Chapter 12 / Multiple Roles of SKI in 211 12 The Multiple Roles of the Oncogenic Protein SKI in Human Malignant Melanoma

Dahu Chen, Qiushi Lin, I. Saira Mian, Jon Reed, and Estela E. Medrano

CONTENTS INTRODUCTION DECONSTRUCTING SKI THE REPRESSOR FUNCTIONS OF SKI THE ACTIVATOR FUNCTIONS OF SKI IN THE WNT-SIGNALING PATHWAY CONCLUSIONS AND PERSPECTIVES REFERENCES

Summary Cellular localization, association with different protein partners, and posttranslational modifica- tions can dramatically change protein function. SKI and the highly homologous protein snoN are potent repressors of transforming growth factor-E signaling through their association with the Smad proteins. In fact, SKI can act as molecular switch converting the Smad proteins from an activating to a repressing entity on chromatin. SKI also plays additional roles in : in association with the LIM protein FHL2 activates E-catenin signaling, a pathway associated with cancer progression. This chapter reviews the transcriptional co-repressor and co-activator activities of SKI and discusses their biological signifi- cance for melanoma tumor progression. Key Words: SnoN; Smad2; Smad3; FHL2; E-catenin; MITF.

INTRODUCTION The protein products of the proto- SKI and the SKI-related novel gene (sno) are implicated in processes as diverse as differentiation and transformation (1–4). Overexpression of SKI correlates with progression of melanoma (5) and esophageal squamous cell carcinoma (6). Although SKI was believed to be a nuclear protein, mela- nomas display aberrant cellular trafficking of this protein (5). In preinvasive melanomas in situ, SKI is observed predominantly in the nucleus of intraepidermal melanoma cells (Fig. 1A). However, primary invasive melanomas display both nuclear and cytoplasmic

From: From Melanocytes to Melanoma: The Progression to Malignancy Edited by: V. J. Hearing and S. P. L. Leong © Humana Press Inc., Totowa, NJ

211 212 From Melanocytes to Melanoma localization of SKI. In melanoma metastasis, SKI localizes to both the nuclear and cytoplasmic compartments, or predominantly to the cytoplasmic compartment (Fig. 1B,C). Interestingly, cytoplasmic SnoN is associated with poor prognosis in breast cancer (7). It remains to be established whether cytoplasmic SKI in preinvasive melanomas also has prognostic value. Here, we review the oncogenic activities associated with overexpression of SKI and discuss possible novel activities of this protein that could lead to silencing and inactiva- tion of tumor suppressors and repression of .

DECONSTRUCTING SKI Ski was originally identified as the transforming protein of Sloan Kettering Viruses (8). The ski protein family includes chicken c-ski; mouse Ski; human SKI; sno, two paralogs (skiA and skiB) in zebra fish (9), and the recently identified Caenorhabditis elegans homolog, Daf-5 (10). The Ski proteins were initially considered transcription factors; however, it was later demonstrated that they do not directly bind to DNA (11,12). The identification of proteins associated with SKI has been critical for unraveling its activities, which include repression of transforming growth factor-E (TGF-E) and acti- vation of Wnt-signaling pathways (discussed in detail in “The Repressor and Activator Functions of SKI”). Structural domains located in the human SKI and mouse Ski proteins include, from the amino-terminus, a rich area (amino acids 61–89), helix-loop-helix motifs, a / rich area, a region of basic amino acids, and a zipper-like domain (Fig. 2A). The Dach domain, common to the SKI/Sno/Dachshund family of proteins, contains a putative domain of 100 amino acids that contains a conserved CLPQ motif. SKI and snoN also share a region of homology at the carboxy-terminus (13–17). Computational analysis using MARCOIL suggests that the C-terminus positions 532–710 adopt a coiled-coil conformation. However, it appears that it is not a single contiguous coiled-coil, but two to three coils separated by one to two hinge regions (Fig. 2A). The amino-terminal residues 75–304 are sufficient for the transforming activity of c-Ski (17). Homodimerization of Ski, mediated by a bipartite C-terminal domain consisting of five tandem repeats and a (Fig. 2A), correlates with efficient DNA binding and cellular transformation (18). However, coexpression of c-ski and c-SnoN results in the preferential formation of heterodimers. Tethered c-ski:Sno heterodimers that lack tandem repeat/leucine zipper domains are more active in cellular transformation than either of their monomeric counterparts, tethered ski:ski homodimers or full-length SnoN and c-ski (2). Ski transcripts are detected in the mouse embryo at 8.5 to 9.5 d post-coitum, during migration of neural crest cells, including melanocytes and dorsal root ganglia (19). SKI and SnoN share a large region of homology in the amino-terminus, and the biological activities of these related appear to be similar (20). However, Ski and snoN play different roles during development. Whereas Ski null mice die shortly after birth, displaying defects in neurulation, craniofacial patterning, and skeletal development (21,22), mice lacking sno die at an early stage of embryogenesis, indicating that sno is required for blastocyst formation (23). Ski activities appear to be required for the expansion of a subset of precursors in the neuroepithelial and skeletal muscle lineages, because no major defects were detected in other neural crest-derived cells, including melanocytes. Chapter 12 / Multiple Roles of SKI in Melanoma 213

