Thomas J. Hornyak

Interferon-γ links ultraviolet radiation to melanomagenesis in mice.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, the incidence of which continues to rise. Epidemiological studies show that the major aetiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and aetiological criteria, but only when irradiated as neonatal pups with UVB, not UVA. However, the mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, identifying a novel candidate therapeutic target for a subset of melanoma patients.

Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf

A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing beta-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants, Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitf(mi), with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of beta-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitf(mi)/+ embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quantification and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitf(mi)/Mitf(mi) embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration.

EZH2-dependent suppression of a cellular senescence phenotype in melanoma cells by inhibition of p21/CDKN1A expression

Polycomb group (PcG) proteins such as Enhancer of zeste homolog 2 (EZH2) are epigenetic transcriptional repressors that function through recognition and modification of histone methylation and chromatin structure. Targets of PcG include cell cycle regulatory proteins which govern cell cycle progression and cellular senescence. Senescence is a characteristic of melanocytic nevi, benign melanocytic proliferations that can be precursors of malignant melanoma. In this study, we report that EZH2, which we find absent in melanocytic nevi but expressed in many or most metastatic melanoma cells, functionally suppresses the senescent state in human melanoma cells. EZH2 depletion in melanoma cells inhibits cell proliferation, restores features of a cellular senescence phenotype, and inhibits growth of melanoma xenografts in vivo. p21/CDKN1A is activated upon EZH2 knockdown in a p53-independent manner and contributes substantially to cell cycle arrest and induction of a senescence phenotype. EZH2 depletion removes histone deacetylase 1 (HDAC1) from the CDKN1A transcriptional start site and downstream region, enhancing histone 3 acetylation globally and at CDKN1A. This results in recruitment of RNA polymerase II, leading to p21/CDKN1A activation. Depletion of EZH2 synergistically activates p21/CDKN1A expression in combination with the HDAC inhibitor trichostatin A. Since melanomas often retain wild-type p53 function activating p21, our findings describe a novel mechanism whereby EZH2 activation during tumor progression represses p21, leading to suppression of cellular senescence and enhanced tumorigenicity. Mol Cancer Res; 9(4); 418–29. ©2011 AACR.

Evidence of ultraviolet type mutations in xeroderma pigmentosum melanomas

To look for a direct role of ultraviolet radiation (UV) exposure in cutaneous melanoma induction, we studied xeroderma pigmentosum (XP) patients who have defective DNA repair resulting in a 1000-fold increase in melanoma risk. These XP melanomas have the same anatomic distribution as melanomas in the general population. We analyzed laser capture microdissection samples of skin melanomas from XP patients studied at the National Institutes of Health. The tumor suppressor gene PTEN was sequenced and analyzed for UV-induced mutations. Samples from 59 melanomas (47 melanomas in situ and 12 invasive melanomas) from 8 XP patients showed mutations in the PTEN tumor suppressor gene in 56% of the melanomas. Further, 91% of the melanomas with mutations had 1 to 4 UV type base substitution mutations (occurring at adjacent pyrimidines) (P < 0.0001 compared to random mutations). We found a high frequency of amino-acid-altering mutations in the melanomas and demonstrated that these mutations impaired PTEN function; UV damage plays a direct role in induction of mutations and in inactivation of the PTEN gene in XP melanomas including in situ, the earliest stage of melanoma. This gene is known to be a key regulator of carcinogenesis and therefore these data provide solid mechanistic support for UV protection for prevention of melanoma.

Sorafenib-induced eruptive melanocytic lesions.

We report 2 cases of eruptive melanocytic lesions associated with sorafenib, a multikinase inhibitor FDA-approved for the treatment of advanced renal cell carcinoma. Understanding the development of this previously unknown side effect may provide further insight into sorafenib’s mechanistic effects on tumors and normal tissues and into the development of pigmented lesions.

Diagnostic Value of HMB-45 and Anti-Melan A Staining of Sentinel Lymph Nodes with Isolated Positive Cells

Numerous immunohistochemical stains have been employed to detect metastatic melanoma in sentinel lymph node (SLN) biopsies. HMB-45 is considered by some as a specific tool to detect early metastatic melanoma (1). Occasionally, one or two isolated HMB-45–positive cells may cause complications in diagnostic interpretation. The goal of this study was to evaluate the reliability of HMB-45 staining of SLNs with sparse isolated positive cells and to compare its staining with anti–Melan A antibody. HMB-45 and anti–Melan A antibody immunostaining was performed on (Group A) 15 histologically negative SLNs excised from patients with malignant melanoma (MM) and on (Group B) 15 histologically negative SLNs excised from patients with breast carcinoma (BC). None of the patients had clinical evidence of systemic metastasis at the time of SLN biopsy. Five cutaneous biopsies with changes of postinflammatory hyperpigmentation (PIHP) were also stained with both antibodies. HMB-45 staining was repeated in all Group B SLNs after blocking endogenous biotins. Electron-microscopic studies were performed on all cases of PIHP. Isolated HMB-45–stained cells were present in 6 of 15 SLNs removed for MM; 8 of 15 for BC; and 3 of 5 cutaneous biopsies of PIHP. HMB-45 reactivity persisted after blocking endogenous biotins in 6 of 8 positive SLNs from Group B. Anti–Melan A antibody was negative in all SLNs of group A and B and in dermal melanophages of all five cases of PIHP. HMB-45 positivity was demonstrated in histologically negative SLNs and cutaneous biopsies, especially in the milieu of aggregated melanophages. Phagocytosis of premelanosomes by macrophages in the draining lymph nodes may account for isolated cell positivity and can hinder correct diagnostic interpretation. HMB-45 may not be a reliable marker for the detection of micro-metastasis of MM and requires correlation with other immunohistochemical markers, such as anti–Melan A antibody, to enhance specificity.

