The scientific program of the 11th Annual PASPCR meeting opened with the session on melanocortins/MSH/Agouti protein. The keynote speaker was Greg Barsh (Stanford Univ.) who presented on the 'genetics and genomics of pigment type-switching'. In his presentation, Greg described the roles of alpha-MSH and Agouti signaling protein as antagonists that switch to eumelanin and pheomelanin synthesis, respectively. He also discussed the roles of Attractin and Mahoganin in regulating the signaling of Agouti via the Mc1r. Greg presented new data on coat color mutations in dogs. His new results ascribed dominant black coat color to a dominant allele of Agouti, variegation of black and yellow coat color to an allele of Mc1r, and the dark coat color of the German Shepherd pedigree to a loss-of-function of Agouti. The highlight of the presentation was that the Barsh laboratory might have discovered a new pigmentary gene that might be responsible for the dominant black color. By crossing Labrador and Greyhound, Greg and his group found that the dominant black mapped to a region of dog chromosome 16 that has not been known to regulate pigmentation. Future identification of this putative gene will extend the ever-growing list of pigmentary genes, and will shed further light on the complexity of regulation of eumelanin and pheomelanin synthesis in mammals.
Zalfa Abdel-Malek (Univ. of Cincinnati) presented on the impact of loss-of function mutations in the MC1R gene on the response of human melanocytes to UV radiation. Three human melanocyte cultures that were refractory to alpha-MSH exhibited a marked increase in apoptosis after UV exposure, compared to melanocytes with similar melanin content and functional MC1R. These three cultures were either homozygous or compound heterozygous for allelic variants of the MC1R that are known to be associated with red hair phenotype, poor tanning ability and increased risk for melanoma. Recently, her laboratory identified two additional cultures with loss-of-function MC1R and aberrant response to UV. New results obtained in the Abdel-Malek laboratory revealed that alpha-MSH promotes the survival of human melanocytes following UV exposure by inhibiting apoptosis. The survival effect of alpha-MSH, however, was absent in melanocytes with loss-of-function MC1R. Increased apoptosis of the melanocytes is interpreted as an indication of inefficient repair of UV-induced DNA damage, which results in apoptosis or mutagenesis. The possibility that loss-of-function mutations in MC1R increase mutagenesis might explain why these mutations increase the risk for melanoma.
Francois Rouzaud (NIH) provided evidence for a new variant of MC1R, namely MC1R 350, which differs from the known MC1R 317 by an additional 33 amino acids in the carboxy terminal domain of the protein. This 350 variant results from alternative splicing of the open reading frame that leads to a longer MC1R mRNA. Expression of this variant by a subset of various human melanocyte cultures did not correlate with their pigmentation. Included in the additional 33 amino acids are 5 cysteine residues that are thought to confer stability to the receptor by anchoring it more firmly in the membrane. Results obtained from 125 I-labeled -MSH and autoradiography suggested thatMC1R, as other G protein coupled receptors, might exist as a dimer. The MC1R 350 variant adds another level of complexity of the MC1R and its function as a regulator of human pigmentation.
Catherine Van Raamsdonk (Stanford Univ.) reported on the role of Gq signaling in the regulation of dermal melanocytes. By investigating specific mouse mutants identified by dark skin (DSK), two dominant mutants, DSK1 and DSK 7 were found to cause an abnormal expansion of melanocytes in the dermis during early embryonic development. DSK1 and DSK2 resulted in missense alterations in the G alpha q genes Gnaq and Gna11, respectively, and interacted in an additive manner. Mice that are null for the endothelin B receptor (that signals through Gq) and DSK7 mutant totally lacked pigment. Further investigation of the loss and gain of function mutations in G alpha q genes revealed that signaling through Gq determines the extent of dermal pigmentation. The effects of DSK7 on eumelanin synthesis were independent of MC1R signaling. The significance of this study is that it highlights a new signaling pathway through Gq that regulates dermal pigment cells and possibly contributes to known pigmentary abnormalities in humans, such as blue nevus or Mongolian spot.
