Review

Oncogene (2003) 22, 3113–3122. doi:10.1038/sj.onc.1206451

PTEN signaling pathways in melanoma

Heng Wu1, Vikas Goel1 and Frank G Haluska1

1Department of Hematology/Oncology, Massachusetts General Hospital, GRJ1021, 55 Fruit Street, Boston, MA 02114, USA

Correspondence: FG Haluska, E-mail: haluska.frank@mgh.harvard.edu

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Abstract

Phosphatase and tensin homolog deleted in from chromosome ten (PTEN), initially also known as mutated in multiple advanced cancers or TGF-beta-regulated and epithelia cell-enriched phosphatase, is a tumor suppressor gene that is mutated in a large fraction of human melanomas. A broad variety of human cancers carry PTEN alterations, including glioblastomas, endometrial, breast, thyroid and prostate cancers. The PTEN protein has at least two biochemical functions: it has both lipid phosphatase and protein phosphatase activity. The lipid phosphatase activity of PTEN decreases intracellular PtdIns(3,4,5)P3 level and downstream Akt activity. Cell-cycle progression is arrested at G1/S, mediated at least partially through the upregulation of the cyclin-dependent kinase inhibitor p27. In addition, agonist-induced apoptosis is mediated by PTEN, through the upregulation of proapoptotic machinery involving caspases and BID, and the downregulation of antiapoptotic proteins such as Bcl2. The protein phosphatase activity of PTEN is apparently less central to its involvement in tumorigenesis. It is involved in the inhibition of focal adhesion formation, cell spreading and migration, as well as the inhibition of growth factor-stimulated MAPK signaling. Therefore, the combined effects of the loss of PTEN lipid and protein phosphatase activity may result in aberrant cell growth and escape from apoptosis, as well as abnormal cell spreading and migration. In melanoma, PTEN loss has been mostly observed as a late event, although a dose-dependent loss of PTEN protein and function has been implicated in early stages of tumorigenesis as well. In addition, loss of PTEN and oncogenic activation of RAS seem to occur in a reciprocal fashion, both of which could cooperate with CDKN2A loss in contribution to melanoma tumorigenesis.

Keywords:

PTEN, melanoma, signaling

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Introduction

PTEN was first identified as a candidate tumor suppressor gene in 1997 after its positional cloning from a region of chromosome 10q24 known to exhibit loss in a wide spectrum of tumor types. Since then mutations of PTEN have been detected in a variety of human cancer including glioblastomas, endometrial, breast, thyroid, prostate cancer and melanoma. Inherited mutations in the gene also predispose carriers to develop Cowden's disease (CD), a heritable cancer risk syndrome, and several related conditions. Studies on the biochemistry of PTEN have provided a great deal of insight into the basis for its involvement in tumor suppression. The PTEN protein has both lipid phosphatase and protein phosphatase activity. Although the tumor-suppressive function of PTEN has mainly been attributed to its lipid phosphatase activity, a role for PTEN protein phosphatase activity in cell-cycle regulation has been suggested as well. In this review, signaling pathways mediated by the lipid and protein phosphatase activities of PTEN, and the implication of PTEN loss in melanoma tumorigenesis, will be discussed.

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PTEN as a tumor suppressor in melanoma

The most convincing initial insight into the potential involvement of chromosome 10 in melanoma, was summarized by Fountain et al. (1990). Studies on the relative frequency of chromosomal aberrations revealed that several chromosomes were more commonly altered than 10; however, chromosomes 9 and 10 were unique in their early alteration in 'benign' and dysplastic lesions. The presence of a tumor suppressor gene(s) on chromosome 10q had long been suspected, since LOH on regions of chromosome 10q was observed frequently in a number of cancer types (Fults and Pedone, 1993; Isshiki et al., 1993; Herbst et al., 1994; Healy et al., 1995; Ittmann, 1996). And in melanoma, loss of chromosome 10 was first reported by Parmiter et al. (1988). Since then LOH of chromosome 10q has been studied extensively and a frequency of 30–50% has been found in melanoma, suggesting the presence of tumor suppressor gene(s) on chromosome 10q critical for melanoma tumorigenesis (Herbst et al., 1994; Healy et al., 1995). However, LOH studies in melanoma did not eventually yield the identification of a tumor suppressor gene on chromosome 10q. In 1997, by homozygous deletion mapping in gliomas and breast tumors, PTEN was finally identified as a candidate tumor suppressor gene on chromosome 10q. PTEN mutations in melanoma were reported shortly after its cloning. Initial studies demonstrated a mutation rate of approx30–40% in melanoma cell lines and approx10% in primary melanomas (Guldberg et al., 1997; Tsao et al., 1998). Functional studies supported the hypothesis that PTEN played an important role in melanoma: in PTEN-deficient melanoma cells, ectopic expression of PTEN was able to reduce melanoma tumorigenecity and metastasis (Hwang et al., 2001), implicating PTEN as a critical tumor suppressor in melanoma tumorigenesis. However, it is not yet clear whether PTEN function loss occurs as an early or late event, and how PTEN loss has contributed to melanoma development. To elucidate the role of PTEN in melanoma tumorigenesis, a thorough understanding of the functions of PTEN on the molecular level and PTEN-mediated signaling events is necessary.

