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663 PIKFYVE regulates the maturation of the melanosome
Pigmentation & Melanoma | ABSTRACTS 660
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Phenformin targets the stem cell compartment in melanoma K Dallaglio5, A Romagnani1, T Petrachi7, G Grisendi2, M Dominici2, C Longo3, G Argenziano3, S Piana4, A Albini6 and A Ciarrocchi7 1 Oncology, ASMN-IRCCS, Reggio Emilia, Italy, 2 Division of Oncology, University of Modena and Reggio Emilia, Modena, Italy, 3 Skin Cancer Unit, ASMN-IRCCS, Reggio Emilia, Italy, 4 Pathology Unit, ASMN-IRCCS, Reggio Emilia, Italy, 5 Scientific Direction, Translational Research Lab., Arcispedale S. Maria Nuova ASMN-IRCCS, Reggio Emilia, Italy, 6 Scientific and Technology Pole, IRCCS MultiMedica, Milan, Milan, Italy and 7 Scientific Direction, Translational Research Lab., ASMN-IRCCS, Reggio Emilia, Italy Melanoma, the deadliest form of skin cancer, is highly resistant to conventional therapies due to the persistence in the patients of stem-like tumor cells, namely cancer stem cells (CSC). Therapies targeting CSCs are warranted. A promising therapeutic strategy is to target cell metabolism, as resistant cells display a specific metabolic asset. Phenformin (Phe) is an anti-diabetic drug perturbing cell metabolism and displaying anti-cancer activity. To date, the effect of Phe on melanoma CSC is unknown. We evaluated the ability of Phe to target melanoma CSC (FACS sorted ALDHhigh cells) and stem cell traits using melanoma cell lines, patient-derived primary cells in monolayer cell cultures and 3D spheroids. We show that Phe abrogates cell viability and growth in monolayer cell cultures and spheroids. In parallel, it reduces the levels of the pro-survival factor MITF in these cells. Phe blocks melanoma spheroids invasion in matrigel, independently from Phe-induced reduction of cell growth. Once sorted, ALDHhigh melanoma cells generate slightly bigger, more viable and less necrotic spheres as compared to ALDHlow cells. While paclitaxel kills mainly ALDHlow cells, Phe reduces ALDHhigh cell viability and the number of viable cells in ALDHhigh -derived spheroids. Treatment of melanoma cells with Phe significantly decreases the expression of the stemness markers ALDH1A and SOX2. Consistently, Phe reduces cell viability and growth in a model of A375 genetically modified to constitutively overexpress SOX2. Our results show that Phe decreases melanoma stem cell traits in melanoma and suggest its potential therapeutic use.
UCHL1 inhibits melanogenesis via regulation of MITF protein level in primary human epidermal melanocyte S Jin1,2,3, E Seo1,2, Y Lee1,2,3, Y Kim1,2,3, D Lee1,2 and J Chung1,2,3 1 Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2 Institute of Human-Environment Interface Biology, Medical Research Center, Seoul National University, Seoul, Korea (the Republic of) and 3 Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Korea (the Republic of) MITF (microphthalmia-associated transcription factor) plays as a master regulator in skin pigmentation. Its protein level is controlled by post-transcriptional regulation, which is such as phosphorylation and ubiquitination. However, it is to be elucidated that which enzymes catalyze MITF ubiquitination. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is an important member of ubiquitin proteasome system and has an E3 ligase activity. Although UCHL1 is expressed in human melanocytes, there has been no reports of its functional role in skin pigmentation. In this study, we examined whether UCHL1 is involved in the regulation of melanogenesis in normal human epidermal melanocytes. Employing in vitro and in vivo studies, we found UCHL1 inhibits melanin synthesis, as well as tyrosinase and tyrosinaserelated protein (TRP) levels were down-regulated. These are results from reduced MITF protein levels which is mediated by overexpression of UCHL1. Taken together, we provide an evidence that MITF is a new targets for UCHL1 E3 ligase activity and UCHL1 may be a biological effector for the developments of hyperpigmented disorders.
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Numb is induced by GSK3 inhibition and inhibits melanoma migration, invasion and metastasis D Hristova1, X Hua1, J Wang1, L Li1, M Beqiri1, A Watters1, A Vultur1, Z Wei2, M Herlyn1 and M Fukunaga-Kalabis1 1 Wistar Institute, Philadelphia, PA and 2 New Jersey Institute of Technology, Newark, NJ The up-regulation of adaptor protein Numb by the canonical Wnt pathway plays a role in triggering melanocytic differentiation from multipotent skin stem cells, which share many properties with aggressive melanoma cells. Numb acts as a tumor suppressor in various human cancer types, however little is known about its role in melanoma. Here, we investigated the role of Numb in melanoma progression and the mechanism of its regulation. An analysis of The Cancer Genome Atlas (TCGA) melanoma dataset revealed that high levels of Numb expression in melanoma tissues correlate with improved patient survival. In the majority of melanoma cell lines, Numb was not up-regulated by canonical Wnt ligand treatment, while the activation of Wnt pathway by a GSK3 inhibitor BIO was able to increase Numb expression, even in the Wnt ligand-refractory cells. BIO treatment reduced migration of melanoma cells in a 3D context in Numb-dependent manner. Numb-depleted melanoma cells significantly increased tumor metastasis nodules in lung tissues by tail vein injection compared to control cells. These results suggest that Numb acts as a metastatic suppressor in melanoma, and the targeted inhibition of GSK3 may have therapeutic effects through up-regulating Numb.
