Monoclonal Antibodies to PRAME Protein Slow the Development of PRAME-Expressing Tumor.

Two monoclonal antibodies recognizing non-overlapping epitopes of the PRAME protein were injected into immunocompetent mice to study their influence on the growth of subcutaneous tumor nodes. The B16F10 murine melanoma line, either expressing human PRAME protein or bearing only a vector without PRAME gene, were used as transplants. Each of the antibodies showed the ability to suppress tumor growth of a PRAME-expressing tumour, but not a tumor without PRAME.

Protective role of histone deacetylase 4 from ultraviolet radiation-induced DNA lesions.

Ultraviolet B (UVB) exposure is a core factor that leads to skin disease or carcinogenesis through the insufficient repair of DNA lesions. UVB-induced DNA lesions are mainly removed by the nucleotide excision repair (NER) mechanism. The expression of histone deacetylase 4 (HDAC4) is altered in the skin upon UVB exposure, indicating its possible implication in UVB-induced DNA lesions repair. Here, we investigated the role of HDAC4 in the NER removal of the main classes of UVB-induced DNA lesions consisting of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). We found that UVB irradiation increased HDAC4 expression at both the mRNA and protein levels. HDAC4 interacted with NER factor XPC, which played an important role in effectively removing the UVB-induced DNA lesions. This study provides an understanding of the HDAC4 function in DNA repair, which will allow the development of efficient strategies to protect the skin from UVR-induced diseases.

Hmgb1-IL-23-IL-17-IL-6-Stat3 axis promotes tumor growth in murine models of melanoma.

In order to understand how tumor cells can escape immune surveillance mechanisms and thus develop antitumor therapies, it is critically important to investigate the mechanisms by which the immune system interacts with the tumor microenvironment. In our current study, IL-17 deficiency results in reduced melanoma tumor size, diminished numbers of proliferating cells and blood vessels, and decreased percentage of CD11b(+)Gr-1(+) MDSCs in tumor tissues. IL-17 promotes IL-6 induction and Stat3 activation. Treatment of Stat3 inhibitor WP1066 in B16-F10 tumor cells inoculated wild-type mice inhibits tumor growth. Additional administration of recombinant IL-6 into B16-F10 tumor-bearing IL-17(-/-) mice results in markedly increased tumor size and p-Stat3 expression, whereas additional recombinant IL-17 administration into B16-F10 tumor-bearing wild-type mice treated with anti-IL-6 mAb does not significantly alter the tumor growth and p-Stat3 expression. In our further study, blockade of Hmgb1-RAGE pathway inhibits melanoma tumor growth and reduces production of IL-23 and IL-17. All these data suggest that Hmgb1-IL-23-IL-17-IL-6-Stat3 axis plays a pivotal role in tumor development in murine models of melanoma, and blocking any portion of this axis will attenuate melanoma tumor growth.

Ultraviolet A does not induce melanomas in a Xiphophorus hybrid fish model.

We examined the wavelength dependence of ultraviolet (UV) ra-diation (UVR)-induced melanoma in a Xiphophorus backcross hybrid model previously reported to be susceptible to melanoma induction by ultraviolet A (UVA) and visible light. Whereas ultraviolet B (UVB) irradiation of neonates yielded high frequencies of melanomas in pigmented fish, UVA irradiation resulted in melanoma frequencies that were not significantly different from unirradiated fish. Spontaneous and UV-induced melanoma frequencies correlated with the degree of pigmentation as expected from previous studies, and the histopathology phenotypes of the melanomas were not found in significantly different proportions in UV-treated and -untreated tumor-bearing fish. Our results support the conclusion that a brief early-life exposure to UVB radiation causes melanoma formation in this animal model. These data are consistent with an essential role for direct DNA damage, including cyclobutane dimers and (6-4) photoproducts, in the etiology of melanoma.