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Cloning and characterization of PTEN

In 1997, three research groups independently reported the cloning of PTEN, MMAC1 and TEP1, which turned out to be the same tumor suppressor gene. Parsons' group first isolated PTEN by mapping of homozygous deletions on chromosome 10q23 in breast tumors (Li et al., 1997). The predicted PTEN protein contained the phosphatase consensus motif and had approx40% homology with the focal adhesion protein tensin. It was named PTEN (phosphatase and tensin homolog deleted in from chromosome ten). Similarly, MMAC1 was cloned based on homozygous deletion studies in glioma tumor cells by Steck and colleagues (Steck et al., 1997). Coding region mutations of this gene were observed in numerous cancer types including glioblastomas, prostate, kidney and breast cancers, thus it was named MMAC1 (mutated in multiple advanced cancers). TEP1, on the other hand, was identified as a protein tyrosine phosphatase by searching Genebank sequences containing phosphatase consensus motifs. The expression level of this gene was found to be altered in a number of tumor cells and it was rapidly downregulated by transforming growth factor-beta (TGF-beta). Therefore, it was called TEP1 (TGF-beta-regulated and epithelial cell-enriched phosphatase) (Li and Sun, 1997). Sequence identity between PTEN, MMAC1 and TEP1 confirmed that they were of the same gene. Subsequently, a high frequency of PTEN mutations have been reported in malignant melanoma, squamous cell carcinoma, endometrial, and thyroid tumors in addition to glioma, prostate and breast tumors. These findings placed PTEN among the most mutated tumor suppressor genes in cancer (Dahia et al., 1997; Guldberg et al., 1997; Tashiro et al., 1997; Poetsch et al., 2002).

When PTEN was first cloned, it was predicted to be a protein phosphatase since PTEN contained (I/V)-H-C-X-A-G-X-X-R-(S/T)-G, the critical motif found in protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs) (Tonks and Neel, 1996; Yuvaniyama et al., 1996; Li et al., 1997). However, the recombinant PTEN protein exhibited higher catalytic activity towards negatively charged phosphorylated polypeptides than phosphoproteins (Myers et al., 1997). PtdIns(3,4,5)P3 (PIP3) was then identified as a substrate of PTEN (Maehama and Dixon, 1998). In 1999, the crystal structure of PTEN was solved, showing an overall phosphatase domain structure similar to that of the DSP VHR. However, the active site pocket of PTEN appeared deeper and wider, and two basic residues (Lys125 and Lys128) were localized within the active site loop, which were absent in PTP and VHR (Lee et al., 1999). It was suggested that PTEN, as a phosphatase, might have preference towards PIP3 and highly acidic residues present in polypeptides, although it could use both protein and lipid as substrates.

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Function and signaling of PTEN