Fig. 1. Aberrant cellular trafficking of SKI in melanoma progression. (A) Elevated expression of SKI with predominant nuclear localization in a melanoma in situ (intra-epidermal malignant melanoma). (B) Increased levels of predominantly nuclear SKI in many (but not all) of the melanoma cells in this metastatic lesion. (C) Elevated, predominantly cytoplasmic, expression of SKI in a different metastatic lesion. All panels immunolabeled for SKI as described previously (5); original magnification ϫ250. 214 From Melanocytes to Melanoma

Fig. 2. The human SKI protein. (A) Cartoon depicting motifs and domains required for different protein–protein association. Pro, a proline rich domain; Zn, a leucine zipper-like domain; AHs, helix-loop-helix motifs; Basic, a region of basic amino acids; DH, a unique tandem repeats of D-helical domains that is involved in the dimerization of the SKI family through coiled-coil interactions. Arrowheads indicate 3 tandem repeats of 25 amino acids located at residues 572–645. SKI-HD, SKI homodimerization domain. SKI domains required for association with multiple proteins are indicated by a double arrow line. (B) sequence. Serine, threo- nine, and tyrosine residues are indicated in bold.

ski–/– melanocytes isolated from the skin of pups delivered by cesarian section at embryonic day 18.5 can be readily established in culture (24); indicating that Ski is not essential for melanoblast migration, proliferation, or differentiation. The ski and snoN heterozygous mice are an example of as yet unexplained paradoxes regarding function of the Ski and sno proteins. When challenged with carcinogens, heterozygous sno+/– mice show increased number of lymphomas compared with wild-type mice (25); a phenotype shared by ski+/– animals (26). These activities apparently contradict previous Chapter 12 / Multiple Roles of SKI in Melanoma 215 data showing that Ski is a transforming protein (18,27). However, Ski protein levels may be critical determinants of its function. For example, Ski is required for the transcrip- tional repression mediated by the retinoblastoma protein RB (28); in contrast, a high level of SKI suppresses RB function. Thus, SKI could function either as a tumor suppres- sor or as an oncogene/tumor promoter. Little is known regarding posttranslational modifications of the SKI protein. The chicken c-ski protein is phosphorylated on serine residues; the carboxy-terminal region is either the site of phosphorylation or is required for phosphorylation (15). The human SKI protein contains multiple serines, threonines, and tyrosine residues (Fig. 1B). Com- putational analysis of the human SKI protein suggests that it is also a phosphoprotein, because it contains 39 serine, 7 threonine, and 2 tyrosine potential phosphorylation sites. The human SKI maps to 1p36 (29), a common region of alterations in human cancers, including melanomas (30). Because an early study did not find alter- ations in SKI restriction enzyme patterns or increases in gene dosage in human melanoma cell lines (31), its overexpression in human melanoma tissues (5,32) likely results from yet to be defined transcriptional and/or posttranscriptional events.