Dan Hebert (U Mass) presented work done in his laboratory and in collaboration with Ruth Halaban's lab regarding the early maturation and processing of tyrosinase. Tyrosinase undergoes extensive glycosylation requiring an extended sojourn in the early secretory pathway, involving the participation of chaperones such as calnexin and Erp57. Disulphide bond formation and oligomerization are also involved. Mutations in tyrosinase and perhaps Tyrp-1 can both interfere with proper Tyr folding. John Hammer (NIH) reviewed the interactions of myosin Va with rab 27a and melanophilin. Myosin Va?s interaction with melanosomes depends upon the tail sequences in the melanocytic splice form. Clearly rabGTPases and their interacting proteins play key role in the movement of vesicular cargo by molecular motors.
Dick Swank (Roswell Park) reviewed the 13 mouse loci associated with Hermansky-Pudlak syndrome (HPS)-like phenotypes. He and his collaborators have been able to reproduce the storage-disorder-like lung disease typically lacking in any of the mouse HPS homozygotes by creating double mutants, thus offering a tractable small animal model to understand the pathogenesis of the oft-fatal lung involvement and a means to test potential therapies. J.C. Valencia (Hearing lab, NIH) presented data on the complex processing of gp100. gp100 appears to be transported from the Golgi via the AP-1 adaptor complex, and is extremely highly represented in coated vesicle fractions from a human melanoma cell line MNT-1.
Reena Rupani (Yale) spoke on metastatic human melanomas, spontaneous nonmetastatic mouse melanoma and a constructed mouse melanoma x macrophage hybrid. The human melanoma and the hybrid each exhibited coarse melanin granules that have previously been observed in a very high percentage of human melanomas in tissue sections. These organelles contained the endolysosomal marker CD63, the melanosomal matrix protein gp100, and stained for the glycan recognized by the lectin LPHA. EM analysis is consistent with an autophagosomal origin for these organelles.
H. Watabe (NIH) presented data on aberrant tyrosinase processing in amelanotic SKMel28 cells. Misfolded Tyr is trapped in the ER and its levels can be increased via proteasomal inhibitors consistent with them being involved in degradation of the misfolded protein. Treatment of the cells with vacuolar proton pump inhibitors such as bafilomycin results in increased expression of Tyr in stage II melanosomes and Tyr exit from the ER, but the effects may be complex and may actually inhibit exit from the Golgi.
D. T. Spaulding (Univ Oklahoma) reviewed differences between Caucasian and Black melanocytes. Tyrosinase levels (8-10 fold higher in Black melanocytes) exhibit similar levels of Tyr protein. However by use of acridine orange, fluorescence in Black melanocytes is much lower than in Caucasian, consistent with a much higher internal pH. Na/H antiporters may well be involved, as several are expressed in melanocytes, and amiloride suppresses pigmentation by Black melanocytes. One particular exchanger, NHE3, may be expressed at higher levels in Black melanocytes, suggesting that it plays a role in alkalinizing the melanosome.
Dick King (Univ. Minnesota) reviewed the question of tyrosinase gene mutations in patients presenting with congenital white hair. While in general those born with white hair are more likely to have OCA1, mutations in OCA2 are also a possibility and need to be looked for if a molecular diagnosis is sought.
Finally, M. Endo (Jimbow lab, Sapporo) reported upon investigations into the effects of Tyrp-1 on Tyr mediated cytotoxicity. While coexpression of Tyrp1 with Tyr can indeed increase tyrosinase activity and melanin production in both melanocytic and non-melanocytic cells, Tyrp1 can suppress the cytotoxicity of ?unprotected? Tyr expression. This toxicity is apparently non-apoptotic in mechanism. Mutations in the domains of Tyrp1 homologous to Tyr?s copper binding domains has more dramatic effects on Tyrp1?s ability to stimulate pigmentation with little effect on its cytotoxicity-protective abilities. Conversely, missense Tyrp1 mutations had greater effects on cytotoxicity protection than on pigmentation. Deletions affected both. These data suggest that different sites on Tyrp1 may be responsible for the observed pigment enhancing vs. cytotoxicity protecting effects.