PTEN knockout mouse models

To evaluate the function of PTEN in vivo, PTEN-deficient mice were generated. Three groups independently reported generation of PTEN knockout mice (Di Cristofano et al., 1998; Stambolic et al., 1998; Podsypanina et al., 1999). The homozygous deletion of PTEN in mice was an embryonic lethal trait, and PTEN-/+ mice developed neoplasms in various organs, including breast, prostate, endometrium, adrenal, lymphoid, skin, colon, thyroid, liver and thymus. Stambolic et al. (1998) generated PTEN-deficient mice by targeted disruption of exons 3–5 of the PTEN gene. Homozygous mutant mice died by day 9.5 of development. Although no obvious difference in apoptosis was observed between wild-type and mutant embryos, immortalized mouse embryonic fibroblast cells derived from homozygous mutant embryos showed a significantly reduced sensitivity to agonist-induced apoptosis (Stambolic et al., 1998). PTEN knockout mice generated by Pandolfi's group carried a targeted deletion of exons 4 and 5 of PTEN gene. PTEN-/- embryos did not survive past day 7.5 (Di Cristofano et al., 1998). Parsons and colleagues generated PTEN knockout mice with disrupted exon 5 only. Homozygous mutant embryos died between days 3.5 and 6.5. Defects of apoptosis in B cells and macrophages were also observed in these mice (Podsypanina et al., 1999). Overall, these mouse models displayed similar phenotypes including development of neoplasms in various organs of PTEN-deficient mice, and increased proliferative capacity as well as reduced sensitivity to agonist-induced apoptosis in PTEN-deficient cells. These data confirmed that PTEN functions as a critical tumor suppressor gene. They also suggested that PTEN plays important roles in regulating cell-cycle progression and cell death.

PTEN as a phosphatase

PTEN has been implicated not only in suppressing cancer growth but also in regulating embryonic development, cell adhesion and migration, apoptosis, stem cell growth and differentiation (Di Cristofano and Pandolfi, 2000; Penninger and Woodgett, 2001; Yamada and Araki, 2001). In all cases, the phosphatase activity of PTEN is essential. The phosphatase domain is encoded by exon 5, mutations of which have comprised 20–30% of both germline and somatic PTEN mutations (Bonneau and Longy, 2000). C124R, a mutation first identified in CD, an autosomal dominant inherited cancer syndrome, results from a single nucleotide change within exon 5 (Nelen et al., 1997). This mutation renders PTEN phosphatase inactive. C124S, another phosphatase-inactivating mutation of PTEN, was identified with high frequency in tumor cells (Kurose et al., 1998). G129E, a germline mutation isolated from a CD patient, also results from a single nucleotide change in exon 5, resulting in the retention of PTEN's activity on protein and polypeptide substrates, but loss of its activity on PIP3 (Liaw et al., 1997). These PTEN mutants, along with wild-type PTEN, have been reintroduced back into PTEN-deficient cells to examine their tumor-suppressive functions in numerous studies. In a breast cancer line, MCF-7, when wild-type PTEN, but not C124S, was overexpressed, cell-cycle arrest at G1 was observed initially, followed by a combination of G1 arrest and cell death, which contributed to the observed cancer cell growth suppression (Weng et al., 1999). Similar results have been reported in endometrial cancer cells, thyroid cancer cells and glioma cells (Adachi et al., 1999; Lilja et al., 2001; Weng et al., 2001c). It is worth noting that glioma and melanoma share a common developmental origin and exhibit several genetic similarities. Both glia cells and melanocytes are derived from the neural crest. In addition, loss of 10q including PTEN locus is frequent in gliomas and melanomas.

PTEN signaling as a lipid phosphatase

Extensive studies with PTEN mutants, especially G129E (retaining only protein phosphatase activity), have begun to unravel the signaling pathways mediated by lipid and protein phosphatase activity of PTEN, respectively. Ectopic expression of G129E into PTEN-null 786-O renal carcinoma cells was unable to arrest cells at G1, like the wild-type PTEN did, suggesting that the lipid phosphatase, but not the protein phosphatase activity of PTEN, was involved in inducing cell-cycle arrest in these cells (Ramaswamy et al., 1999). The G129E mutant of PTEN is unable to dephospharylate PIP3, resulting in elevated intracellular PIP3 levels. PIP3, as an important second messenger, transduces signals from growth factors, hormones and extracellular matrix components. One of the best-studied downstream targets of PIP3 is Akt, also known as protein kinase B. When cells are stimulated, Akt is recruited by PIP3 to the plasma membrane, where Akt is phosphorylated at Thr308/Ser473 and activated (Nicholson and Anderson, 2002). Since Akt is involved in promoting cell survival, proliferation and migration, loss of PTEN and consequent overactivation of Akt result in loss of proliferative and apoptotic control.

