To identify microRNAs potentially involved in melanomagenesis, we compared microRNA expression profiles between melanoma cell lines and cultured melanocytes. The most differentially expressed microRNA between the normal and tumor cell lines was miR-211. We focused on this pigment-cell-enriched miRNA as it is derived from the microphthalmia-associated transcription factor (MITF)-regulated gene, TRPM1 (melastatin). We find that miR-211 expression is greatly decreased in melanoma cells and melanoblasts compared to melanocytes. Bioinformatic analysis identified a large number of potential targets of miR-211, including POU3F2 (BRN2). Inhibition of miR-211 in normal melanocytes resulted in increased BRN2 protein, indicating that endogenous miR-211 represses BRN2 in differentiated cells. Over-expression of miR-211 in melanoma cell lines changed the invasive potential of the cells in vitro through directly targeting BRN2 translation. We propose a model for the apparent non-overlapping expression levels of BRN2 and MITF in melanoma, mediated by miR-211 expression.

Summary The individuals carrying melanocortin-1-receptor (MC1R) variants, especially those associated with red hair color, fair skin and poor tanning ability (RHC-trait), are more prone to melanoma while the underlying mechanism is poorly defined. Here, we report that UVB exposure triggers PTEN interaction with wild-type (WT), but not RHC-associated MC1R variants, which protects PTEN from WWP2-mediated degradation, leading to AKT inactivation. Strikingly, the biological consequences of the failure of MC1R variants to suppress PI3K/AKT signaling are highly context dependent. In primary melanocytes, hyperactivation of PI3K/AKT signaling leads to premature senescence; in the presence of BRAFV600E, MC1R deficiency-induced elevated PI3K/AKT signaling drives oncogenic transformation. These studies establish the MC1R-PTEN axis as a central regulator for melanocytes’ response to UVB exposure, and reveal the molecular basis underlying the association between MC1R variants and melanomagenesis.

The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared to adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression respectively. This links BRN2 as an activator and conversely MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense ablated cell lines decreased melanoma sphere forming capability, cell adhesion during 3D-spheroid formation, and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than growth as adherent melanoma cells.

There is increasing epidemiological and molecular evidence that cutaneous melanomas arise through multiple causal pathways. The purpose of this study was to explore the relationship between germline and somatic mutations in a population-based series of melanoma patients to reshape and refine the divergent pathway model for melanoma. Melanomas collected from 123 Australian patients were analyzed for melanocortin-1 receptor (MC1R) variants and mutations in the BRAF and NRAS genes. Detailed phenotypic and sun exposure data were systematically collected from all patients. We found that BRAF-mutant melanomas were significantly more likely from younger patients and those with high nevus counts, and were more likely in melanomas with adjacent neval remnants. Conversely, BRAF-mutant melanomas were significantly less likely in people with high levels of lifetime sun exposure. We observed no association between germline MC1R status and somatic BRAF mutations in melanomas from this population. BRAF-mutant melanomas have different origins from other cutaneous melanomas. These data support the divergent pathways hypothesis for melanoma, which may require a reappraisal of targeted cancer prevention activities.

Melanocytes respond to UVR not only by producing melanin, but also by proliferating. This is essentially a protective response. We have studied the melanocyte proliferative response after a single UVR exposure to neonatal mice. At 3 days post-UVR in wild-type neonates we observed a marked melanocyte activation not seen in adults. Melanocytes migrated to the epidermal basal layer, their numbers peaking at 3-5 days after UVR then diminishing. They appeared to emanate from the hair follicle, migrating to the epidermis via the outer root sheath. In melanoma-prone mice with melanocyte-specific overexpression of Hras(G12V), basal layer melanocytes were increased in size and dendricity compared to UVR-treated wild-type mice. Melanocytes in mice carrying a pRb pathway cell-cycle defect (oncogenic Cdk4(R24C)) did not show an enhanced response to UVR such as those carrying Hras(G12V). The exquisite sensitivity to UVR-induced proliferation and migration that characterizes neonatal mouse melanocytes may partly explain the utility of this form of exposure for inducing melanoma in mice that carry oncogenic mutations.

SummaryWe report on a systematic analysis of genotype-specific melanocyte (MC) UVR responses in transgenic mouse melanoma models along with tumour penetrance and comparative histopathology. pRb or p53 pathway mutations cooperated with Nras Q61K to transform MCs. We previously reported that MCs migrate from the follicular outer root sheath into the epidermis after neonatal UVR. Here, we found that Arf or p53 loss markedly diminished this response. Despite this, mice carrying these mutations developed melanoma with very early age of onset after neonatal UVR. Cdk4 R24C did not affect the MC migration. Instead, independent of UVR exposure, interfollicular dermal MCs were more prevalent in Cdk4 R24C mice. Subsequently, in adulthood, these mutants developed dermal MC proliferations reminiscent of superficial congenital naevi. Two types of melanoma were observed in this model. The location and growth pattern of the first was consistent with derivation from the naevi, while the second appeared to be of deep dermal origin. In animals carrying the Arf or p53 defects, no naevi were detected, with all tumours ostensibly skipping the benign precursor stage in progression.

