Different genetic mechanisms mediate spontaneous versus UVR-induced malignant melanoma.

Genetic variation conferring resistance and susceptibility to carcinogen-induced tumorigenesis is frequently studied in mice. We have now turned this idea to melanoma using the collaborative cross (CC), a resource of mouse strains designed to discover genes for complex diseases. We studied melanoma-prone transgenic progeny across seventy CC genetic backgrounds. We mapped a strong quantitative trait locus for rapid onset spontaneous melanoma onset to Prkdc, a gene involved in detection and repair of DNA damage. In contrast, rapid onset UVR-induced melanoma was linked to the ribosomal subunit gene Rrp15. Ribosome biogenesis was upregulated in skin shortly after UVR exposure. Mechanistically, variation in the 'usual suspects' by which UVR may exacerbate melanoma, defective DNA repair, melanocyte proliferation, or inflammatory cell infiltration, did not explain melanoma susceptibility or resistance across the CC. Instead, events occurring soon after exposure, such as dysregulation of ribosome function, which alters many aspects of cellular metabolism, may be important.

Keratinocyte Cytokine Networks Associated with Human Melanocytic Nevus Development.

Melanocytes can group together in nevi, commonly thought to form because of intrinsic somatic mutations involving MAPK pathway activation. However, the role of the microenvironment, in particular keratinocytes, in nevogenesis is rarely studied. Melanocytes proliferate during the hair follicle growth phase and in some basal cell carcinomas, allowing us to construct keratinocyte gene expression clusters correlated with melanocyte activation. We asked whether such correlations are evident in the more subtle context of regulation of melanocyte behavior in normal skin. We considered genes which, when mutated in keratinocytes in mice, lead to nevogenesis. Across the human GTEx normal skin database, their expression was correlated with that of keratinocyte cytokines KITLG, HGF, FGF2, EDN1, and melanocyte markers. These cytokines have pleiotropic effects on melanocyte-specific and pigmentation genes and also influence mast cell gene expression. We show five classes of keratinocyte genes that, via germline genetic variation, influence melanocyte activity. These include genes involved in SHH signaling, structural keratins, ribosomal biogenesis, and stem cell governance. In agreement with the finding of KITLG linked to nevogenesis in human genome-wide association studies, we provide evidence that specific keratinocyte cytokines are components of networks that may drive or exacerbate nevus development.

Keratinocyte Sonic Hedgehog Upregulation Drives the Development of Giant Congenital Nevi via Paracrine Endothelin-1 Secretion.

Giant congenital nevi are associated with clinical complications such as neurocutaneous melanosis and melanoma. Virtually nothing is known about why some individuals develop these lesions. We previously identified the sonic hedgehog (Shh) pathway regulator Cdon as a candidate nevus modifier gene. Here we validate this by studying Cdon knockout mice, and go on to establishing the mechanism by which Shh exacerbates nevogenesis. Cdon knockout mice develop blue nevi without the need for somatic melanocyte oncogenic mutation. In a mouse model carrying melanocyte NRASQ61K, we found that strain backgrounds that carry genetic variants that cause increased keratinocyte Shh pathway activity, as measured by Gli1 and Gli2 expression, develop giant congenital nevi. Shh components are also active adjacent to human congenital nevi. Mechanistically, this exacerbation of nevogenesis is driven via the release of the melanocyte mitogen endothelin-1 from keratinocytes. We then suppressed nevus development in mice using Shh and endothelin antagonists. Our work suggests an aspect of nevus development whereby keratinocyte cytokines such as endothelin-1 can exacerbate nevogenesis, and provides potential therapeutic approaches for giant congenital nevi. Furthermore, it highlights the notion that germline genetic variation, in addition to somatic melanocyte mutation, can strongly influence the histopathological features of melanocytic nevi.

Targeting Adenosine in BRAF-Mutant Melanoma Reduces Tumor Growth and Metastasis.

