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J Cutan Pathol 2008: 35 (Suppl. 2): 11–15doi: 10.1111/j.1600-0560.2008.01119.xBlackwell Munksgaard. Printed in Singapore
Copyright # Blackwell Munksgaard 2008
Journal of
Cutaneous Pathology
Deciphering the melanoma interactome
Late-stage malignant melanoma continues to pose a significanttherapeutic challenge, despite numerous recent advances in ourunderstanding of the molecular and genetic pathways leading to tumordevelopment and progression. Dr Scott McNutt was among the firstresearchers to employ the cutting edge technology, electronmicroscopy, to the study of cutaneous neoplasms. This work providedthe foundation for more recent studies using molecular pathology toexamine disease in the context of aberrant interactions betweencellular signaling pathways in the so-called �interactome’.Understanding the functional interrelationships of aberrant signalingnetworks in melanoma may lead to the development of novel therapiesfor advanced disease. This mini review will focus on few of the proteinsthat likely significantly contribute to the melanoma diseaseinteractome.
Reed JA. Deciphering the melanoma interactome.J Cutan Pathol 2008; 35 (Suppl. 2): 11–15. # Blackwell Munksgaard2008.
Jon A. Reed1,2
Section of Dermatopathology,1Department of Pathology, and2Department of Dermatology, Baylor Collegeof Medicine, Houston, TX, USA
Conflicts of interest: none declared.
Jon A. Reed, MD, Department of Pathology, BaylorCollege of Medicine, One Baylor Plaza, Room 286A,MS 315, Houston, TX 77030, USATel: 713 798 1478Fax: 713 798 5838e-mail: [email protected]
Accepted for publication June 17, 2008
Cutaneous melanoma continues to be a public healthcare challenge for much of the world. The AmericanCancer Society estimates that there will be 62,480new cases of melanoma and 8420 melanoma-relateddeaths in the USA in 2008 (http://www.cancer.org/downloads/STT/2008CAFFfinalsecured.pdf).These predictions place melanoma as the sixth mostcommon form of cancer accounting for approxi-mately 4–5% of all newly diagnosed malignancies inboth sexes, excluding the seldom lethal cutaneoussquamous cell carcinoma, cutaneous basal cellcarcinoma and in situ carcinomas of other organsexcluding urinary bladder. Deaths caused by mela-noma also continue to increase in number annually,despite better public awareness and continuingimprovements in efforts directed toward earlierdiagnosis and treatment. This troubling increase isdirectly attributable to the lack of predictive markersand effective therapies for advanced stage disease. Assuch, considerable emphasis has been placed onidentifying specific biochemical, molecular, geneticand immunological abnormalities that could beexploited in the development of novel therapies forpatients with advanced disease.Much attention has been given recently to aberra-
tions of key cellular signal transduction pathwaysidentified in a majority of sporadic melanomas.1–6
Among these, greatest focus has been placed upon
mutations affecting the mitogen-activated protein(MAP) kinase pathway. Most notably, a specificmutation of BRAF (BRAFV600E) results in a singleamino acid substitution that putatively leads toconstitutive activation of this protein kinase.7,8 Thesubsequent activation of protein kinases furtherdownstream in the MAP kinase pathway is believedto result in a dysregulated cell cycle that contributesdirectly to the pathogenesis of melanoma.9 This workhas in turn led to the development and use of severalchemical MAP kinase inhibitors in vitro10 that to datehave producedmixed results in early clinical trials.3,11
It is important to note, however, that the identicalactivating mutation of BRAF occurs in the melano-cytes of an even greater percentage of ordinary banalacquiredmelanocytic nevi.12Clearly, thismutation byitself is insufficient to drive melanomagenesis. Func-tional analysis reveals that theMAP kinase pathway isextremely complex and tied to several other signalingpathways with which normal cross talk establishesa precise balance of cellular proliferation, differenti-ation, senescence and apoptosis.13,14 Emergent fromthese and similar observations in other types of cancerhas been the continuing evolution of functionalgenomics and the development of a new field inmolecular biology focused on intracellular proteintrafficking, the interactions between proteins indifferent signaling pathways and the interactions of
11
proteins with nucleic acids. This so-called �interac-tome’ is a functional network of interacting moleculesthat in different temporal and spatial arrangementscan have different biological effects.15–18 Work hasonly recently begun on the immense task of decipher-ing the complex global interactome of cancercells.16,18 This brief mini review will focus on a fewspecific examples in which changes within theinteractome appear to play an important role in thepathogenesis and progression ofmalignantmelanoma.