The role of PTEN in cell-cycle regulation has also been explored extensively. Results from numerous studies have placed p27 (Kip1), the cyclin-dependent kinase inhibitor, as a downstream target of PTEN. Overexpression of PTEN in PTEN-deficient glioblastoma cell lines U87MG, U373MG and U251MG leads to the recruitment of p27 to cyclin E/CDK2, resulting in decreased cyclin E/CDK2 activity and reduced phosphorylation of retinoblastoma protein (pRB). As a consequence, cell-cycle entry is blocked at S phase (Cheney et al., 1999). In the two thyroid tumor cell lines FB-1 and ARO, ectopic PTEN expression was able to inhibit tumor cell growth, which was correlated with decreased Akt phosphorylation and induced p27 expression. Interestingly, growth inhibition by PTEN overexpression was more effective in FB-1 cells in which endogenous PTEN level was lower, comparing with that in ARO cells (Bruni et al., 2000). Regulation of p27 by PTEN may occur on both transcriptional and post-translational levels. Forkhead transcription factors have been shown to mediate PTEN-induced p27 upregulation transcriptionally (Nakamura et al., 2000; Kops et al., 2002). p27 protein degradation was also found to be regulated by PTEN. As shown recently, PTEN-induced p27 accumulation in U87MG could be reversed by overexpression of SKP2, a key component of ubiquitin E3 ligase complex SCF (Mamillapalli et al., 2001). More recently, a direct interaction between Akt and p27 was suggested by Fujita et al. (2002). They found that Akt directly bound and phosphorylated p27. Phosphorylation at a newly identified site Thr198 was critical for the binding of p27 to 14-3-3 proteins, a family of molecules involved in cytoplasmic translocalization and degradation of many proteins. Aside from p27, other cell-cycle-regulated proteins including cyclin A, cyclin D1, cyclin D3, p15 and p21 have also been implicated in mediating PTEN-induced cell-cycle arrest, although results have not been as consistent (Wu et al., 2000; Gottschalk et al., 2001; Matsushima-Nishiu et al., 2001; Weng et al., 2001b; Zhu et al., 2001). Utilization of tumor cells as model systems may have contributed to the inconsistency. Undefined mutations may exist in cancer cell lines, and these uncharacterized mutations may well belong to the signaling pathways being studied; thus, caution ought to be observed when analysing data obtained from these cells.

The role of PTEN in cell survival has been attributed to its lipid phosphatase activity as well. Stambolic et al. (1998) showed that PTEN knockout MEFs exhibited reduced sensitivity to apoptosis, and wild-type PTEN, but not G129E mutant, could restore the sensitivity to agonist-induced apoptosis. PTEN was able to sensitize cells to a variety of apoptotic stimuli such as death receptors, kinase inhibitors and chemotherapeutic agents. It was demonstrated that PTEN expression sensitized LNCaP prostate cancer cells to apoptosis induced by chemotherapeutic agents such as stauro-sporine, doxorubicin and vincristine. Through a FADD-dependent pathway, PTEN overexpression induced caspase-8 activation, which in turn facilitated the cleavage of BID, leading to the release of cytochrome c and activation of downstream caspases (Yuan and Whang, 2002). PTEN has also been suggested to downregulate the level of antiapoptotic protein Bcl2. Regulation of Bcl2 by PTEN was on the transcriptional level and exogenous expression of Bcl2 was able to attenuate PTEN-induced chemosensitivity in LNCaP cells (Huang et al., 2001). Similar results have been observed in thyroid cancer cells, breast carcinoma cells and malignant mesothelioma cells (Ghosh et al., 1999; Weng et al., 2001a, 2001c; Mohiuddin et al., 2002).

PTEN signaling as a protein phosphatase

Although PTEN was first cloned as a tyrosine and dual-specificity protein phosphatase, much less attention has been drawn towards PTEN's protein phosphatase activity. One of the reasons for this is that the lipid, but not protein phosphatase activity of PTEN, has been associated with its tumor-suppressive function in most if not all cases. However, it has been suggested that the protein phosphatase activity may be linked to its ability to induce cell-cycle arrest. Weng et al. (2001b) showed that in MCF7 breast cancer cells, ectopic expression of G129E decreased the level of cyclin D1 and arrested cells at G1, despite the absence of PTEN lipid phosphatase activity. When cells were treated with PD980059, a MEK inhibitor, similar results were observed. In the same cells, PTEN ectopic expression inhibited insulin growth factor 1-induced IRS-1 phosphorylation and IRS-Grb2/Sos association, as well as MAPK phosphorylation (Weng et al., 2001d). Moreover, PTEN expression resulted in a decrease in MEK1/2 and ERK1/2 phosphorylation levels in the absence of extracellular stimuli. These data suggested that the MAPK pathway might mediate signaling of the PTEN protein phosphatase. Examing the role of PTEN in growth signal-induced MAPK activation has yielded more supporting evidence. MAPK activation induced by growth signals such as epidermal growth factor (EGF) and platelet-derived growth factor was blocked by exogenous PTEN expression in U87MG glioblastoma cells. The adapter protein Shc was dephosphorylated by PTEN protein phosphatase and association of Shc with Grb2/Sos induced by EGF was abolished by PTEN overexpression in these cells (Gu et al., 1998, 1999). It is interesting to note that PTEN did not dephosphorylate receptor tyrosinase kinases such as EGFR and insulin receptor. Therefore, PTEN, as a protein phosphatase, may intersect with MAPK-mediated growth factor signaling pathway by dephosphorylating adapter proteins such as Shc and IRS, leading to complex dissociation between adapter proteins and Grb2-Sos, resulting in reduced phosphorylation of MEK and MAPK.