Lynn Margulis (U Mass) discussed symbiogenesis, a cellular evolutionary process of acquisition of genomes by merger, which led to the creation of ?new features? like cellular organelles. She claimed that the accumulation of mutations alone cannot lead to the generation of a new species, and is more likely to result in a sick or dead organism, while genome acquisitions could clearly result in the creation of new species. The talk was centered on the creation of the first eukaryotic, nucleated cell, from a merger between an eubacterium and an archaebacterium. The origin of the nucleus and its cytoskeletal system (nucleus + nuclear connector + motile structures like kinetosome and undulipodia) were discussed.
Natalie Ahn (Univ. of Colorado-Boulder) provided an overview on functional proteomics, and discussed the pros and cons of two technical approaches, the 2D-PAGE/MS and the multidimensional LC/MS/MS methods. One example demonstrated how the Rho signaling pathway was followed with 2D gels, identifying posttranslational modifications involved in Rho-induced migrations. Another example described the search for melanoma markers for disease progression. Technical issues in peptide sequence assignment during data analysis were discussed. An improvement in the validation of peptide assignment was achieved through the integration of multiple search algorithms.
The first Aaron B Lerner Honorary Lecture, given by Ruth Halaban, (Yale) was titled ?Tyrosinase: not only black and white?. Ruth had reviewed the many activities of tyrosinase: catalysis of the production of melanin precursors, proton/pH sensing, self regulatory quality control, coupling of aromatic compounds, and possibly crosslinking. The role of DOPA in the maturation of tyrosinase, resulting in proper folding and movement from the ER to the Golgi was emphasized. Inactive or misfolded tyrosinase is retained in the ER of albino melanocytes and melanoma cells, resulting in proteolytic degradation and no pigment production. Inactive tyrosinase in albino cells was detected using Tyramide cross-linking. An increase in culture media pH could shift tyrosinase of melanoma cells to the Golgi, resulting in pigment production.
VJ Setaluri (Wake Forest) described the role of PI3 kinase and AKT2 signaling in melanosome biogenesis. Inhibition of PI3K blocked the sorting of newly synthesized Tyrp-1 into melanosomes. A yeast two hybrid screen identified a PDZ-domain protein, GIPC, which temporarily interacts with the newly synthesized Tyrp-1. GIPC binds APPL, which is an AKT2-binding protein, and a complex of these three proteins was identified. AKT2 phosphorylation was found stronger in non-pigmented melanocytes.
Ana Luisa Kadekaro (Univ. Cincinnati) discussed endothelin1 and alpha MSH as melanocyte survival factors following UV irradiation. An increase in viable cells, and a reduction in apoptotic cells, was demonstrated following treatment with either ET1 or alpha MSH. Forskolin could mimic the MSH effect, suggesting the involvement of cAMP in the survival mechanism. ET-R antagonists and MC1-R mutants abrogated the protective effects of their respective ligands, demonstrating that the UV-survival signaling involves these receptors. Melanin synthesis per se is not involved in the protective effect, as was documented by rescuing tyrosinase-negative, albino melanocytes, with these agents. The AKT/PKB pathway was shown to be involved in the process.
Two lectures provided an overview on recent advances in the epidemiology of melanoma and novel drugs that target simultaneously apoptotic and survival pathways. Dr. Marianne Berwick (Memorial Sloan-Kettering Cancer Center) discussed new epidemiological data showing that the biological behavior of melanoma differs according to whether it developed under conditions of high or low exposure to UV irradiation. Intriguingly, prognosis of melanoma is better in high incidence populations than low incidence populations and improves in a population as incidence increases. This behavior likely reflects the fact that populations in areas with higher levels of UV have increased awareness of melanoma risk and so detect melanoma earlier, leading to better prognosis.
