John D'Orazio

Melanoma is the deadliest form of skin cancer because of its propensity to spread beyond the primary site of disease and because it resists many forms of treatment. Incidence of melanoma has been increasing for decades. Though ultraviolet radiation (UV) has been identified as the most important environmental causative factor for melanoma development, UV-protective strategies have had limited efficacy in melanoma prevention. UV mutational burden correlates with melanoma development and tumor progression, underscoring the importance of UV in melanomagenesis. However, besides amount of UV exposure, melanocyte UV mutational load is influenced by the robustness of nucleotide excision repair, the genome maintenance pathway charged with removing UV photoproducts before they cause permanent mutations in the genome. In this review, we highlight the importance of the melanocortin hormonal signaling axis on regulating efficiency of nucleotide excision repair in melanocytes. By understanding th...

Loss-of-function in melanocortin 1 receptor (MC1R), a GS protein-coupled receptor that regulates signal transduction through cAMP and protein kinase A (PKA) in melanocytes, is a major inherited melanoma risk factor. Herein, we report a novel cAMP-mediated response for sensing and responding to UV-induced DNA damage regulated by A-kinase-anchoring protein 12 (AKAP12). AKAP12 is identified as a necessary participant in PKA-mediated phosphorylation of ataxia telangiectasia mutated and Rad3-related (ATR) at S435, a post-translational event required for cAMP-enhanced nucleotide excision repair (NER). Moreover, UV exposure promotes ATR-directed phosphorylation of AKAP12 at S732, which promotes nuclear translocation of AKAP12-ATR-pS435. This complex subsequently recruits XPA to UV DNA damage and enhances 5' strand incision. Preventing AKAP12's interaction with PKA or with ATR abrogates ATR-pS435 accumulation, delays recruitment of XPA to UV-damaged DNA, impairs NER and increases UV...

The melanocortin 1 receptor (MC1R) is a melanocytic Gs protein coupled receptor that regulates skin pigmentation, UV responses, and melanoma risk. It is a highly polymorphic gene, and loss of function correlates with a fair, UV-sensitive, and melanoma-prone phenotype due to defective epidermal melanization and sub-optimal DNA repair. MC1R signaling, achieved through adenylyl cyclase activation and generation of the second messenger cAMP, is hormonally controlled by the positive agonist melanocortin, the negative agonist agouti signaling protein, and the neutral antagonist β-defensin 3. Activation of cAMP signaling up-regulates melanin production and deposition in the epidermis which functions to limit UV penetration into the skin and enhances nucleotide excision repair (NER), the genomic stability pathway responsible for clearing UV photolesions from DNA to avoid mutagenesis. Herein we review MC1R structure and function and summarize our laboratory's findings on the molecular mechanisms by which MC1R signaling impacts NER.

The melanocortin 1 receptor (MC1R), a GS-coupled receptor that signals through cAMP and protein kinase A (PKA), regulates pigmentation, adaptive tanning, and melanoma resistance. MC1R-cAMP signaling promotes PKA-mediated phosphorylation of ataxia telangiectasia and rad3-related (ATR) at Ser435 (ATR-pS435), a modification that enhances nucleotide excision repair (NER) by facilitating recruitment of the XPA protein to sites of UV-induced DNA damage. High-throughput methods were developed to quantify ATR-pS435, measure XPA-photodamage interactions, and assess NER function. We report that melanocyte-stimulating hormone (α-MSH) or ACTH induce ATR-pS435, enhance XPA's association with UV-damaged DNA and optimize melanocytic NER. In contrast, MC1R antagonists agouti signaling protein (ASIP) or human β-defensin 3 (HBD3) interfere with ATR-pS435 generation, impair the XPA-DNA interaction, and reduce DNA repair. Although ASIP and HBD3 each blocked α-MSH-mediated induction of the signaling pathway, only ASIP depleted basal ATR-pS435. Our findings confirm that ASIP diminishes agonist-independent MC1R basal signaling whereas HBD3 is a neutral MC1R antagonist that blocks activation by melanocortins. Furthermore, our data suggest that ATR-pS435 may be a useful biomarker for the DNA repair-deficient MC1R phenotype.

This thesis examined the effects of two superantigens (sAgs), staphylococcal enterotoxin B (SEB) and Mycoplasma arthritidis mitogen (MAM) on human natural killer (NK) cells. Incubation of peripheral blood mononuclear cells (PBMC) with either sAg augmented NK cytotoxicity against NK-sensitive targets in a dose-dependent manner with the greatest augmentation occurring within the first 24 hours. Additionally, incubation of PBMC with SEB

As a group, malignancies of the skin are the most commonly diagnosed cancers, with over a million cases each year identified in the United States alone. While the keratinocyte malignancies – basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) – represent the majority of clinical cases of skin cancer, malignant melanoma is the most deadly form of skin cancer. Each of these skin malignancies is clearly linked to UV radiation, with chronic cumulative UV exposure being most relevant for keratinocyte malignancies and intense, blistering sunburns most relevant for melanoma. In this review, we describe the epidemiology of skin cancer, the link with UV radiation, and the innate defenses used to resist UV damage with particular attention to the nucleotide excision DNA repair pathway. We also focus on the role of skin pigmentation and the molecular events that control melanization of the skin, since these signaling pathways appear to be major determinants of skin cancer risk. We are ...