The ubiquitin ligase Siah2 regulates tumorigenesis and metastasis by HIF-dependent and -independent pathways.

The ubiquitin ligase Siah2 has been shown to regulate prolyl hydroxylase 3 (PHD3) stability with concomitant effect on HIF-1alpha availability. Because HIF-1alpha is implicated in tumorigenesis and metastasis, we used SW1 mouse melanoma cells, which develop primary tumors with a propensity to metastasize, in a syngeneic mouse model to assess a possible role for Siah2 in these processes. Inhibiting Siah2 activity by expressing a peptide designed to outcompete association of Siah2-interacting proteins reduced metastasis through HIF-1alpha without affecting tumorigenesis. Conversely, inhibiting Siah2 activity by means of a dominant-negative Siah2 RING mutant primarily reduced tumorigenesis through the action of Sprouty 2, a negative regulator of Ras signaling. Consistent with our findings, reduced expression of PHD3 and Sprouty2 was observed in more advanced stages of melanoma tumors. Using complementary approaches, our data establish the role of Siah2 in tumorigenesis and metastasis by HIF-dependent and -independent mechanisms.

Psychologic Stress Drives Progression of Malignant Tumors via DRD2/HIF1α Signaling.

Although it is established that the sustained psychologic stress conditions under which patients with tumors often reside accelerates malignant progression of tumors, the molecular mechanism behind this association is unclear. In this work, the effect of psychologic stress on tumor progression was verified using a stress-stimulated tumor-bearing mouse model (Str-tumor). Both D2 dopamine receptor (DRD2) and hypoxia-inducible factor-1α (HIF1α) were highly expressed in the nucleus of Str-tumors. Treatment with trifluoperazine (TFP), a DRD2 inhibitor, elicited better antitumor effects in Str-tumors than the control group. These results indicate that DRD2 may mediate stress-induced malignant tumor progression. DRD2 interacted with von Hippel-Lindau (VHL) in the nucleus, and competitive binding of DRD2 and HIF1α to VHL resulted in reduced ubiquitination-mediated degradation of HIF1α, enhancing the epithelial-mesenchymal transition of tumor cells. TFP acted as an interface inhibitor between DRD2 and VHL to promote the degradation of HIF1α. In conclusion, DRD2 may promote the progression of malignant tumors induced by psychologic stress via activation of the oxygen-independent HIF1α pathway, and TFP may serve as a therapeutic strategy for stress management in patients with cancer. SIGNIFICANCE: This work identifies DRD2 regulation of HIF1α as a mechanism underlying the progression of malignant tumors stimulated by psychologic stress and suggests that DRD2 inhibition can mitigate these stress conditions in patients.See related commentary by Bernabé, p. 5144.

Megakaryocytes Mediate Hyperglycemia-Induced Tumor Metastasis.

High blood glucose has long been established as a risk factor for tumor metastasis, yet the molecular mechanisms underlying this association have not been elucidated. Here we describe that hyperglycemia promotes tumor metastasis via increased platelet activity. Administration of glucose, but not fructose, reprogrammed the metabolism of megakaryocytes to indirectly prime platelets into a prometastatic phenotype with increased adherence to tumor cells. In megakaryocytes, a glucose metabolism-related gene array identified the mitochondrial molecular chaperone glucose-regulated protein 75 (GRP75) as a trigger for platelet activation and aggregation by stimulating the Ca2+-PKCα pathway. Genetic depletion of Glut1 in megakaryocytes blocked MYC-induced GRP75 expression. Pharmacologic blockade of platelet GRP75 compromised tumor-induced platelet activation and reduced metastasis. Moreover, in a pilot clinical study, drinking a 5% glucose solution elevated platelet GRP75 expression and activated platelets in healthy volunteers. Platelets from these volunteers promoted tumor metastasis in a platelet-adoptive transfer mouse model. Together, under hyperglycemic conditions, MYC-induced upregulation of GRP75 in megakaryocytes increases platelet activation via the Ca2+-PKCα pathway to promote cancer metastasis, providing a potential new therapeutic target for preventing metastasis. SIGNIFICANCE: This study provides mechanistic insights into a glucose-megakaryocyte-platelet axis that promotes metastasis and proposes an antimetastatic therapeutic approach by targeting the mitochondrial protein GRP75.