Focal adhesion kinase (FAK) was identified as another substrate of PTEN protein phosphatase. A phosphatase-inactive mutant of PTEN, D92A, was found to bind to FAK, suggesting a direct interaction of PTEN with FAK (Tamura et al., 1999). Expression of wild-type PTEN, or G129E, downregulated phosphorylated FAK level in U87MG cells. In these cells, PTEN was demonstrated to inhibit cell migration, and spreading, as well as focal adhesion formation (Tamura et al., 1998). Interestingly, it was shown that activated FAK was also associated with PI3 K. PTEN overexpression inhibited association between FAK and PI3 K, which resulted in a decrease in PI3 K activity (Tamura et al., 1999). Therefore, a synergistic effect on reducing intracellular PIP3 level and Akt signaling may be achieved by the protein and lipid phosphatase activity of PTEN.

Other aspects of PTEN signaling

Recently, a potential implication of the intracellular localization of PTEN in the pathogenesis of melanoma has been reported (Whiteman et al., 2002). The sequence of PTEN contains no nuclear localization signal, and it is predicted to be a cytoplasmic protein. However, a few research groups have reported detection of PTEN expression in the nucleus. By immunohistochemistry, strong nuclear staining was observed in normal follicular thyroid cells and endothelial cells, as well as in some tumor cells including melanoma cells (Gimm et al., 2000). On the other hand, in fibroblasts, prostate cancer xenografts and hepatocellular carcinoma, predominantly cytoplasmic PTEN staining was detected (Li and Sun, 1997; Tamura et al., 1998; Whang et al., 1998). This cytoplasmic/nuclear dual localization pattern has been noticed for signaling molecules such as MAPK, PI3 K, Akt, and beta-catenin, which carry out their distinct functions at distinct intracellular locations. Little is known about the function of PTEN in the nucleus. PIPs have been shown to be present within the nucleus, so PIP3 may serve as a substrate for PTEN in the nucleus. Nevertheless, PTEN may be playing some undefined role in the nucleus, which is yet to be determined. Owing to the lack of nuclear translocalization signal, it has been hypothesized that PTEN may be shuttled into the nucleus by another molecule. Such a mechanism has been described for translocalization of p53 by mdm2. It is yet to be determined whether such a molecule exists for PTEN, how PTEN activity is regulated by this translocalization, and whether it is involved in tumorigenic process. It is interesting to note that in melanoma, an association between loss of the PTEN nuclear expression (in 84 in 92 samples) and clinical features of melanoma such as mitotic index and anatomical site has been found. Such an association did not exist for cytoplasmic expression of PTEN (Whiteman et al., 2002).

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PTEN mutations in human cancer: implications of PTEN in melanoma tumorigenesis

Both germline and somatic mutations of PTEN have been extensively characterized. PTEN germline mutations have been found in three linked autosomal dominant cancer predisposition syndromes: CD, Lhermitte–Duclos disease and Bannayan–Zonana syndrome. These cancer syndromes share similar phenotypic characteristics including mental retardation, gastrointestinal hamartomas, thyroid adenomas, breast fibroadenomas, macrocephaly, and mucocutaneous lesions (Vazquez and Sellers, 2000). Over 80% of patients with CD harbored germline PTEN mutations. LOH studies in 20 hamartomas using markers flanking and within PTEN showed that wild-type PTEN locus was indeed lost in two breast fibroadenomas, one thyroid adenoma, and one pulmonary hamartoma, confirming that PTEN functions as a tumor suppressor gene in CD (Nelen et al., 1997). A great number of PTEN germline mutations (approx30%) were found in exon 5, with the rest scattered along the entire PTEN gene. These mutations included point missense mutations small deletions, insertions, splice site mutations and gross deletions of the gene. The majority of these mutations lead to premature termination or functional inactivation of the protein.