Dr. Maria Soengas (Univ. Michigan) presented the results of a study using the proteasome inhibitor Bortezomib (VelcadeTM, previously known as PS-341) as a putative candidate to bypass melanoma chemoresistance. Bortezomib induced massive cell death in 24 aggressive melanoma cell lines without compromising the viability of normal melanocytes. Biochemical studies indicated that Bortezomib activates both non-apoptotic and apoptotic programs in melanoma cells. Thus, casp-8, casp9, -3 and -7 were activated even in cells with low Apaf-1 and high survivin, Bcl-xL, Bcl-2, Mcl-1, FLIP or XIAP. Moreover, the cytotoxic effect of Bortezomib was also independent on the functional status of p53, p19, p16 and the Ras/BRAF/ERK pathway. Fluorescence-based tumor imaging techniques revealed a significant effect of Bortezomib in vivo (mouse xenografts), particularly in controlling melanoma metastasis. This effect was significantly enhanced by small molecule inhibitors of Bcl-xL.
The morning symposium then continued with three talks selected from the submitted abstracts. Dr. V Alexeev (Jefferson Medical College) discussed the role of activating mutations in the c-kit gene in the survival of mouse melanocytes and melanoma cells. Expression of a c-Kit mutant receptor did not induce features of transformation of the mouse melanocyte cell line Melan-C, either in vitro or in vivo, or increased apoptosis. Conversely, a constitutively active c-Kit receptor induced apoptosis in melanoma. These results highlight the divergent biology or normal and tumorigenic melanocytes.
Dr. Dorothy Bennett (St. George's Hospital Medical School, England) discussed the role of the Inkk4a-Arf locus in the generation of immortal mutant mouse melanocyte cell lines. Melanocytes isolated from mice with one or two Ink4a-Arf copies deleted fail to senescence in culture and form immortal cell lines without going into crisis. Dr. Bennett has been able to generate 15 immortal mutant mouse melanocyte cell lines by crossing C57BL6/J mutant mice, carrying mutations for Hermandsky-Pudlak, MITF and others, with Ink4a-ARF null mice, and isolation and culture of epidermal melanocytes. Availability of such cell lines will be a useful tool for the biological characterization and in-depth molecular analysis of the respective phenotypes.
Finally, Dr. K. Fitch (Stanford Univ.) presented results from a large-scale ENU mouse mutagenesis project. One of the mutants analyzed (Dsk5) was an hypermorphic mutation of the Egfr gene. These animals display excess epidermal pigmentation in the footpads that progresses with age. Pigmentation is associated with hyperkeratosis and melanocytosis. Dr. Fitch postulated that the mutation likely affects keratinocytes and secondarily the melanocytes. The Dsk5 mouse model could be used to better understand the pathological link between keratinocyte activation and melanocyte proliferation.
One of the rewarding aspects of pigment cell research is the breadth and depth of the resources that are available to us. Session # 5, Genetic and Comparative Pigment Cell Biology, sampled a range of animal systems, from human to insect, with special attention to the mouse, and including chickens and chameleons. Unfortunately mentioned only in passing were the fish models that carry so much potential for pigment cell biologists.
Seth Orlow (NYU School of Medicine) reviewed two 'Multipass Membrane Proteins that Control Pigmentation'. The p locus of mice encodes a 12 TM protein whose absence results in tyrosinase positive albinism, with much reduced pigmentation of eumelanic pigmentation of the coat and eyes, and also causes albinism in man. It seems the protein encoded at the p locus controls trafficking of tyrosinase, but it is not clear how this is accomplished. Dr. Orlow reported studies in yeast that suggest the protein may facilitate intracellular glutathione transport that might play a role in folding of cysteine-rich tyrosinase. The underwhite locus in the mouse encodes MATP, another 12 TM membrane. Mutation at this locus results in pale pigmentation of the hair coat of the mouse that seems superficially similar to that caused by pinkeye, but does not strongly affect eye pigmentation. The effect of the mutation upon melanosome structure and melanization in mouse hair bulb melanocytes is extreme, and mutation in the human can also cause albinism.