Altered trafficking of proteins in melanoma
Onemechanism by which a protein’s functionmay bealtered is for it to have anabnormal locationwithin thecell. In its new atypical location, loss of function mayresult from the absence of a normal binding partneror, in the case of an enzyme, absence of a normalsubstrate. Function may also be altered by a protein’sassociation with another protein with which it doesnot normally interact or with which it would normallyinteract only in another subcellular compartment.One such example in melanoma occurs with theaberrant trafficking of the oncogenic protein SKI.19
SKI
Human SKI is a 95 kDa, 728 amino acid nuclearprotein that is normally expressed at very low levels inmelanocytes.19 SKI does not bind DNA directly butacts as either a transcriptional co-activator or co-repressor depending on its association with otherfactors within a transcriptional complex. During theearliest stages of melanoma progression, SKI proteinis upregulated probably by transcriptional or post-transcriptional events20 and found in a strictly nucleardistribution (Fig. 1A).21,22 In melanoma cells, SKIcan suppress the downstream growth inhibitoryeffects of transforming growth factor-beta by associ-ating with Smad family transcription factors (Smad2,Smad3 and Smad4) in the nucleus.22,23 This resultsin the repression of downstream growth inhibitoryactivities of genes normally activated by Smad in-cluding the cell cycle inhibitor p21Waf-1. In addition,SKI candirectly suppress the growth inhibitory effectsof the retinoblastoma protein producing a phenotypethat mimics that associated with loss of expression ofthe cyclin-dependent kinase inhibitor p16INK4a.24
Intracellular trafficking of SKI becomes moreimportant in the later stages of melanoma pro-gression. More advanced primary invasive melano-mas and metastatic melanomas often display analtered cellular location of SKI.21 In these lesions,a significant amount of SKI can be found in thecytoplasmic fraction of tumor cells (Fig. 1B). In thecytoplasm, SKI also binds to Smad family members,
thereby inhibiting their translocation to the nucleus toaffect downstream signaling. As such, SKI can act asa molecular �sink’ for Smad proteins.19 By over-whelming the pool of available Smad familymembersavailable for normal transcriptional activation in thenucleus, the stoichiometric excess of SKI protein isfree to act as co-activator or co-repressor in avariety ofother transcription factor complexes that would notform under normal circumstances.
Fig. 1. Expression of the oncogenic protein Ski in cutaneous
melanoma. A) Intraepidermal melanoma (melanoma in situ)
showing exclusively nuclear expression of Ski (arrows). B) Metastatic
melanoma displaying predominantly cytoplasmic expression of Ski.
Both panels, immunohistochemistry as previously described.21
Original magnification 3200.
Reed
12
One example of alternative signaling caused byexcess SKI in melanoma results in the aberrantactivation of the Wnt/beta-catenin pathway bydisplacing co-repressors normally found in the beta-catenin transcriptional complex.25 Upregulation ofbeta-catenin and several of its downstream transcrip-tional targets, includingmarkers of tumor progressionsuch as microphthalmia transcription factor andneural cell adhesion molecule (Nr-CAM), also hasbeen shown in a significant percentage of melanomasin vivo.19,26,27 Thus, it appears that the subcellulardistribution and expression level of SKI can havea direct impact on its choice of binding partners andtheir resultant biological effects in the cell.
PKA
Another example of altered protein trafficking inmelanoma occurs among the various isoforms of thecAMP-dependent protein kinase/protein kinase A(PKA). PKA exists as a heterotetrameric holoen-zyme having two monomeric catalytic subunits thatare activated and released upon conformationalchanges induced by the binding of cAMP to theregulatory subunit dimmer.28 Class-specific iso-forms of PKA regulatory subunits (RI-alpha, RI-beta, RII-alpha and RII-beta) are anchored todifferent subcellular locations in differing ratioswhere their signaling is involved in diverse biologicalfunctions ranging from proliferation, differentiation-related processes such as melanin synthesis and insenescence.28–30
PKA regulatory subunit isoform switching hasbeen described in cancer.31 In melanoma, it wasshown recently that the PKA RI-alpha isoform ratiotypically associated with cellular proliferation iselevated compared with that of normal melanocytesdisplaying a higher RII isoform ratio.32 Altered PKAsignaling has also been reported in melanomasharboring activating RAS mutations, thus revealingan aberrant cross talk pathway linked to MAP kinasesignaling.13
Furthermore, the differentiation-associated PKARII isoforms predominate in a complex with specificanchoring proteins in the nucleus and nuclearmembrane33 where they are tethered to the regula-tion of chromatin remodeling and G1/S cyclinactivities.34,35 The activity of one of the G1/S cyclins,Cyclin E, is consistently upregulated in melanoma.36
As such, PKA RII isoform expression seen in thenuclear membrane of non-proliferative melanocyticnevus cells in vivo are more typically localized to thecytoplasm ofmelanoma cells in vivo and in vitro (Fig. 2).As such, upregulation and isoform switching seem toplay a role in the subcellular distribution, traffickingand activity of PKA in melanoma.
