AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis.

Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here ß1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

Distinguishing migration from isolation using genes with intragenic recombination: detecting introgression in the Drosophila simulans species complex.

BACKGROUND: Determining the presence or absence of gene flow between populations is the target of some statistical methods in population genetics. Until recently, these methods either avoided the use of recombining genes, or treated recombination as a nuisance parameter. However, genes with recombination contribute additional information for the detection of gene flow (i.e. through linkage disequilibrium). METHODS: We present three summary statistics based on the spatial arrangement of fixed differences, and shared and exclusive polymorphisms that are sensitive to the presence and direction of gene flow. Power and false positive rate for tests based on these statistics are studied by simulation. RESULTS: The application of these tests to populations from the Drosophila simulans species complex yielded results consistent with migration between D. simulans and its two endemic sister species D. mauritiana and D. sechellia, and between populations D. mauritiana on the islands of the Mauritius and Rodrigues. CONCLUSIONS: We demonstrate the sensitivity of the developed statistics to the presence and direction of gene flow, and characterize their power as a function of differentiation level and recombination rate. The properties of these statistics make them especially suitable for analyzing high-throughput sequencing data or for their integration within the approximate Bayesian computation framework.

Constitutive activation of the ERK pathway in melanoma and skin melanocytes in Grey horses.

BACKGROUND: Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses. METHODS: Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting. RESULTS: We found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses. CONCLUSIONS: These findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this pathway during melanomagenesis. These findings are consistent with the universal importance of the ERK pathway in melanomagenesis and may have valuable implications for human melanoma research.

RACK1, a clue to the diagnosis of cutaneous melanomas in horses.

BACKGROUND: Melanocytic proliferations are common in horses but the diagnosis of malignancy is not always straightforward. To improve diagnosis and prognosis, markers of malignancy are needed. Receptor for activated C kinase 1 (RACK1) protein may be such a marker. RACK1 was originally found to characterize malignant melanocytic lesions in the Melanoblastoma-bearing Libechov minipig (MeLiM) and, later, in human patients. Our purpose was to investigate the value of RACK1 in the classification of cutaneous melanocytic proliferations in horses. RESULTS: Using immunofluorescence, we report here that both MITF (Microphthalmia-associated transcription factor) and PAX3 (Paired box 3) allow the identification of melanocytic cells in horse skin samples. Importantly, RACK1 was detected in melanocytic lesions but not in healthy skin melanocytes. Finally, we found that RACK1 labeling can be used in horses to distinguish benign melanocytic tumors from melanomas. Indeed, RACK1 labeling appeared more informative to assess malignancy than individual histomorphological features. CONCLUSIONS: This study confirms that horses provide an interesting model for melanoma genesis studies. It establishes MITF and PAX3 as markers of horse melanocytic cells. RACK1 emerges as an important marker of malignancy which may contribute to progress in the diagnosis of melanomas in both human and veterinary medicine.

Single-dose radiotherapy disables tumor cell homologous recombination via ischemia/reperfusion injury.

Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.

Haplosufficiency of PAX3 for melanoma development in Tyr: NRASQ61K; Cdkn2a-/- mice allows identification and sorting of melanoma cells using a Pax3GFP reporter allele.

The role of the Pax3 gene in embryonic development of pigment cells is well characterized. By contrast, the function of Pax3 in melanoma development is controversial. Indeed, data obtained from cultured cells suggest that PAX3 may contribute to melanomagenesis. PAX3 is found to be overexpressed in melanomas and also in nevi compared with normal skin samples. Pax3 homozygous loss of function is embryonic lethal. To assess the role of Pax3 in melanoma development in vivo, we analyzed Pax3 haploinsufficiency in a mouse model of melanoma predisposition. The Pax3(GFP/+) knock-in reporter system was combined with the Tyr::NRAS(Q61K); Cdkn2a(-/-) mouse melanoma model. Melanoma development was followed over 18 months. Histopathological, immunohistochemical, and molecular analyses of lesions at different stages of melanoma progression were carried out. Fluorescence-activated cell sorting on GFP of cells from primary or metastatic melanoma was followed by ex-vivo transformation tests and in-vivo passaging. We report here that Tyr::NRAS(Q61K); Cdkn2a(-/-); Pax3(GFP/+) mice developed metastasizing melanoma as their Tyr::NRAS(Q61K); Cdkn2a(-/-); littermates. Histopathology showed no differences between the two genotypes, although Pax3 mRNA and PAX3 protein levels in Pax3(GFP/+) lesions were reduced by half. The Pax3(GFP) allele proved to be a convenient marker to identify and directly sort heterogeneous populations of melanoma cells within the tumor bulk at each stage of melanoma progression. This new mouse model represents an accurate and reproducible means for identifying melanoma cells in vivo to study the mechanisms of melanoma development.

Pax3( GFP ) , a new reporter for the melanocyte lineage, highlights novel aspects of PAX3 expression in the skin.

The paired box gene 3 (Pax3) is expressed during pigment cell development. We tested whether the targeted allele Pax3(GFP) can be used as a reporter gene for pigment cells in the mouse. We found that enhanced green fluorescent protein (GFP) can be seen readily in every melanoblast and melanocyte in the epidermis and hair follicles of Pax3(GFP/+) heterozygotes. The GFP was detected at all differentiation stages, including melanocyte stem cells. In the dermis, Schwann cells and nestin-positive cells of the piloneural collars resembling the nestin-positive hair follicle multipotent stem cells exhibited a weaker GFP signal. Pigment cells could be purified by fluorescent activated cell sorting and grown in vitro without feeder cells, giving pure cultures of melanocytes. The Schwann cells and nestin-positive cells of the piloneural collars were FACS-isolated based on their weak expression of GFP. Thus Pax3(GFP) can discriminate distinct populations of cells in the skin.