This MATP mutation is known also in Medaka fish where it has been referred to as b, and also greatly reduces melanization on the body but not the eyes, and in humans as the AIM-1 locus, implicated in OCA4 albinism.
Yasu Tomita (Hokkaido University, Japan) reported the identification of several Japanese patients exhibiting 'An Autosomal Dominant Oculocutaneous Albinism Caused by a Mutation in OCA4 Gene, AIM-1'. He compared phenotypes and genotypes. Unlike other types of albinism, this exhibits a dominant negative phenotype in the patients examined.
Marjan Huising (NIH) reported 'New Findings in Hermansky-Pudlak Syndrome', a disorder or biogenesis of lysosomes and related organelles, of course including melanocytes. She reviewed the six genes that are now identified to cause HPS in humans (HPS1 to HPS6) and described the protein complexes of which they are a part. These include the adaptor complex AP3 and six different protein complexes known as BLOCs (biogenesis of lysosome related complexes).
Emelia Costin (Texas A&M) spoke on the 'Characterization of Two new Mouse Melanocyte Lines Carrying the Slaty and Slaty Light Mutations'. The slaty locus encodes DOPAchrome tautomerase (Dct), an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin and the rapid metabolism of its toxic intermediates. In both of these cell lines, Dct activity was reduced compared with wild type cell lines. Confocal microscopy is being used to evaluate intracellular trafficking.
There were two reports of the effect of 4-TBP on melanocytes.
Prashiela Manga (Univ. Cincinnati) discussed 'A Role of Tyrp1 in Determining Melanocyte Sensitivity to 4-(tert)-butypylphenol', a chemical that can cause chemical leukoderman and vitiligo. Their research had previously shown that exposure to 4-TBP in culture is preferentially cytotoxic to melanocytes in culture and results in dose dependent initiation of apoptosis and that Tyrp1 expression increased the sensitivity of transfected cells to 4-TBP. This study compared melanocytes obtained from normal individuals with those from an individual with OCA3 (mutant Tyrp1) and concluded that upregulation of Tyrp1 expression increases sensitivity of melanocytes to 4TPB. Tyrp1 may utilize increased amounts of 4TBP as substrate and produce toxic intermediates at concentrations that eventually cause melanocyte death.
Caroline Le Poole (Loyola University) concentrated on the relationship between stress proteins and apoptosis in 'HSP70 and the Response of Melanocytes to 4-TBP', using normal and immortalized melanocytes and fibroblasts. Overexpression of HSP70 enhances MHc Class I expression and sensitizes melanocytes to T-cell-mediated cytotoxicity. Exposure to 4-TPB sensitized melanocyte killing by HLA-matched, melanocyte reactive T cells. Thus it appears that exposure to 4-TBP elevates HSP70 expression by melanocytes which can elicit enhanced cellular immune responses to melanocytes, thus contributing to vitiligo.
Gisela Erf (Univ. Arkansas) used Smyth Chickens to study vitiligo at the organismal level, Evaluating environmental impact on 'Circulating Melanocyte-Specific Auto-Antibodies and Feather Infiltrating Lymphocytes in young Smyth Line Chickens prior to Visible onset of Vitiligo'. Turkey herpesvirus (HVT) administration at hatch is associated with a high incidence of vitiligo in these chickens. Groups of chicks were seperately innoculated with HVT or with control. Antibodies to the HVT were identified in feathers several weeks before the onset of vitiligo.