Dysfunction of CD8 + PD-1 + T cells in type 2 diabetes caused by the impairment of metabolism-immune axis.

The metabolic changes and dysfunction in CD8 + T cells may be involved in tumor progression and susceptibility to virus infection in type 2 diabetes (T2D). In C57BL/6JJcl mice fed with high fat-high sucrose chow (HFS), multifunctionality of CD8 + splenic and tumor-infiltrating lymphocytes (TILs) was impaired and associated with enhanced tumor growth, which were inhibited by metformin. In CD8 + splenic T cells from the HFS mice, glycolysis/basal respiration ratio was significantly reduced and reversed by metformin. In the patients with T2D (DM), multifunctionality of circulating CD8 + PD-1 + T cells stimulated with PMA/ionomycin as well as with HLA-A*24:02 CMV peptide was dampened, while metformin recovered multifunctionality. Both glycolysis and basal respiration were reduced in DM, and glycolysis was increased by metformin. The disturbance of the link between metabolism and immune function in CD8 + PD-1 + T cells in T2D was proved by recovery of antigen-specific and non-specific cytokine production via metformin-mediated increase in glycolytic activity.

Intravital Imaging of Adoptive T-Cell Morphology, Mobility and Trafficking Following Immune Checkpoint Inhibition in a Mouse Melanoma Model.

Efficient T-cell targeting, infiltration and activation within tumors is crucial for successful adoptive T-cell therapy. Intravital microscopy is a powerful tool for the visualization of T-cell behavior within tumors, as well as spatial and temporal heterogeneity in response to immunotherapy. Here we describe an experimental approach for intravital imaging of adoptive T-cell morphology, mobility and trafficking in a skin-flap tumor model, following immune modulation with immune checkpoint inhibitors (ICIs) targeting PD-L1 and CTLA-4. A syngeneic model of ovalbumin and mCherry-expressing amelanotic mouse melanoma was used in conjunction with adoptively transferred OT-1+ cytotoxic T-cells expressing GFP to image antigen-specific live T-cell behavior within the tumor microenvironment. Dynamic image analysis of T-cell motility showed distinct CD8+ T-cell migration patterns and morpho-dynamics within different tumor compartments in response to ICIs: this approach was used to cluster T-cell behavior into four groups based on velocity and meandering index. The results showed that most T-cells within the tumor periphery demonstrated Lévy-like trajectories, consistent with tumor cell searching strategies. T-cells adjacent to tumor cells had reduced velocity and appeared to probe the local environment, consistent with cell-cell interactions. An increased number of T-cells were detected following treatment, traveling at lower mean velocities than controls, and demonstrating reduced displacement consistent with target engagement. Histogram-based analysis of immunofluorescent images from harvested tumors showed that in the ICI-treated mice there was a higher density of CD31+ vessels compared to untreated controls and a greater infiltration of T-cells towards the tumor core, consistent with increased cellular trafficking post-treatment.

Melanocyte expression of survivin promotes development and metastasis of UV-induced melanoma in HGF-transgenic mice.