THE REPRESSOR FUNCTIONS OF SKI The nuclear receptor co-repressor (N-CoR) protein contains a conserved bipartite nuclear receptor interaction domain (NRID) and three independent repressor domains that can actively repress a heterologous DNA-binding domain (reviewed in ref. 33). N- CoR can interact with the Sin3 co-repressor, which, in turn, binds to the histone deacetylase (HDAC), HDAC1 (34). Different HDAC proteins, including HDAC1, HDAC2, and HDAC3, are found in N-CoR complexes. HDACs deacetylate the H-amino group of lysyl residues in histones, resulting in nonpermissive, compact heterochroma- tin structures. High Levels of SKI Repress TGF-E Signaling: A Major Pathway Involved in Tumor Progression N-CoR plays a critical role in the transcriptional repression of some but not all SKI complexes. For example, SKI/N-CoR association is required for repression of Mad, the thyroid hormone receptor (35), vitamin D receptor, and bone morphogenetic protein signaling, but not for Smad signaling (36). High levels of SKI repress TGF-E signaling; a major pathway involved in tumor progression. Binding of TGF-E to its receptors initiates a signaling cascade transduced by the Smad family of transcriptional coactivators (reviewed in refs. 37–39). Ligand-mediated receptor activation results in carboxy-ter- minal phosphorylation of Smad2 and Smad3, formation of heterotrimeric complexes with the common partner Smad4, nuclear translocation, and transcriptional activation of TGF-E target genes. The GTCTAGAC sequence, which is bound by c-ski-containing proteins (39a), was also identified as a Smad binding element (SBE). The Smad proteins cooperate with a diverse number of transcription factors in response to TGF-E. SBEs contain the four basepairs (5'AGAC-3' or its reverse complement 5'AGAC-3') that are directly bound by Smad3 and Smad4 proteins (40). Several groups including ours demonstrated, through a variety of approaches that included affinity chromatography, GST pull-downs, and yeast two-hybrid screening (41–43), that mouse Ski and human SKI associate with a 216 From Melanocytes to Melanoma multi-Smad complex that specifically bind the SBE. Nuclear SKI binds the MH2 domains of Smad2 and Smad3, forming repressor complexes that curtail TGF-E signaling in melanomas and other cell types (32). Increased levels of p21Waf-1 appear to be essential for TGF-E-mediated inhibition of the cyclin-dependent kinase CDK2 and growth inhibition (reviewed in ref. 44). High levels of SKI prevent p21Waf-1 induction through a Smad-dependent mechanism that involves transcriptional repression (5). Thus, elevated expression of SKI facilitates cell cycle progression by targeting the RB pathway in at least two ways: high levels can directly repress retinoblastoma protein (RB) activity, and, indirectly, increase CDK2 activity by repressing TGF-E-mediated induction of p21Waf-1. In turn, cytoplasmic SKI associates with Smad3 and prevents its nuclear translocation in response to TGF-E(5). SKI retains Smads in the cytoplasm by the formation of Smads inactive complexes though the suppression of Smad2 phosphorylation (45) and associa- tion with the protein C184M (46). Thus, the biological consequence of SKI/Smad inter- action in the cytoplasm appears to be similar to NLS mutations in Smad3, because this mutant remains in the cytoplasm and functions as dominant-negative inhibitor of TGF-E signaling (47). High Levels of SKI Repress RB Function: A Lesion Reciprocal to p16INK4a Loss RB functions as a potent repressor of genes required in the S phase of the cell cycle. The RB protein family associate via the pocket domain with mSin3-HDAC complexes containing exclusively class I HDACs. These proteins do not interact directly with RB family proteins; they use the protein RBP1 to target the pocket (48). c-Ski directly interacts with RB, forming complexes containing mSin3 and HDAC. Overexpression of SKI can partially represses RB activity (28). However, SKI in association with the Ski- interacting protein 1 (Skip1) can completely overcome the G1 arrest and flat cell phe- notype induced by RB (49). Thus, SKI can cause lesions in the RB pathway similar to deletions or mutations of the cyclin-dependent kinase inhibitor p16INK4a, an event associated with mouse and human melanoma formation and progression (reviewed in refs. 50–52). The SKI Protein Is Required for Methyl CpG-Mediated Transcriptional Repression MeCP2, a member of the familyof methyl-CpG-binding proteins, directly binds to the co-repressor mSin3, which also interacts with class I histone deacetylase, recruiting them to methyl-CpG regions to suppress transcription. c-Ski and SnoN are required for MeCP2-mediated transcriptional repression (29). Recent data demonstrated that MeCP2 associates with and facilitates histone methylation at Lys9 of histone H3, a key epige- netic modification involved in gene silencing (53). Increased cellular proliferation associated with high levels of SKI may be the result of enhanced methyl CpG gene silencing of growth inhibitory genes (29). It has been proposed that association of MeCP2 with DNA could serve to identify novel targets of epigenetic inactivation in human cancer (54). Determining whether SKI participates in such activity could help to identify global patterns of required for immortality and cellular transformation of melanoma and other tumors overexpressing SKI or snoN. Chapter 12 / Multiple Roles of SKI in Melanoma 217