Beautiful pictures of chameleons accompanied Randy Morrison's (McDaniel College) report, 'An Analysis of Skin Color in Panther Chameleons from Different Regions of Madagascar using Reflectance Spectrophotometry'. The colors are interesting both because of their creation upon the body of the animal, studied by wave-length spectra obtained using reflectance spectrophotometry, and because of the evolutionary implications of geographical distribution of the color forms in the wild. Data were analyzed in terms of hue, brightness, saturation. This approach offers the potential to correlate the spectral (noninvasive) data with ultrastructure of the multiple chromatophores that make up a complex and highly specialized phenotype.
Insects are one of the earliest models of pigment formation. Manickam Sugumaran, (U Mass) in 'Molecular Interactions of Insect Phenol Oxidases', described the significance of phenol oxidase to both the sclerotization pathway, or hardening of the exoskeleton, and the melanogenic pathway. Melanin biosynthesis is associated with three physiologically important processes in the insect: immune response, wound healing, and pigment formation. Thus the phenol oxidases in insects are required for a complexity of processes such that their absence is life threatening. To perform these multiple tasks, phenol oxidase participates in a number of protein complexes, three of which were discussed.
The Melanoma, Genetics, Animal Models and Angiogenesis Session was held on Saturday afternoon at the PASPCR meeting. The turnout was excellent and there was ample discussion following each of the talks, which are summarized as follows:
Boris Bastian (Univ. of California-San Francisco) began the session discussing the use of array comparative genomic hybridization (CGH) to examine gene expression in nevi (with limited proliferation) and melanoma (with unlimited proliferation). His studies were aimed to characterize the genetic differences between those 2 populations, and they performed their analyses on microdissected paraffin-embedded tissue sections. In brief, they found that melanomas had many differently expressed genes while the nevi had relatively few. Cyclin D1 was a gene commonly up-regulated in the melanomas, and they found that amplifications of different genes varied by their type and location on the body, e.g. acral, mucosal and non-acral melanomas. Mutations in bRAF also varied significantly by melanoma type, but there was not a correlation in that expression and disease outcome/ survival. They confirmed the results obtained with the array CGH by fluorescence in situ hybridization (FISH). Interestingly, they found aberrant melanocytes often 1 – 2 cm outside of the malignant area, and these are considered precursor cells of melanoma in situ, herein termed field cells. Bastian provided a mechanistic basis for these findings in relation to genetic hits from the environment (e.g. UV) and stated that wider margins might be necessary during surgical excision of melanoma outside of the standard excision margins.
Lynda Chin (Dana-Farber Cancer Center) then followed with a lecture about the genetically engineered mouse models for melanoma, and discussed the standard melanoma-signature mutations in CDK4, INK4a, bARF and Myc. They developed a model based on transgenic mice generated via the Ras gene linked to the tyrosinase promoter to obtain specific expression in melanocytes, and then they activated p16/INK4a or p19/ARF in knockout mice to induce melanoma-genesis. The question arose as to whether those tumors harbored secondary mutations, and they used the array CGH technique to assess that, and found that the answer was yes. That also found similar amplification regions as were found in the human melanoma studies discussed earlier by Bastian. The induced mouse tumors had similar histopathologic properties and characteristics to human melanomas. UVB cooperates with ARF but not with p16 in melanoma-genesis, i.e. UVB targets the Rb pathway for melanoma promotion. When Ras was put on a tetracycline-inducible promoter, they found that continued expression of Ras was required for tumor growth, which reflects in part angiogenic factors secreted by these cells that is required for the continued development of the malignant cell population.