We previously found the apoptosis inhibitor Survivin to be expressed in melanocytic nevi and melanoma but not in normal melanocytes. To investigate the role of Survivin in melanoma development and progression, we examined the consequences of forced Survivin expression in melanocytes in vivo. Transgenic (Tg) mouse lines (Dct-Survivin) were generated with melanocyte-specific expression of Survivin, and melanocytes grown from Dct-Survivin mice expressed Survivin. Dct-Survivin melanocytes exhibited decreased susceptibility to UV-induced apoptosis but no difference in proliferative capacity compared with melanocytes derived from non-Tg littermates. Induction of nevi in Dct-Survivin and non-Tg mice by topical application of 7,12-dimethylbenz(a)anthracene did not reveal significant differences in lesion onset (median, 10 weeks) or density (4 lesions per mouse after 15 weeks). Dct-Survivin mice were bred with melanoma-prone MH19/HGF-B6 Tg mice, and all progeny expressing either individual, neither, or both (Survivin/HGF) transgenes were UV-treated as neonates and then monitored for 43 weeks. Melanocytes in neonatal Survivin+/HGF+ mouse skin were less susceptible to UV-induced apoptosis than those from Survivin-/HGF+ mice. Onset of melanocytic tumors was earlier (median, 18 versus 24 weeks; P = 0.01, log-rank test), and overall tumor density was greater (7.7 versus 5.2 tumors per mouse; P = 0.04) in Survivin+/HGF+ compared with Survivin-/HGF+ mice. Strikingly, melanomas arising in Survivin+/HGF+ mice showed a greater tendency for lymph node (35% versus 0%; P = 0.04) and lung (53% versus 22%) metastasis and lower rates of spontaneous apoptosis than those in Survivin-/HGF+ mice. These studies show a role for Survivin in promoting both early and late events of UV-induced melanoma development in vivo.

Photoperiod and testosterone modulate growth and melanogenesis of s91 murine melanoma.

In vivo and in vitro assays were performed with S91 murine melanoma cells aiming to investigate the effects of testosterone and photoperiod on tumor growth and melanogenesis (tyrosinase activity). In vivo assays were performed by inducing melanoma tumors in castrated mice receiving increasing concentrations of testosterone and submitted to varying photoperiod regimens. The results demonstrated that the increase of melanin content was higher in animals submitted to the longest days, thus demonstrating the importance of photoperiod length in melanin synthesis. Increase in tumor growth and protein content was observed in testosterone-treated animals submitted to 12L:12D; in testosterone-treated animals submitted to 4L:20D and 20L:4D tumor growth was significantly smaller. In S91 cultured cells, testosterone increased cell proliferation and reduced tyrosinase activity in a dose-dependent manner. Radioactive binding assays demonstrated that the hormone was acting through low affinity testosterone receptors, since the presence of aromatase inhibitor did not affect the binding assay in a statistically significant way, and all the in vitro experiments were performed in the presence of the inhibitor. Our in vivo data added to the in vitro results corroborate the hypothesis that S91 melanoma cells directly respond to testosterone and that this effect is modulated by light.

Spontaneous and UV radiation-induced multiple metastatic melanomas in Cdk4R24C/R24C/TPras mice.

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 UVR-induced melanoma development whereas the cell cycle defect introduced by mutant Cdk4 contributes to tumor progression, producing more aggressive, metastatic tumors.

Induction of nevi and skin tumors in Ink4a/Arf Xpa knockout mice by neonatal, intermittent, or chronic UVB exposures.