We describe the case of a 5-year-old girl whose abdominal pain and distension were caused by Wilms tumor of the kidney. Because of the bilateral nature of her disease, she was spared biopsy or initial nephrectomy as part of her treatment course. Rather, she was treated presumptively for Wilms tumor based primarily on radiologic findings. Neoadjuvant chemotherapy consisting of vincristine, dactinomycin and doxorubicin was given to facilitate nephron-sparing surgery for tumor resection. Her initial chemotherapeutic course was complicated by tumor lysis syndrome manifested by elevated serum uric acid and was treated effectively with hyperhydration and alkalization of intravenous fluids. The patient's disease responded well to chemotherapy, and she underwent successful tumor excision after 12 weeks of chemotherapy. The resected tumor was identified as anaplastic Wilms tumor, illustrating that pathologic identification of Wilms tumor is possible even after multiple cycles of neoadjuv...

ABSTRACT The V600E gain-of-function mutation in the BRAF serine/threonine kinase is a driver mutation in roughly 50% of human melanomas, in nearly all hairy cell leukemias and in certain subsets of colorectal, ovarian, lung, breast and papillary thyroid cancers. BRAF(V) (600E) is generated as a result of a 1799T>A missense change in exon 15 of the BRAF gene, causing valine to be replaced by a phosphomimetic moiety (glutamic acid) at the 600(th) residue of the mature protein. This amino acid substitution endows BRAF(V) (600E) with exaggerated and unregulated signaling resulting in continuous activation of the MAPK signaling cascade on which melanomagenesis seems to depend. This article is protected by copyright. All rights reserved.

While the effects of superantigens on T lymphocytes are well characterized, how superantigens interact with other immune cells is less clear. This report examines the effects of Mycoplasma arthritidis mitogen (MAM) on human natural killer (NK) cell activity. Incubation of peripheral blood mononuclear cells (PBMC) with MAM for 16 to 20 h augmented NK cytotoxicity (against K562) in a dose-dependent manner (P < or = 0.05). Superantigen-dependent cellular cytotoxicity, an activity of superantigen-activated cytotoxic T cells, was not involved in lysis of K562 cells because the erythroleukemic tumor target cells expressed no class II major histocompatibility complex by fluorescence-activated cell sorter analysis. Kinetic experiments showed that the largest increase in NK activity induced by MAM occurred within 48 h. Incubation with MAM caused a portion of NK cells to become adherent to tissue culture flasks, a quality associated with activation, and augmented NK activity was found in b...

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection (1). Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Two siblings presenting with exudative retinopathy, thrombocytopenia, and macrocytosis were found to have markedly shortened telomeres and a previously unreported inherited mutation in TERT, c.2603A>G. Revesz syndrome, a subtype of dyskeratosis congenita (DC) caused by TINF2 mutation, combines marrow failure with exudative retinopathy, intracranial calcifications, and neurocognitive impairment. As our patients manifested neither intracranial calcification nor significant neurocognitive impairment, we conclude that the c.2603A>G TERT mutation may define a subtype of DC manifesting first as exudative retinopathy without other signs of DC. Children with exudative retinopathy should be periodically screened for macrocytosis and cytopenias to evaluate for underlying DC.

We previously reported that topical application of forskolin to the skin of fair-skinned MC1R-defective mice with epidermal melanocytes resulted in accumulation of eumelanin in the epidermis and was highly protective against UV-mediated cutaneous injury. In this report, we describe the long-term effects of chronic topical forskolin treatment in this animal model. Forskolin-induced eumelanin production persisted through 3 months of daily applications, and forskolin-induced eumelanin remained protective against UV damage as assessed by minimal erythematous dose (MED). No obvious toxic changes were noted in the skin or overall health of animals exposed to prolonged forskolin therapy. Body weights were maintained throughout the course of topical forskolin application. Topical application of forskolin was associated with an increase in the number of melanocytes in the epidermis and thickening of the epidermis due, at least in part, to an accumulation of nucleated keratinocytes. Together, these data suggest in this animal model, short-term topical regular application of forskolin promotes eumelanin induction and that over time, topical forskolin treatment is associated with persistent melanization, epidermal cell accumulation, and skin thickening.

Though infantile leukemia has a historically poor prognosis, there may be a subset of patients with cutaneous disease whose disease will resolve without therapy. The authors report a case of infantile leukemia cutis who presented with a single subcutaneous chloroma that spontaneously resolved over the course of several weeks and who remains without evidence of disease nearly two years later. After reviewing the literature of congenital leukemia cutis, the authors conclude that withholding chemotherapy in infants with cutaneous myeloid leukemia in the absence of known negative prognostic factors (MLL or BCR-ABL translocations) or progressive disease is clinically indicated.

People with pale skin, red hair, freckles and an inability to tan--the 'red hair/fair skin' phenotype--are at highest risk of developing melanoma, compared to all other pigmentation types. Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAF(V600E), into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced…

A 2-year-old boy presented to his primary care provider with a 2-week history of daily fevers (to 39 C), malaise, increasing pallor, decreased appetite, and a 3-lb weight loss. Review of systems was otherwise negative (no history of cough, vomiting, diarrhea, or rash). The ...

UV-induced pigmentation (suntanning) requires induction of alpha-melanocyte-stimulating hormone (alpha-MSH) secretion by keratinocytes. alpha-MSH and other bioactive peptides are cleavage products of pro-opiomelanocortin (POMC). Here we provide biochemical and genetic evidence demonstrating that UV induction of POMC/MSH in skin is directly controlled by p53. Whereas p53 potently stimulates the POMC promoter in response to UV, the absence of p53, as in knockout mice, is associated with absence of the UV-tanning response. The same pathway produces beta-endorphin, another POMC derivative, which potentially contributes to sun-seeking behaviors. Furthermore, several instances of UV-independent pathologic pigmentation are shown to involve p53 "mimicking" the tanning response. p53 thus functions as a sensor/effector for UV pigmentation, which is a nearly constant environmental exposure. Moreover, this pathway is activated in numerous conditions of pathologic pigmentation and thus mimics the tanning response.