Increasing evidence exists for the role of immunosuppressive adenosine in promoting tumor growth and spread in a number of cancer types, resulting in poor clinical outcomes. In this study, we assessed whether the CD73-adenosinergic pathway is active in melanoma patients and whether adenosine restricts the efficacy of clinically approved targeted therapies for commonly mutated BRAFV600E melanoma. In AJCC stage III melanoma patients, CD73 expression (the enzyme that generates adenosine) correlated significantly with patients presenting nodal metastatic melanoma, suggesting that targeting this pathway may be effective in advanced stage disease. In addition, dabrafenib and trametinib treatment of CD73+ BRAFV600E-mutant melanomas caused profound CD73 downregulation in tumor cells. Inhibition of BRAF and MEK in combination with the A2A adenosine receptor provided significant protection against tumor initiation and metastasis formation in mice. Our results suggest that targeting adenosine may enhance therapeutic responses for melanoma patients receiving targeted or immune-based therapies. Cancer Res; 77(17); 4684-96. ©2017 AACR.

A mutation in the Cdon gene potentiates congenital nevus development mediated by NRAS(Q61K).

Congenital nevi develop before birth and sometimes cover large areas of the body. They are presumed to arise from the acquisition of a gene mutation in an embryonic melanocyte that becomes trapped in the dermis during development. Mice bearing the Cdk4(R24C) ::Tyr-NRAS(Q) (61K) transgenes develop congenital nevus-like lesions by post-natal day 10, from melanocytes escaping the confines of hair follicles. We interbred these mice with the collaborative cross (CC), a resource that enables identification of modifier genes for complex diseases (those where multiple genes are involved). We examined variation in nevus cell density in 66 CC strains and mapped a large-effect quantitative trait locus (QTL) controlling nevus cell density to murine chromosome 9. The best candidate for a gene that exacerbates congenital nevus development in the context of an NRAS mutation is Cdon, a positive regulator of sonic hedgehog (Shh) that is expressed mainly in keratinocytes.

Fluorometric study of fluoxetine DNA binding.

Fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI), is commonly prescribed to treat depression. The interaction between FLX antidepressant and calf thymus DNA (ctDNA) was investigated under simulated physiological conditions (Tris-HCl buffer at pH 7.4) using methylene blue [3,7-bis(dimethylamino) pheno-thrazin-5-ium chloride] (MB) dye as a probe using fluorescence spectroscopy. A strong fluorescence quenching reaction of DNA to FLX was observed. The corresponding numbers of binding sites (n) and binding constants (K(f)) of DNA with FLX at 281, 310 and 318K were calculated 2.1×10(5), 6.7×10(5) and 9.7×10(5) respectively. It can be concluded that FLX molecules could interact with ctDNA via outside, non-intercalative, binding as evidenced by quenching study with I(-), ionic strength with NaCl and competitive investigation with MB. Thermodynamic parameters, enthalpy (ΔH) and entropy (ΔS) changes were calculated according to Van't Hoff equation, which indicated that reaction is entropically driven. Furthermore, the interaction of FLX with poly A-T and poly C-G were carried out in order to comprehend the binding location of drug to DNA.

DNA-binding studies of fluoxetine antidepressant.

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) antidepressant that is widely prescribed. The DNA-binding behavior of fluoxetine antidepressant and calf thymus DNA was investigated in Tris-HCl buffer at physiological pH 7.4 with a series of techniques, including UV-Vis and circular dichroism spectroscopies, competitive study with Hoechst 33258, viscometry, and cyclic voltammetry. Fluoxetine molecules bind to DNA via groove mode as illustrated by hypochromism with no red shift in the UV absorption band of fluoxetine, decrease in Hoechst-DNA solution fluorescence, and no significant changes in viscosity of DNA. The CD spectra of DNA molecules show a little change in stacking mode of base pair but no modification changes in DNA conformation, for example, from B-DNA to A or C-DNA. The binding constant (K(b)) of DNA with fluoxetine was calculated to be 6.7 × 10(4) M(-1), which is in the range of reported and known groove binders, such as distamycin. All results showed the groove-binding mode of interaction of fluoxetine with DNA.