Nevi and melanomas correlate to childhood and intermittent solar UV exposure, xeroderma pigmentosum patients run increased risk, and p16(Ink4a) expression is often lost in malignant progression. To ascertain the effect of these risk factors, pigmented hairless Ink4a/Arf-, Xpa- knockout mice were subjected to various combinations of neonatal [7,12-dimethylbenz(a)anthracene (DMBA) or UVB exposure] and adult treatments (12-O-tetradecanoylphorbol-13-acetate or subacute daily UVB exposure or intermittent overexposure). Nevi occurred earliest, grew largest, and were most numerous in mice exposed to DMBA followed by intermittent UVB overexposure [effect of six minimal edemal doses (MED), 1 x /2 weeks > 4 MED 1 x /wk]. Neonatal UV exposure enhanced nevus induction but lost its effect after 200 days. The Xpa(-/-) mice proved exquisitely sensitive to UV-driven nevus induction, indicating the involvement of pyrimidine dimer DNA lesions, but Xpa(+/+) mice developed many more nevi (>40 per mouse) at high UV dosages not tolerated by Xpa(-/-) mice. Ink4a/Arf(-/-) mice developed most skin tumors faster, but surprisingly developed nevi slower than their heterozygous counterparts especially after neonatal UV exposure. Despite raising >1,600 nevi, only six melanomas arose in our experiments with Ink4a/Arf knockout mice (five of which in Xpa(+/+) mice at high UV dosages). In contrast to human nevi, these nevi lacked hotspot mutations in Braf or Ras genes, possibly explaining the lack of malignant progression in the Ink4a/Arf(-/-) mice. Hence, although our experiments did not effectively emulate human melanoma, they provided clear evidence that intermittent UV overexposure strongly stimulates and the Ink4a/Arf(-/-) genotype may actually impair nevus development.

The potential for enhancement of mouse melanoma metastasis by diagnostic and high-amplitude ultrasound.

The potential for enhancement of the metastatic spread of cells from mouse melanoma tumors was examined for exposure to diagnostic ultrasound (DUS) and high-amplitude ultrasound (HAUS) without and with ultrasound (US) contrast agent. The melanoma cell line B16-D5, which is metastatic specifically to lung, was cultured and implanted on a hind leg of female C57/bl6 mice. For DUS, tumors were scanned using 1.5-MHz harmonic B-mode imaging with 1-Hz intermittent frame triggering at 2.1 MPa (equivalent MI = 1.7) in a 37 degrees C water bath. For HAUS, a 1.35-MHz focused transducer directed 1-ms bursts at 5 MPa to the tumor at a 1-Hz rate. A total dose of 1 mL/kg Optison was injected during exposures. Exposure without contrast agent received the same exposure followed by the contrast agent with the US off. The primary tumor was removed surgically one day after US. Lungs were removed after four weeks for evaluation of metastases. Experiments involved exposure without and with contrast agent in groups of 20 mice. For DUS, mean counts of 0.8 +/- 0.3 (standard error) and 1.3 +/- 0.9 (P = 0.62) metastases were found for groups exposed without and with contrast, respectively. For HAUS, mean counts of 3.4 +/- 1.2 and 5.9 +/- 1.7 (P = 0.35) metastases were found for groups exposed without and with contrast, respectively. The lack of effect of DUS exposure with contrast confirms a previous finding. However, the HAUS counts and incidence were significantly larger than the DUS results (P < 0.05) in a two-way analysis of variance. This indicates a potential for HAUS to enhance metastasis.

Interleukin 1-dependent and -independent mouse melanoma metastases.