Juan Recio (George Washington University) then presented another mouse model for melanomas that was developed in Glenn Merlino's laboratory. In that model, hepatocyte growth factor (HGF)-transgenic mice were produced and then were exposed to UVB to induce tumorigenesis. Neonatal UVB exposure was strictly required to generate melanomas, which is consistent with observations of the role of UV on melanoma-genesis in humans (i.e. dependency on UV doses in early life). The question was posed as to what genetic mutations contributed to the development of malignant melanoma. They found that INK4a/ARF gene expression is lost in the mouse melanomas produced in this system, and this loss correlated well with the UV-induction of the tumors. Array CGH was used to show the loss of INK4a in mouse melanomas. These tumor lines are proving useful to dissect the cMet-signal pathway, and Recio described a pathway in which CD44 interacts with cMet to stimulate ERK1/2 to then stimulate Egr1 and proliferation. HGF activates ATF2 and CRE transcription factors. This model will be quite useful to further characterize the signaling pathways involved and also to elucidate mechanisms involved in UV-induced melanoma-genesis.
Y.E. Marin (Rutgers University) then presented the first of 2 papers from Suzie Chen's group on the role of Grm1 in mouse melanoma cells. She discussed the involvement of NFkappaB (a transcription factor frequently deregulated in many types of cancers) in Grm1 gene expression. Ectopic expression of Grm1 is sufficient for melanoma-genesis in mice (Grm1 is the receptor for glutamate). An important question is what are the cellular signaling events involved in this process. They created a transgenic albino mouse (termed TG-3) which then gave rise to amelanotic melanomas. NFkappaB is an excellent candidate, and they found that it was constitutively active in this system. It is normally regulated by IKK phosphorylation.
Suzie Chen (Rutgers University) then continued this theme, explaining that Grm1 is a G protein coupled receptor, and that its altered function results in melanomas in mice. They linked the Dct promoter to the Grm1 cDNA to allow for specific expression in melanocytes and generated a transgenic animal. They found that it worked through the phosphorylation of ERK1/2 and their next goals are to establish the intermediate links between Grm1 and ERK1/2 that are important to this process.
Michael Detmar (Mass General Hospital) then presented another overview lecture on angiogenesis and lymphangiogenesis. Those 2 processes are induced by hypoxia and by hyperplasia of keratinocytes which stimulates release of VEGF (vascular endothelial growth factor) which in turn stimulates endothelial cell proliferation. He mentioned that PlGF (placental growth factor) was also a minor player in these processes but wouldn?t be discussed further. VEGF can facilitate skin inflammation, which can be very persistent. VEGF-transgenic mice develop severe and persistent inflammation which eventually develops into skin lesions. This can be abrogated in thrombospondin 1 transgenic mice. The inflammatory response to UV can also be abrogated in those transgenic mice. Many therapies have been developed which are based on inhibiting angiogenesis to reduce melanoma outgrowth; those have been tested in mouse models and a phase I clinical trial is currently underway on melanoma patients. But is there a distinct relationship between lymphangiogenesis and melanoma? Actually there is no clear correlation between blood vessel size or number and metastatic or nonmetastatic melanomas. However, there is a large (~2-fold) difference in lymphatics between those 2 types of melanomas suggesting that lymphangiogenesis may play a more important role than angiogenesis. This turns out to be an excellent prognostic indicator.
John Pawelek (Yale) then presented his laboratory?s work on coarse melanin as an specific indicator of malignancy and corroborating evidence of the hybrid melanoma theory. Coarse melanin occurs in virtually 100% of metastases and not so frequently in primary tumors. They used tissue microarray to show that LPHA staining is commonly positive in metastases of many types of tumors, not just melanomas. Pawelek also discussed a patient who had a bone marrow transplant and who subsequently developed renal cell carcinoma. The metastases in that patient had genetic markers not only for the patient for also for the donor, proving that at least in those metastatic tumor cells, hybridization of cells from both sources had occurred. For those interested, a paper will be published in the Sept 1 issue of Cancer Research detailing some of the above.