A similar mutational profile has been found for PTEN somatic mutations in cancer. The highest frequency of PTEN mutations is found in endometrial carcinomas and glioblastomas. PTEN somatic mutations are also found in lymphoma, thyroid, breast, prostate carcinomas and melanomas (Bonneau and Longy, 2000). In melanoma, a PTEN mutation rate of 30–50% in melanoma cell lines and 5–20% in uncultured melanomas have been reported (summarized in Table 1). In 1997, Guldberg et al. (1997) first reported that 43% (15/35) of examined melanoma cell lines harbored PTEN mutations. Nine of these cell lines showed homozygous deletion of PTEN gene, and six lines had mutations in one allele in combination with the loss of the second. Tsao et al. (1998) examined 45 melanoma cell lines and found PTEN mutations in 29% of the melanoma cell lines, including nine homozygous deletions and four frameshift, nonsense and intronic splice mutations. Similar results were reported by Teng et al. (1997). In their study, seven PTEN mutations in 14 melanoma cell lines including four cases (28%) of homozygous deletions were identified (Teng et al., 1997).


PTEN mutations in uncultured melanoma samples are rarer. Tsao et al. (1998) examined 17 uncultured metastatic melanoma samples; only one case of homozygous deletion and another case of premature stop mutation were identified. They also examined 28 samples from patients with familial melanoma; no mutations were found in these cases (Tsao et al., 1998). Teng et al. (1997) found one missense mutation of PTEN in 10 primary melanoma tumors. Birck et al. (2000) screened a panel of 77 uncultured melanoma samples including 16 primary and 61 metastatic tumors. By examing two intragenic biallelic polymorphisms, 21 out of 39 (54%) informative specimens showed loss of one PTEN allele. PTEN mutations were identified in four of the metastatic tumors (Birck et al., 2000). Reifenberger et al. (2000) identified four cases of somatic PTEN mutations (11%) out of 37 metastatic melanomas. Celebi et al. (2000) also detected PTEN sequence alterations in four of 21 (19%) metastatic melanoma samples. Taken together, these data supported the notion that PTEN alterations occur in melanomas and loss of PTEN contributes to melanoma development.

Functional studies support a role for PTEN in melanoma tumor suppression. An in vitro LOH study by Robertson et al. (1998) showed that PTEN was indeed targeted for LOH in melanoma. A melanoma cell line UACC903 with duplicated mutant chromosome 10 was used to build the in vitro LOH model. A wild-type chromosome 10 was transferred into the cells and underwent spontaneous breakage and deletions over time in culture. During this process, the introduced wild-type copy of PTEN was lost. In parallel, another melanoma cell line with wild-type PTEN gene maintained a transferred 10q23–24 region that contained the exogenous PTEN gene. Ectopic expression of PTEN into UACC903 cells was also demonstrated to suppress tumor cell growth (Robertson et al., 1998). Similar findings have been reported by Tsao et al. (2000) as well. In a number of melanoma cell lines, overexpression of PTEN uniformly inhibited colony formation, implicating a tumor-suppressive function of PTEN in melanoma (Tsao et al., 2000).

Three groups, in contrast, have failed to detect significant PTEN mutation rates in melanoma. Boni et al. (1998) used two microsatellite markers flanking PTEN gene to search for LOH surrounding the PTEN locus, and found no LOH for either of the markers in 40 (23 primary and 17 metastatic) melanoma tissue. Further SSCP analysis for exons of PTEN gene did not yield any abnormal bands (Boni et al., 1998). Herbst et al. (1999) analysed LOH at loci closely linked or intragenic to PTEN in 65 melanomas. A rate of LOH of lower than 16% with eight different polymorphism markers led to the conclusion that it rather represented random genetic events than indicating that PTEN was the target in melanoma (Herbst et al., 1999). Poetsch et al. (2001) screened 25 primary and 25 metastatic melanomas for PTEN mutation, and found neither nonsense mutations nor deletion of the entire gene or any exon.