Human melanoma susceptibility is often characterized by germ-line inactivating CDKN2A (INK4A/ARF) mutations, or mutations that activate CDK4 by preventing its binding to and inhibition by INK4A. We have previously shown that a single neonatal UV radiation (UVR) dose delivered to mice that carry melanocyte-specific activation of Hras (TPras) increases melanoma penetrance from 0% to 57%. Here, we report that activated Cdk4 cooperates with activated Hras to enhance susceptibility to melanoma in mice. Whereas UVR treatment failed to induce melanomas in Cdk4 R24C/R24C mice, it greatly increased the penetrance and decreased the age of onset of melanoma development in Cdk4 R24C/R24C /TPras animals compared with TPras alone. This increased penetrance was dependent on the threshold of Cdk4 activation as Cdk4 R24C/+ / TPras animals did not show an increase in UVR-induced melanoma penetrance compared with TPras alone. In addition, Cdk4 R24C/R24C /TPras mice invariably developed multiple lesions, which occurred rarely in TPras mice. These results indicate that germ-line defects abrogating the pRb pathway may enhance UVR-induced melanoma. TPras and Cdk4 R24C/R24C /TPras tumors were comparable histopathologically but the latter were larger and more aggressive and cultured cells derived from such melanomas were also larger and had higher levels of nuclear atypia. Moreover, the melanomas in Cdk4 R24C/R24C /TPras mice, but not in TPras mice, readily metastasized to regional lymph nodes. Thus, it seems that in the mouse, Hras activation initiates UVRinduced melanoma development whereas the cell cycle defect introduced by mutant Cdk4 contributes to tumor progression, producing more aggressive, metastatic tumors.

BackgroundSkin cancer is the most prevalent and most preventable cancer in Australia. Despite Australia’s long-running public health campaigns, young Australian adults continue to report high levels of ultraviolet radiation (UVR) exposure and frequent sunburns. Young people are now increasingly turning away from traditional media, such as newspapers and TV, favoring Web-based streaming, which is challenging the health care sector to develop new ways to reach this group with targeted, personalized health promotion messages. Advances in technology have enabled delivery of time- and context-relevant health interventions.ObjectiveThe primary aim of this randomized controlled trial was to test the effect of UVR feedback from a smartphone app or a UVR dosimeter feedback device on sun protection habits, sun exposure behaviors, sunburn, and physical activity levels in young adults.MethodsYoung adults aged 18-35 years (n=124) were recruited from Queensland, Australia, between September 2015 and April 2016, via social or traditional media campaigns and outreach activities in the local community. Participants were randomized into 3 groups for a 4-week intervention: (1) no intervention control group; (2) UVR monitor group, who were asked to wear a UVR dosimeter feedback device set to their skin type; and (3) a SunSmart app group, who were asked to download and use the SunSmart phone app. Data were self-assessed through Web-based surveys at baseline and 1 week and 3 months postintervention.ResultsComplete data were available for 86.2% (107/124) of participants (control group, n=36; UVR monitor group, n=36; and SunSmart app group, n=35). Intervention uptake in the UVR monitor group was high, with 94% (34/36) of participants using the device all or some of the time when outdoors. All SunSmart app group participants downloaded the app on their smartphone. There was no significant difference in the change in the sun protection habits (SPH) index (main outcome measure) across the 3 groups. However, compared with the control group, a significantly greater proportion of the participants in the UVR monitor group reduced their time unprotected and exposed to UVR on weekends during the intervention compared with the baseline (odds ratio [OR]: 2.706, 95% CI 1.047-6.992, P=.04). This significant effect was sustained with greater reductions observed up to 3 months postintervention (OR: 3.130, 95% CI 1.196-8.190, P=.02). There were no significant differences between the groups in weekday sun exposure, sunscreen use, sunburn, suntan, or physical activity.ConclusionsUsing technology such as apps and personal UVR monitoring devices may improve some sun exposure behaviors among young adults, but as the SPH index did not increase in this study, further research is required to achieve consistent uptake of sun protection in young people.Trial RegistrationThe Australian and New Zealand Clinical Trials register ACTRN12615001296527; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=368458 (Archived by WebCite at http://www.webcitation....