BACKGROUND: The adhesion of cancer cells to the endothelial lining of blood vessels, which is important for metastasis, is promoted by the action of interleukin 1 (IL-1) and other cytokines. PURPOSE: IL-1-producing melanoma cells were used to induce metastases in mice to test whether melanoma metastasis--wherever it occurs--depends on the action of IL-1. METHODS: We used the following experimental designs in this study: 1) Male C57BL/6J mice were inoculated in the left cardiac ventricle with 5 x 10(4) murine B16 melanoma cells, and no treatment was given (control animals). 2) Mice received an intraperitoneal injection of either saline (control animals) or recombinant human IL-1 receptor antagonist (rHuIL-1Ra) 2 hours before the injection of cancer cells; thereafter, they received an additional injection of saline or rHuIL-1Ra daily for 20 days. 3) Mice received an intravenous injection of either saline or rHuIL-1Ra; 15 minutes later, mice that received saline were given either a second injection of saline (control animals) or an injection of bacterial lipopolysaccharide (LPS) to stimulate host IL-1 production and endothelial cell activation. The mice that received rHuIL-1Ra were also given an injection of LPS at this time. Six hours later, all mice were inoculated with cancer cells, followed by no further treatment. In all experiments, the mice were killed 20 days after the injection of cancer cells, and metastases were counted in multiple organs and bones. Metastasis incidence values (relating to the frequency that a given site was positive for metastasis) and metastasis development index values (relating to the extent of metastasis at a given site) were calculated. A hierarchical cluster analysis was performed to determine whether groups of organs exhibited characteristic changes in their metastasis development index values in response to the three treatments given (i.e., rHuIL-1Ra, LPS, or rHuIL-1Ra plus LPS). Reported P values are two-sided. RESULTS AND CONCLUSIONS: Treatment with rHuIL-1Ra alone significantly (P<.05) reduced the occurrence of metastasis in the bone marrow, spleen, liver, lung, pancreas, skeletal muscle, adrenal gland, and heart, indicating that host- and/or melanoma-derived IL-1 promoted metastasis in these organs; treatment with rHuIL-1Ra had no effect on metastasis in the kidney, testis, brain, skin, and gastrointestinal tract, suggesting that metastasis in these latter organs was IL-1 independent. Treatment with LPS alone significantly (P<.05) enhanced metastasis in the same organs for which rHuIL-1Ra treatment reduced metastasis, except for the heart and the adrenal gland. Treatment with rHuIL-1Ra 15 minutes before LPS treatment abrogated the LPS-mediated enhancement of metastasis. Two independent organ groups for which IL-1 promoted melanoma metastasis were identified in the cluster analysis.

Differences in latency and inducibility of mouse skin melanomas depending on the age and anatomic site of the skin.

To determine whether the occurrence of skin melanoma is influenced by the age or the anatomic source of the skin in melanoma-susceptible transgenic mouse models, skin was grafted from donors of different ages or from different anatomic sites to a standard (lateral trunk) site in adult recipients of the same transgenic strain. In 27 grafts of neonatal body skin, melanomas arose with a significantly shorter latency than in 37 grafts of older body skin. The difference may reflect not only the larger number of extrafollicular melanocytes in a given area of neonatal skin but also their unusually high mitotic activity shortly after birth and the influence of other growing skin cells nearby. Each of these body-skin grafts usually developed a single tumor situated near the graft edge. Because maximal wound healing occurs at the edge of such full-thickness skin grafts, melanocytes near the edge would receive the highest exposure to growth factors and cytokines associated with wound healing. In contrast to these results, grafts of snout skin yielded many melanomas, each originating from melanocytes within a vibrissa follicle rather than at the graft edge. The relatively strong local tumorigenic stimulus may be attributable to intrafollicular growth factors normally involved in whisker growth. The above-described experiments support the conclusion that agents in the immediate skin environment of the melanocyte, in addition to the state of the melanocyte itself, contribute to melanoma formation.

Ultraviolet B but not ultraviolet A radiation initiates melanoma.

Cutaneous malignant melanoma is one of the fastest increasing cancers with an incidence that has more than doubled in the last 25 years. Sunlight exposure is strongly implicated in the etiology of cutaneous malignant melanoma and the UV portion of the sunlight spectrum is considered responsible. Data are, however, conflicting on the roles of ultraviolet B [UVB; 280-320 nanometers (nm)] and ultraviolet A (UVA; 320-400 nm), which differ in their ability to initiate DNA damage, cell signaling pathways and immune alterations. To address this issue, we have used specialized optical sources, emitting isolated or combined UVB or UVA wavebands or solar simulating radiation, together with our hepatocyte growth factor/scatter factor-transgenic mouse model of UV-induced melanoma that uniquely recapitulates human disease. Only UVB-containing sources initiated melanoma. These were the isolated UVB waveband (>96% 280-320 nm), the unfiltered F40 sunlamp (250-800 nm) and the solar simulator (290-800 nm). Kaplan-Meier survival analysis indicated that the isolated UVB waveband was more effective in initiating melanoma than either the F40 sunlamp or the solar simulator (modified log rank P < 0.02). The latter two sources showed similar melanoma effectiveness (P = 0.38). In contrast, transgenic mice irradiated with either the isolated UVA waveband (>99.9% 320-400 nm, 150 kJ/m2), or an F40 sunlamp filtered to remove > 96% of the UVB, responded like unirradiated control animals. We conclude that, within the constraints of this animal model, UVB is responsible for the induction of mammalian cutaneous malignant melanoma whereas UVA is ineffective even at doses considered physiologically relevant. This finding may have major implications with respect both to risk assessment from exposure to solar and artificial UVB, and to development of effective protection strategies against melanoma induction by UVB. Moreover, these differences in wavelength effectiveness can now be exploited to identify UV pathways relevant to melanomagenesis.

