Faculty Bibliography

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to alpha-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to alpha-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to alpha-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by alpha-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes

Summary Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways

Background: Most skin cancer-related deaths are due to malignant melanoma. Risk assessment criteria for melanoma currently include skin phenotype, family and sun exposure history, factors that are subject to observer and recall bias. Genetic markers of susceptibility have been identified in association studies; however little progress has been made in developing them to improve screening and identification of individuals at risk of melanoma. Tyrosinase (TYR), a known susceptibility gene and a determinant of skin pigmentation, was thus investigated further to characterize its association with melanoma susceptibility and to identify markers which can be used in a risk assessment model. Methods: The cohort consisted of 326 individuals diagnosed with melanoma and 400 control subjects. TYR was interrogated using fifteen tag single nucleotide polymorphisms (SNPs) spanning the gene and statistical association tests performed. Additionally, ancestry informative markers were utilized to correct for population genetic sub-structure. Haplotype analysis was performed to determine if specific regions of the gene contributed more significantly to susceptibility. Coding regions of the gene are currently being sequenced and identified variants will be tested for impact on enzymatic function. Results: Of the 15 SNPs, 8 were associated with melanoma; 4 with decreased risk (Odds ratios 0.41-0.71) and 4 with increased risk (Odds ratios 1.43-1.96). SNPs localized to 2 regions of the gene (spanning exon 1 to intron 2 and intron 3 to 4) with markers of increased as well as decreased susceptibility present in both areas. With the exception of one coding region variant, SNPs were localized to introns. Conclusions: SNPs localized to TYR may serve as useful biomarkers for determining susceptibility to melanoma. We are currently sequencing the gene in our population in order to identify additional and potentially more potent markers of melanoma susceptibility. Coding region variants are being characterized for their effect on protein stability and enzyme activity such that functional active variants (most likely to affect susceptibility to melanoma) can be identified and assessed for their utility in melanoma risk assessment

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) to enable cells to recover from ER stress. If the UPR fails to restore normal ER function, apoptosis is induced instead. We have demonstrated that melanocytes have the capacity to adapt to chronic ER stress and escape from UPR-induced cell death. Mutations in the pinkeyed dilution/oculocutaneous albinism type 2 (Oca2)-gene result in altered tyrosinase processing and trafficking in melanocytes, accompanied by accumulation of misfolded tyrosinase in the ER. Despite this chronic overload of ER-retained tyrosinase, Oca2-mutant melanocytes do not show diminished viability suggesting that they have adapted to the ER stress. The aim of this study is to elucidate the process of adaptation to ER stress in melanocytes. Differential protein expression between wildtype (melan-a) and Oca2-melanocytes (melan-p) was analyzed using microarray assays and Western blotting. Adaptation to chronic ER stress was studied by comparing wildtype murine melanocytes dosed with the ER stressor thapsigargin to Oca2-mutants. We observed increased Ire1 expression in Oca2-melanocytes compared to wildtype, indicating that this UPR pathway is activated. Prolonged Ire1 signaling after UPR activation in stressed cells has been found to promote cell survival. In addition, expression of proteins involved in the pro-apoptotic Perk pathway appears to be decreased in Oca2- melanocytes. However, Oca2-mutants also demonstrated up-regulation of Chop, which typically promotes cell death by down-regulating expression of anti-apoptotic Bcl-2. Remarkably, the pro-apoptotic effects of Chop expression appear to be mitigated by up-regulation of Bcl-2 expression. Furthermore, expression of pro-apoptotic proteins such as Bid and caspase 1 were down-regulated in Oca2-mutant melanocytes as compared to wildtype cells. Acute ER stress (0-24 h) in wildtype melanocytes activated all three UPR pathways, but Ire1 signaling was attenuated in chronically stressed cells (12 days), while Perk signaling was maintained. Cell viability did not change during this period. Thus, wildtype melanocytes adapted to chronic ER stress despite sustained activation of pro-apoptotic pathways. These results indicate that chronically stressed wildtype melanocytes and Oca2-mutant melanocytes adapt to ER stress by differential mechanisms. Melanocytes may thus adapt to ER-stress by multiple mechanisms, some of which may account for the increased drug resistance observed in melanocytes and melanoma

Background: Accumulation of immature proteins in the Endoplasmic Reticulum (ER) causes organelle stress that is counteracted by the unfolded protein stress response (UPR). Three pathways compose the UPR and are initiated when Ire1, Perk and Atf6 respectively, are released from heterodimers formed with the ER chaperone BiP. The UPR signals down-regulation of global translation and increased ER-chaperone expression. Ire1 is phosphorylated, activating its nuclease activity, which leads to splicing of the X-box binding protein 1 (Xbp1). The spliced RNA encodes a transcription factor that regulates expression of a subset of genes that comprise one arm of the UPR. Apoptosis is initiated if homeostasis is not re-established following UPR activation. The Ire1 pathway has recently been shown to play arole in the development of vitiligo, while the UPR has been implicated in keratinocyte response to UVB and in drug resistance in melanoma. We therefore investigated the melanocyte response to ER stress induced by chemical ER disruptors and by oxidative stress (the mechanism by which we propose UV exposure perturbs the melanocyte ER). Method: Wild-type mouse melanocytes were treated with thapsigargin, which disrupts the calcium balance in the ER causing UPR induction, and cells harvested at 6, 12 and 24 h. Western blot and microarray analyses were performed and data evaluated to identify pathways activated by thapsigargin treatment. In addition, melanocytes were dosed with compounds that induce oxidative stress. RNA was harvested and evaluated for activation of the UPR by Xbp-1 splicing. Results: IRE1 expression was upregulated within 6 h of treatment with thapsigargin, which promoted splicing of XBP1 mRNA and activation of its transcription factor activity. PERK and its downstream target CHOP were phosphorylated and HA-tagged ATF6 was cleaved within 6-12 h of treatment. Up-regulation of BiP and ER chaperones such as Ero1 and down-regulation of tyrosinase were also observed. In addition, several p53-related pathways were modulated in response to thapsigargin. Induction of oxidative stress was found to induce Xbp1 splicing. Conclusions: The UPR may play an important role in melanocyte response to stress, including response to oxidative stress induced by UV exposure. Dysfunction of this response may contribute to initiation and progression of vitiligo and drug resistance in melanoma