These finding are in contrast to the weight of abundant evidence implicating PTEN as an important tumor suppressor in melanoma and other cancers. They must be taken in context of the fact that although an overall mutation rate of PTEN in cultured melanoma specimens is 5–20%, only one case of PTEN mutation has been detected in over 30 primary melanomas. There are several possible explanations for this observation: (1) although PTEN loss is important in melanoma, it occurs late in melanoma tumorigenesis since mutation is rarely detected in primary melanomas; (2) PTEN loss may in fact be relatively rare in melanoma, and the establishment of cell lines selects for melanomas with PTEN alterations; (3) the biology of PTEN alteration in early melanomas makes detection of alteration difficult (eg, from dosage reduction, epigenetic downregulation of expression or homozygous deletion); (4) or the number of primary melanoma samples examined is small, and the correct histologies or types of tumors have not been examined. These possibilities need to be examined.

The best possible explanation for occasional negative studies is likely the biology of PTEN alteration. The homozygous deletion rate observed by several laboratories may contribute. Homozygous deletion makes LOH of chromosome 9p21 (at CDKN2A) difficult to detect in tumor samples (Cairns et al., 1995). Similarly, chromosome 10q24 PTEN deletions in melanomas may have been underdetected. Moreover, epigenetic studies suggested recently that the involvement of PTEN function loss in melanoma might have been in fact underestimated. Zhou et al. (2000) analysed PTEN protein expression, instead of analysing PTEN gene mutations in melanomas. Using immunohistochemistry, they found no PTEN protein expression in 15% (5/34) and low expression in 50% (17/34) of melanoma samples (four primary and 30 metastatic). Surprisingly, among the five melanomas with no PTEN protein expression, four showed no deletion or mutation of PTEN gene, indicating the presence of an epigenetic mechanism of biallelic functional inactivation of PTEN. Their data strongly supported PTEN as a tumor suppressor in melanoma tumorigenesis (Zhou et al., 2000).

The timing of PTEN alterations in melanoma development is also not understood. Cytogenetic studies, cited above, suggest an early involvement of PTEN. But Birck et al. (2000) examined a relatively large number (16) of primary melanoma samples and detected no PTEN mutations. However, they also found allelic loss of PTEN gene in 38% (3/8) primary melanomas, indicating a decreased PTEN dosage possibly occurring early in melanoma development (Birck et al., 2000).

Thus different types of mutation, coupled with the potential for the accumulation of PTEN mutations with tumor progression, may allow for better establishment of melanoma cell lines carrying PTEN alterations, and provide some explanation for the discrepancies in the literature. Additional work will be needed to accumulate data to allow for the reconciliation of divergent mutation rates from different types of studies.

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Interaction of PTEN alterations with other genes

In addition to PTEN somatic mutations, germline and somatic mutations of CDKN2A, as well as NRAS somatic mutations have been frequently observed in melanoma (Pollock and Trent, 2000; Saida, 2001). CDKN2A is the most often mutated tumor suppressor gene in melanoma, with 60% homozygous deletions and additional 15–20% point mutations (Liu et al., 1995; Flores et al., 1996; Haluska and Hodi, 1998). The two proteins encoded by CDKN2A, p16 and p14ARF, function in the pRB and p53 pathway, respectively. p16 is a cyclin-dependent kinase inhibitor. It binds to and inhibits cyclin D/CDK4, which in turn blocks pRB phosphorylation, leading to G1 cell-cycle arrest. p14, on the other hand, binds mdm2 and relieves p53 from mdm2-mediated p53 degradation. p53 is known to block cell proliferation by inducing cell-cycle arrest or apoptosis.

RAS is the most studied oncogene involved in melanoma tumorigenesis. Like PTEN, it has several functions. RAS gene family members include HRAS, NRAS and KRAS. They encode 21 kDa proteins with GTPase activity. RAS is involved in regulating receptor tyrosine kinase-induced MAPK activation in that RAS activates MEK and MAPK through RAF. RAS also binds and activates lipid kinase PI3K, and therefore activates Akt pathway. Finally, RAS interacts with p53 and p16. In primary mouse embryonic fibroblasts, for example, HRAS was shown to induce premature cell senescence, which was associated with the accumulation of p16 and p53 (Serrano et al., 1997). Pathways controlled by these three elements, RAS, p53 and p16, therefore appear to be central to control of the malignant phenotype.