Epigenetic silencing of protein expressionin melanoma
Functional genomic analysis of melanoma cells hasyielded much information regarding loss or gain ofa cellular function caused bymutations, deletions andamplifications of genes. These genomic alterationshave obvious implications for trafficking and molecular
Fig. 2. Expression of PKA RII-alpha in nevi and in melanoma. A)
Nuclear membrane expression of PKA RII-alpha in intradermal
melanocytic nevus cells (arrows). B) Predominantly cytoplasmic
localization in a primary invasive melanoma. Both panels,
immunohistochemistry as described previously.46
Original magnifi-
cation 3200. C) Western blot as described previously47
using
chemiluminescence detection of PKA RII-alpha subunits in nuclear
(N) and cytoplasmic (C) fractions of a melanoma cell line (SK-Mel
93.3). Note absence of nuclear expression. Pan-actin, lane load
control. PKA, protein kinase A.
Deciphering the melanoma interactome
13
interactions involving the encoded proteins. Otheralterations of the melanoma interactome may involveprotein-nucleic acid interactions in the absence ofsuch gene amplification, loss or mutation. Theemerging field of epigenetics (outside genetics) isfocused on the so-called �epigenome’ of cancercells37,38 and is inexorably tied to the interactome.Examples of epigenetic regulation involve chemicalmodifications (acetylation, methylation, sumoylation,ubiquitination or phosphorylation) of nuclear histoneproteins5,39,40 or methylation of CpG islands in thepromoter sequences of genes.39,41 These modifica-tions are carried out by histone acetyl transferases(HATs), histone deacetylases (HDACs), histonemethyl transferases, histone kinases, histone ubiquitinligases and DNA methyl transferases.39 Globalepigenetic modifications of DNA are often associatedwith stable heterochromatin structure and themaintenance of cellular replicative senescence.29,42,43
Similar epigenetic modifications can occur at thelevel of individual genes or small genomic regions.This can result in the silencing of a specific protein’sexpression without altering the DNA sequence.Indeed, the loss of expression of p16INK4A commonlyseen in melanoma44 may be because of loss ofheterozygosity, homozygous deletion or by promoterhypermethylation.45
The oncogenic protein SKI (see above) playsa significant role in epigenetic regulation of genesrelated to melanoma tumor progression through itsrole as a nuclear transcriptional co-repressor or co-activator. Many of SKI’s effects are mediated by therecruitment of HATs orHDACs to the transcriptionalcomplex, thereby activating or silencing transcriptionat that locus. As such, epigenetic mechanisms couldsilence a protein’s expression directly through pro-moter hypermethylation (e.g. p16INK4a), histone de-acetylation (e.g. SKI-induced repression of p21Waf-1),or indirectly, by similarly silencing the expression ofinteracting proteins needed as substrates, co-activatorsor chaperones required for normal function ortrafficking in the interactome.
Future challenges
These are just a few examples in which thefunction(s) of a protein are altered by trafficking,alternative associations with other molecules in theinteractome or epigenetic control. None of theseaforementioned anomalies are driven directly bygenetic perturbations and as such are not discover-able by commonly employed genomic sequencingtechniques. One of the biggest future challenges willbe to examine the extent of aberrant molecularinteractions (such as those described for SKI andPKA in this review) in the context of the global
landscape of the interactome of melanoma cells. Insilico experiments are being designed that willfacilitate the identification of families or �nodes’ ofproteins likely to interact with each other based upontheir amino acid sequence or known structuralproperties. A better understanding of this interac-tome in melanocytes and in melanoma cells wouldprovide specific information needed for the futuredevelopment of agents specifically designed toreconstitute or bypass dysfunctional interactivenodes and perhaps even be exploited for thepersonalized therapy of individual patients.
Acknowledgements
Cited work performed by the author was funded in part by a grant
from the Public Health Service, National Institutes of Health and
National Cancer Institute CA97872. The author also thanks Dr
Estela Medrano for critical review of this manuscript.
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