Toshihiko Hoashi (Tokyo Koseinenkin Hospital, Japan) then discussed their study on the expression of various MMPs (matrix metalloproteinases) and their inhibitors (TIMPs) in various types of melanomas. He described their expression in lentigo malignant melanoma (LMM), superficial spreading melanoma (SSM), acral lentiginous melanoma (ALM), nodular melanoma (NM) and desmoplastic melanoma (DM). He reviewed the substrate specificity of the different MMPs, noting that MMP9 was particularly interesting because it degraded collagen type IV, a major component of the basement membrane and thus was an advantageous protease to permit metastasis. Many previous studies showed contrasting results on the expression of various MMPs and TIMPs in different types of melanomas, and Hoashi?s study (which examined matched samples of primary and metastatic tumors from the same patients) found that in general there were no dramatic changes in the expression of any of the MMPs or TIMPs between primary and metastatic melanomas in the 16 patients they examined, although there were slight increases in MMP7 and TIMP1 in all lymph node metastases. However, MMP9 expression in DM was dramatically increased in all 6 metastatic tissues examined compared with the expression of MMP9 in primary tumors, suggesting an important role for MMP9 in the metastatic spread of those tumors.
Shosuke Ito (Kumamoto Univ. School of Medicine, Japan) then summarized his group?s research on the presence and analysis of melanogenic intermediates in the serum and urine of melanoma patients. They found that eumelanin and pheomelanin intermediates are partially secreted from melanomas and are then methylated and can be detected in the serum and urine. They found an excellent correlation of 5-SCD content with the progression of disease. In serum, 4-AHP (4-aminohydroxyphenylalanine) is of comparable significance to the presence of 5-SCD (5-S-cysteinyldopa), both being markers of pheomelanin production. In sum, the latter compound is of great potential value in diagnosing the course of disease in melanoma patients.
The symposium entitled ?Transcriptional Regulation? focused on the pioneer field of transcriptional regulation of the embryonic commitment of the melanocytes and their eventual survival. This is an exciting field because many new regulatory molecules/transcription factors have recently been identified and their coordinate interactions are now being elucidated.
The symposium began with a presentation entitled 'Genomic analysis of neural crest-melanocyte development' by William Pavan (NIH). The expression and associated functions of SOX10, PAX3 and MITF during embryogenesis were presented. An analysis of their temporal expression in normal embryogenesis and specific alterations in various mutants of these transcription factors has lead to their proposed sequential interactive role in melanocyte commitment.
The elaborate role of MITF in signaling pathways of the melanocytes was reviewed by David Fisher (Dana-Farber Cancer Center). Two significant associations with MITF have recently been identified. The gene for an associated transcription factor TFEB, when translocated in papillary renal cell carcinomas, results in aberrant expression of several melanocytic antigens. In addition, a downstream target of MITF activation is the anti-apoptic molecule Bcl2. Associated with mutation of MITF has been neurosensory deafness, as exemplified in Waardenburg syndrome type II or Tietz syndrome.
The lack of melanoblast development and the associated otic morphologic/physiologic defect in MitfMi-Wh/+ mice was presented by Thomas Hornyak (Henry Ford Hospital). ?The overexpression of beta-catenin affects the proliferation and migration of melanoblasts? was presented by Lionel LaRue. Catenin appears to both medicate the interaction between cadherins and the cytoskeleton and to function as transcription factors. Aberrant expression of catenin in transgenic mice and melanoma cells demonstrated the role of this molecule in regulating proliferation and migration of melanoblasts.
Finally, the role a unique transcription factor, FOXN1, in pigmentation was presented by Loren Weiner (Mass General Hospital, Harvard Univ.). Mutations of FOXN1 are responsible for the immunocompromised, hairless ?nude? mouse. A transgenic mouse expressing FOXN1 in the basal epidermis via the keratin 5 promoter resulted in the development of interfollicular melanocytes. It was proposed that from the population of hair follicle destined melanoblast there was recruited cells that homed into the interfollicular epidermis via signaling cytokines regulated by FOXN1. This symposium clearly demonstrated the numerous intricate and interactive roles of known and unknown molecules regulating melanocyte development.
This page was modified last on September 22, 2003.