Promyelocytic Leukemia Protein Complexes Promyelocytic leukemia protein (PML) is a critical component of the senescence response (55). Overexpression of PML results in senescence of human diploid fibro- blasts, which is characterized by a modest increase in p53 levels and activity, accumu- lation of hypophosphorylated RB and a reduced expression of E2F-dependent genes (56). PML interaction with the co-repressors c-Ski, N-CoR, mSin3A, and HDAC1 is required for transcriptional repression mediated by RB. However, high levels of SKI could alter the stoichiometry of the complexes because it interacts with PML and RB through the same amino acid sequence (Fig. 2A). Altered complex composition could, in turn, lead to altered PML function. If this hypothesis were confirmed, high levels of SKI could result in lesions similar to loss of PML protein expression, which is associated with tumor grade and progression in a variety of human tumors (57), and with decreased apoptosis (58). Because pml–/– mice and cells are protected from apoptosis triggered by a number of stimuli, including ionizing radiation, interferon, ceramide, Fas, and TNF, repression of PML function by SKI could lead to the well-known resistance of melanoma tumors to apoptosis signals (57).

THE ACTIVATOR FUNCTIONS OF SKI IN THE WNT-SIGNALING PATHWAY The Wnt-signaling pathway controls cell fate determination in neural crest cells, which give rise to melanocytes (59). Activation of Wnt signaling involves the inhibition of E-catenin degradation by the proteasome, which results in its nuclear accumulation and transcriptional activation of LEF/TCF target genes (reviewed in refs. 60 and 61). Aberrant activation of E-catenin signaling by either mutations in E-catenin (62) or by the elevation in wild-type E-catenin nuclear content (63) has been linked to melanoma progression. We have recently demonstrated that SKI interacts with FHL2 (64), a LIM-only protein that functions as a co-repressor or coactivator of E-catenin, depending on the promoter or cellular context (65,66) . LIM domains are characterized by the cysteine-rich consensus CX2CX16–23HX2CX2CX2CX16–21CX2–3(C/H/D) (67), and function as adapters and modifiers in protein interactions (68). The FHL2-interacting domain resides within amino acids 99–274 of the SKI molecule. This same domain is required for its association with N-CoR and the transcriptional repression activities of SKI (35). This suggests that distinct SKI complexes, having repressive or activating activities, may coexist in the cell, and, by targeting different promoters, diversify the functions of SKI. SKI Is an Activator of the MITF and Nr-CAM Genes It was recently demonstrated that E-catenin is a potent mediator of melanoma growth by mechanisms involving MITF (68). MITF can target the expression of Bcl2, and disruption of MITF in melanoma cells induces massive apoptosis (69). Thus, activation of the E-catenin pathway and MITF expression appears to be essential for growth and survival of melanoma cells. In addition, SKI and FHL2 are potent activators of Nr-CAM, a protein involved in melanoma proliferation, motility, and tumorigenicity (70). Overexpression of SKI appears to be sufficient for increasing both MITF and Nr-CAM 218 From Melanocytes to Melanoma

Fig. 3. The multiple functions of SKI in human melanoma. (A) SKI repressive complexes. SKI in association with Smad2 and Smad3 is a potent repressor of TGF-Esignaling in human mela- noma. (B) SKI in association with FHL2 is a potent activator of E-catenin. mRNA over the basal levels. Cutaneous melanomas display nuclear expression of E-catenin (63). However, in contrast with other human cancers, localization of E-catenin in melanomas does not correlate with mutations. We suggest that the nuclear accumu- lation of E-catenin may result from its interaction with active SKI/FHL2 complexes.

CONCLUSIONS AND PERSPECTIVES Is SKI a Likely Target for Melanoma Therapy? SKI is associated with a cascade of events known to increase cell cycle alterations and growth potential, invasion, and cell survival (Fig. 3). The extent of TGF-Eresistance often correlates with metastatic progression (71). However, TGF-E-mediated growth inhibition in melanomas can be restored by downregulation of SKI protein levels (5). But will that help in vivo? Current evidence suggests that that might be the case. Melanoma Chapter 12 / Multiple Roles of SKI in Melanoma 219 tumors are known to secrete large amounts of TGF-Eto the microenvironment (72). In the absence of SKI, TGF-Ecould limit tumor growth by an autocrine mechanism, because no measurable defects at its receptors have been found in melanomas (73). In addition, low levels of SKI could curtail the oncogenic activities of E-catenin by restricting its nuclear localization. In conclusion, SKI may be a valuable target in the treatment of human malignant melanoma, because it regulates two major pathways involved in cancer progression: the Wnt/E-catenin- and the TGF-E-signaling pathways.

ACKNOWLEDGMENT Work mentioned in this review that was performed in the E.E.M. laboratory was supported by an RO-1 grant from the National Cancer Institute.

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