Identification of a Population of Epidermal Squamous Cell Carcinoma Cells with Enhanced Potential for Tumor Formation

Epidermal squamous cell carcinoma is among the most common cancers in humans. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation.

SKI knockdown inhibits human melanoma tumor growth in vivo.

The SKI protein represses the TGF‐β tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease‐progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF‐β signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF‐β signaling including high levels of nuclear Smad3 and p21Waf1, which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain‐ and loss‐of‐function approaches and found that simultaneously to blocking the TGF‐β‐growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF‐β‐mediated downregulation of the oncogenic protein c‐MYC, and for inducing the plasminogen activator inhibitor‐1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF‐β pathway and its deficiency restores TGF‐β tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.

Skin cancers, blindness, and anterior tongue mass in African brothers

HistoryTwo Northern African brothers presented to theNational Institutes of Health for evaluation of severedamage to sun-exposed areas of the skin, eyes, andmucosae; multiple skin cancers; a tongue mass; andphotophobia with loss of vision.The patients were born full term after uncompli-cated pregnancies and achieved age-appropriatedevelopmental milestones. Patient XP393BE (Fig 1,A), 23 years old, was noted to have freckle-likepigmented lesions on his face at 2.5 years andphotophobia by age 3 (Table I). A squamous cellcarcinoma(SCC)hadbeenremovedfromhisnoseatage 13 and the site was grafted with sun-shieldedskin from his thigh. His brother, patient XP394BE(Fig 1, D), 17 years old, developed freckle-likelesions on his face by 8 years of age. At age 13, anSCCwasexcisedfromhisrightcheek.Bothboyshadbilateral progressive loss of vision with unilateralblindness since the age of 12 to 14 years (Fig 1, B).PatientXP393BEhada10-year history ofaslowlyenlarging, painful, bleeding mass on the tip of histongue (Fig 2, A). He denied weight loss, excessiveconsumption of alcohol, or chewing of tobacco orbetel quid.LivinginKuwait,Sudan,Libya,andEgypt,neitherpatient had a history of sunburns or use of sunprotection. The patients have a 21-year-old unaf-fected brother. Their parents were second cousinsand members of the same tribe in the Sudan. Therewas no family history of cancer.Physical examinationSkin examination of both patients revealed nu-merous 1- to 5-mm hyperpigmented macules on thecheeksandscalp(seeFigs1and2)andsun-exposedportions of the chest and extremities with sparing ofsun-protected sites. Patient XP393BE had a 1.3- 31.7-cm stellate, indurated, black, brown and grayplaque on his left cheek (Fig 1, A). Dermatoscopyrevealed characteristic leaf-like structures and blue-gray ovoid nests that distinguish pigmented basalcell carcinoma from melanoma (Fig 1, C). A 1-cmnodular, ulcerated mass was present on the base ofhis nose, and there were multiple translucent blacklesions on his face. Patient XP394BE had a crusted,darklypigmentedplaqueontheleftalaandtipofthe

Neurofibromin as a regulator of melanocyte development and differentiation

Patients with the genetic disease type I neurofibromatosis (NF1) exhibit characteristic pigmentary lesions associated with loss of a single allele of NF1, encoding the 260 kDa protein neurofibromin. To understand the basis for these pigmentary problems, the properties of melanocytes haploinsufficient for the murine gene Nf1 were studied using Nf1+/– knockout mice. We demonstrate that neurofibromin regulates the Kit-Mitf signaling axis in vivo during melanocyte development. Primary Nf1+/– melanocytes were purified by FACS to measure melanogenic gene expression. We found that Nf1+/– melanocytes exhibit higher levels of melanogenic gene expression than their wild-type counterparts. Both prior to and following Kit stimulation, Nf1+/– melanocytes also exhibit increased activation of the MAP kinase pathway compared with primary cells. The melanogenic response of primary melanocytes to Mek inhibition is consistent with the changes observed with Nf1 haploinsufficiency; however, these changes differ from those observed with their immortalized counterparts. The observation that reduction of neurofibromin, either from haploinsufficiency in the case of primary melanocytes or from neurofibromin knockdown in the case of melan-a cells, enhances melanogenic gene expression suggests that neurofibromin plays a dominant role to MEK activity in controlling melanogenic gene expression in murine melanocytes.