2 in NSCLC (You et al. 2004). Following our same study design in Wnt-1 (He et al. 2004), we demonstrated that inhibition of Wnt-2-mediated signaling by small interfering RNA (siRNA) or a monoclonal antibody induced apoptosis in NSCLC cells (You et al. 2004). Moreover, we most recently found that the anti-Wnt-2 antibody has sufficient potency to inhibit growth in primary human NSCLC tissue cultures (unpublished data).

Recently there has been a suggested role for Wnt-7a in lung cancer. It has been reported that expression of Wnt-7a is downregulated in most lung cancer cell lines and tumor samples (Calvo et al. 2000; Winn et al. 2005). It has been further hypothesized that Wnt-7a upregulates E-cadherin expression in lung cancer cells (Ohira et al. 2003). Interestingly, Wnt-7a functions through a P-catenin-independent pathway during limb development (Kengaku et al. 1998) and through the canonical pathway in lung cancer, as has been suggested, even though TCF/LEF transcriptional activity is not directly targeted (Winn et al. 2005). Further studies from the same group (Winn et al. 2006) indicate that combined expression of Wnt-7a and Frizzled-9 (Fz-9) in NSCLC cell lines inhibits transformed growth and that this antitumorigenic effect of Wnt-7a and Fz-9 is mediated through ERK5-dependent activation of PPARy.

Wnt-5a, which is known to activate the Wnt/Ca2+ noncanonical pathway during development (Moon et al. 1997), has a controversial role in carcinogenesis. Wnt-5a is upregulated in some cancers (Saitoh et al. 2002) and can increase invasion of metastatic melanoma in a P-catenin-

independent manner (Weeraratna et al. 2002). Yet, Wnt-5a also behaves as a tumor suppressor gene in hematopoietic malignancies (Liang et al. 2003). The role of Wnt-5a in human sarcoma and in the development of lung metastases (Nakano et al. 2003) raises interest in its role in primary lung cancer. Indeed, a recent study in 123 patients with NSCLC (Huang et al. 2005) found that Wnt-5a expression in squamous cell carcinoma was significantly higher than that in adenocarcinoma. Furthermore, this study revealed that Wnt-5a overexpression could produce more aggressive NSCLC, especially in squamous cell carcinomas, during tumor progression.

4.2 Wnt Antagonists

There are two groups of Wnt antagonists. The first includes the SFRP family, WIF-1 and Cerberus. They inhibit Wnt signaling by directly binding to Wnt molecules. The second group, which includes the Dickkopf (DKK) family, inhibits Wnt signaling by binding to the LRP5/ LRP6 component of the Wnt receptor complex (Kawano and Kypta 2003). These inhibitors have been extensively studied in developmental experiments, with demonstration of a role in oncogenesis, specifically in cervical (Ko et al. 2002), breast (Ugolini et al. 2001), gastric (To et al. 2001), and colorectal cancers (Suzuki et al. 2002; Suzuki et al. 2004; Caldwell et al. 2004).

The role of Wnt antagonists in lung carcinogenesis has recently been described. WIF-1 was first identified from the human retina and is a highly conserved gene. Overexpression of WIF-1 in Xenopus embryos blocks the Wnt-8 pathway and induces abnormal somitogenesis (Hsieh et al. 1999). Recently, Wissman et al. reported the downregula-tion of WIF-1 in several cancer types including lung cancer by using a chip hybridization assay and immunohistochemistry (Wissmann et al. 2003). We recently demonstrated, by using methylation-specific PCR (MSP) and sequence analysis after bisulfite treatment, that frequent hy-permethylation of CpG islands in the functional WIF-1 promoter region correlated with its transcriptional silencing in human lung cancer cell lines (Mazieres et al. 2004). We also studied WIF-1 expression in freshly resected lung cancers and showed a downregulation in 83% of cases. This silencing also correlates with WIF-1 promoter methylation

(Mazieres et al. 2004). We thus propose that methylation silencing of WIF-1 is a common and likely important mechanism for aberrant activation of Wnt signaling in lung cancer pathogenesis.

SFRP proteins are endogenous modulators of Wnt signaling that compete with Wnt ligands in binding to the frizzled receptors. Previous studies have shown SFRP downregulation in colorectal cancer (Suzuki et al. 2002, 2004; Caldwell et al. 2004), gastric cancer (To et al. 2001), and invasive breast tumors (Wong et al. 2002). Interestingly, Suzuki et al. demonstrated that restoration of SFRP function in colorectal cancer cells attenuates Wnt signaling, even in the presence of P-catenin mutation, and may complement downstream mutations in the development of colorectal cancer (Suzuki et al. 2002). Although somatic mutations were not detected in the SFRP1 coding sequence, a loss of heterozygosity (LOH) analysis found that 38% of informative surgical specimens had LOH in the SFRP1 gene locus (Fukui et al. 2005). It was also found that SFRP was frequently downregulated in NSCLC and mesothelioma cell lines (Lee et al. 2004). Moreover, the SFRP gene promoter was hy-permethylated in more than 80% of mesothelioma primary tissues (Lee et al. 2004) and in approximately 55% of primary lung tumors. More recently, it was found that promoter hypermethylation of the APC, CDH1, SFRP1, and WIF-1 genes may be able to discriminate lung primary ade-nocarcinomas from colorectal metastasis to the lung (Tang et al. 2006).

DKK proteins are a group of secreted glycoproteins that have the ability to antagonize Wnt-mediated signals (Fedi et al. 1999). The DKK-3 gene has been found to be downregulated in many cancer cell lines, including NSCLC (Tsuji et al. 2000), and in 63% of freshly resected NSCLC tissues (Nozaki et al. 2001). It has also been shown that forced expression of DKK-3 can inhibit cell growth (Tsuji et al. 2001). Moreover, no mutation was found within the DKK-3 gene. The gene is instead silenced by promoter hypermethylation in a high proportion of lung cancers (Kobayashi et al. 2002).

4.3 Dishevelled (Dvl)

Dvl proteins are positive mediators of Wnt signaling located downstream of the frizzled receptors and upstream of P-catenin. Three Dishevelled genes have thus far been characterized, Dvl-1 to Dvl-3. Di shevelled proteins possess three conserved domains, an N-terminal DIX domain that binds to Axin (Zeng et al. 1997), a central PDZ domain involved in protein-protein interactions (Ponting et al. 1997), and a C-terminal DEP domain found in proteins that regulate Rho GTPases (Ponting and Bork 1996). We showed that Dvl-3 was overexpressed in 75% of fresh microdissected NSCLC samples compared to autologous matched normal tissues (Uematsu et al. 2003a). Moreover, targeted inhibition of Dvl-1, -2 and -3 decreased P-catenin expression and TCF-dependent transcription and inhibited cell growth in human NSCLC cell lines (Uematsu et al. 2003a). We also demonstrated that a PDZ domain deletion mutant of Dvl could suppress tumorigenesis in pleural malignant mesothelioma (Uematsu et al. 2003b). Collectively, we believe these data support the novel hypothesis that Wnt signaling is activated through Dvl overexpression in thoracic malignancies. Many proteins including Daam-1, casein kinases 1 and 2, Notch, and P-arrestin have been shown to interact with Dvl (Wharton Jr 2003). While their role in carcinogenesis has been shown, their specific function in activating Dvl proteins in lung cancer remains largely unknown.

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