Ink4a/arf deficiency promotes ultraviolet radiation-induced melanomagenesis.

Cutaneous malignant melanoma (CMM), already known for its highly aggressive behavior and resistance to conventional therapy, has evolved into a health crisis by virtue of a dramatic elevation in incidence. The underlying genetic basis for CMM, as well as the fundamental role for UV radiation in its etiology, is now widely accepted. However, the only bona fide genetic locus to emerge from extensive analysis of CMM suppressor candidates is INK4a/ARF at 9p21, which is lost frequently in familial and occasionally in somatic CMM. The functional relationship between INK4a/ARF and UV radiation in the pathogenesis of CMM is largely unknown. Recently, we reported that hepatocyte growth factor/scatter factor (HGF/SF)-transgenic mice develop melanomas after a single erythemal dose of neonatal UV radiation, supporting epidemiological data implicating childhood sunburn in CMM. Here we show that neonatal UV irradiation induces a full spectrum of melanocyte pathology from early premalignant lesions through distant metastases. Cutaneous melanomas arise with histopathological and molecular pathogenetic features remarkably similar to CMM, including loss of ink4a/arf. A role for ink4a/arf in UV-induced melanomagenesis was directly assessed by placing the HGF/SF transgene on a genetic background devoid of ink4a/arf. Median time to melanoma development induced by UV radiation was only 50 days in HGF/SF ink4a/arf(-/-) mice, compared with 152 and 238 days in HGF/SF ink4a/arf(+/-) and HGF/SF ink4a/arf(+/+) mice, respectively. These studies provide experimental evidence that ink4a/arf plays a critical role in UV-induced melanomagenesis and strongly suggest that sunburn is a highly significant risk factor, particularly in families harboring germ-line mutations in INK4a/ARF.

Array comparative genome hybridization for tumor classification and gene discovery in mouse models of malignant melanoma.

Chromosomal numerical aberrations (CNAs), particularly regional amplifications and deletions, are a hallmark of solid tumor genomes. These genomic alterations carry the potential to convey etiologic and clinical significance by virtue of their clonality within a tumor cell population, their distinctive patterns in relation to tumor staging, and their recurrence across different tumor types. In this study, we showed that array-based comparative genomic hybridization (CGH) analysis of genome-wide CNAs can classify tumors on the basis of differing etiologies and provide mechanistic insights to specific biological processes. In a RAS-induced p19(Arf-/-) mouse model that experienced accelerated melanoma formation after UV exposure, array-CGH analysis was effective in distinguishing phenotypically identical melanomas that differed solely by previous UV exposure. Moreover, classification by array-CGH identified key CNAs unique to each class, including amplification of cyclin-dependent kinase 6 in UV-treated cohort, a finding consistent with our recent report that UVB targets components of the p16(INK4a)-cyclin-dependent kinase-RB pathway in melanoma genesis (K. Kannan, et al., Proc. Natl. Acad. Sci. USA, 21: 2003). These results are the first to establish the utility of array-CGH as a means of etiology-based tumor classification in genetically defined cancer-prone models.