PTEN functions as a lipid and protein phosphatase that downregulates Akt and MAPK, potentially suggesting that RAS and PTEN have opposite functions in both protein and lipid kinase signaling pathways (Figure 1). Is it possible that PTEN loss and RAS oncogenic activation are redundant in tumor development? Tsao et al. (2000) reported a reciprocal mutational status for PTEN and NRAS in human melanoma cells. Among 53 cutaneous melanoma cell lines, 16 cell lines (30%) harbored PTEN mutations and 11 lines (21%) had oncogenic NRAS mutations. Only one cell line showed mutations in both the genes, so a total of 50% cell lines had mutations in either PTEN or NRAS (Table 2, from Tsao et al., 2000). Similar reciprocal findings have been reported in endometrial cancer (Ikeda et al., 2000).

Figure 1.
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Signaling pathways mediated by lipid and protein phosphatase activity of PTEN. The lipid phosphatase activity of PTEN decreases intracellular PIP3 level and downstream Akt activity, which promotes agonist-induced apoptosis, mediated though the downregulation of antiapoptotic protein such as Bcl2 and the upregulation of proapoptotic machinery involving Caspases. The PTEN lipid phosphatase also arrests cell-cycle progression at G1/S, which is mediated at least in part by the upregulation of cyclin-dependent kinase p27. The PTEN protein phosphatase dephosphorylates FAK and Shc, which leads to the inhibition of focal adhesion formation and migration, as well as the inhibition of growth factor-stimulated MAPK signaling, respectively

Full figure and legend (30K)


In mouse melanoma models, RAS and CDKN2A loss cooperate to lead to melanoma development (Chin et al., 1997). Recently, it has been shown that CDKN2A loss coupled with PTEN loss lead to melanoma; however, it is not clear that PTEN loss confers greater susceptibility to melanoma development than CDKN2A loss alone (You et al., 2002). Further studies are needed to elucidate the details of PTEN, RAS and CDKN2A interaction in murine models.

Recently, a direct downstream target of RAS, BRAF, has been shown to exhibit a 66% mutation frequency in melanoma (Davies et al., 2002). In all, 20 of 34 (59%) melanoma cell lines, six of nine (67%) primary melanomas and 12 of 15 (80%) melanomas in short-term culture harbored mutated BRAF gene. In this study, NRAS mutations were also detected in 9% (3/34) of the melanoma cell lines, none of which had BRAF mutations. Hence, all together 23 out of 34 melanoma cell lines were detected with oncogenic-activated RAS-RAF-MEK-MAPK pathway. Thus, BRAF is a second gene whose mutations are reciprocally distributed with regard to RAS. Like RAS, RAF can activate PI3K, and PI3K and Akt can directly alter RAF kinase activity (Mograbi et al., 2001; Moelling et al., 2002). Thus, understanding the relation of PTEN, RAS and RAF, in the context of PI3K-Akt and RAS-MAPK pathways will be crucial to understanding melanoma tumorigenesis.

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Summary and future perspectives

In this review, we have discussed function and signaling of PTEN as a lipid phosphatase as well as a protein phosphatase. The consequences of PTEN loss are alterations in the control of cell-cycle progression, of apoptosis, and cell contact and migration. Together these aberrations function to contribute to the malignant cell phenotype (Figure 2).

Figure 2.
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Reciprocal functions of PTEN and RAS in lipid and protein kinase signaling. RAS can activate MAPK signaling pathway through RAF. It can also bind and activate PI3 K, resulting in an increase in intracellular PIP3 level and downstream Akt activity. PTEN, on the other hand, functions as both protein and lipid phosphatase. It dephosphorylates adapter protein such as Shc, leading to decreased MEK and MAPK activity. As a lipid phosphatase, PTEN reduces PIP3 level, thus decreases Akt activity

Full figure and legend (17K)

We have reviewed several lines of evidence implicating PTEN in the development of melanoma. Although the importance of 10q24 loss in melanoma is clear, and studies of PTEN in cultured melanoma lines seem to suggest strongly that PTEN is the target of this loss, much remains to be learned about the precise role of PTEN in melanoma tumorigenesis. Whether PTEN loss occurs as an early or late event in melanoma tumorigenesis is still controversial. The exact frequency of loss in primary tumors, in metastases and the relation of these observations to cell line findings need to be worked out. The inter-relation of PTEN mutation with alterations in RAS, BRAF, CDKN2A and other genes important in melanoma needs to be studied. And finally, modeling of these genetic discoveries in mouse systems promises to provide insights into the precise nature of PTEN function in melanocyte and melanoma.

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References

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