PIKFYVE regulates the maturation of the melanosome MC Liggins1, J Flesher1, S Sandhu2, R Boissy3 and A Ganesan1,2 1 Department of Biological Chemistry, University of California, Irvine, Irvine, CA, 2 Department of Dermatology, University of California, Irvine, Irvine, CA and 3 Department of Dermatology, University of Cincinnati, Cincinnati, OH Phosphoinositide lipids coat the surface of transport vesicles and interact with PI binding proteins on target membranes to direct vesicle fusion events. While specific phosphoinositides involved in autophagy and Golgi trafficking have been identified, it is not currently known what phosphoinositides and phosphoinositide binding proteins regulate melanosome biogenesis. VAC14, SAC3, and PIKFYVE form the only mammalian PtdIns5P kinase complex, which synthesizes PI(3,5)P2 which can then be converted to PI(5)P. Published studies have determined that VAC14 or SAC3 mouse mutants and knockouts have severe neurological defects and dilute coat color, implicating a role for the PIKFYVE complex in regulating melanosome maturation. In this study, we investigated the role of PIKFYVE, the only PtdIns5P kinase, in melanogenesis. Partial depletion of PIKfyve, VAC14, or SAC3 using shRNAs in melanocytes resulted in decreased tyrosinase protein and mRNA accumulation and decreased expression of the melanogenic regulator MITF-M but did not block melanosome maturation. These results suggest that the PIKFYVE complex can regulate the transcription of proteins involved in melanogenesis. More complete inhibition of PIKFYVE using pharmacologic agents inhibited melanin accumulation by blocking the formation of stage III melanosomes and the trafficking of proteins to the melanosome and lysosome, validating that PIKFYVE also controls vesicle maturation. Addition of PI(5)P but not PI(3,5)P2 to cultured cells could induce the expression of tyrosinase, indicating that PI(5)P, which is primarily derived from PI(3,5)P2, regulates melanogenesis. Melanocyte-specific PIKFYVE knockout mice exhibited a coat that contained numerous white hairs, indicating that PIKFYVE regulates pigmentation in vivo. Taken together, these results define a specific and novel role for the phosphoinositide PI(5)P in regulating melanogenesis.
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Vasoactive intestinal peptide (VIP) regulates human melanocyte biology and hair follicle pigmentation M Bertolini1,2, M Ba¨hr3, M Sulk1, L Ponce1,2, JA Hardman4, T Bı´ro´5, D Tobin6 and R Paus1,2,4 1 University of Mu¨nster, Mu¨nster, Germany, 2 Monasterium Laboratory, Mu¨nster, Germany, 3 University of Lu¨beck, Lu¨beck, Germany, 4 University of Manchester, Manchester, United Kingdom, 5 University of Debrecen, Debrecen, Hungary and 6 University of Bradford, Bradford, United Kingdom Exploratory pilot experiments in our lab(s) had raised the possibility that vasoactive intestinal peptide (VIP), a key immunoinhibitory neuropeptide released by perifollicular sensory nerve fibers, may be a novel modulator of human HF pigmentation. This hypothesis was followedup in the current study. After showing that the VIP receptors, VPAC1 and VPAC2, are expressed at gene and protein level in human anagen hair bulbs, we investigated whether VIP (10-7M) modulates human HF pigmentation in situ and in isolated human HF melanocytes (HFiMc). VIP significantly increased both the number of intraepithelial c-Kit+ HF melanocytes (HFMc) in situ and intrafollicular c-Kit transcription. By contrast, VIP down-regulated SCF gene and protein expression in organ-cultured human HFs. Interestingly, c-Kit/gp100 double- immunostaining indicated that most of the VIP-induced c-Kit+ HFMc represent immature/amelanotic HFMc. Intriguingly, VIP up-regulated the total number of gp100+, MITF+, or p-MITF+ HFMc and promoted intrafollicular melanin production in situ. VIP also significantly stimulated melanogenesis in isolated human HFiMc, suggesting that it impacts directly on HFMc, but also slightly inhibited HFMc/HFiMc proliferation in situ and in vitro. Preliminary results obtained with specific antagonists suggested that VPAC1 and VPAC2 mediated the VIP effects on HFMc. Since microarray and qRT-PCR analyses showed an up-regulation of phosphodiesterase 4D7 transcription in VIP-treated HFs, VIP may regulate the cAMP level in HFMc, possibly by utilizing downstream pathways shared with a-MSH signaling. In summary, our results reveal VIP as a novel, complex and clinically-relevant neuroendocrine regulator of human HF pigmentation.
Expression of a-Synuclein in melanocytic cells W Eng1, S Saikaly1, M Jimenez-Capdeville2, I Rodriguez-Leyva2 and RA Norman1 1 Pathology, University of Central Florida Medical School, Lake Nona, FL and 2 Neurology, University of St Luis, St Luis- Potsi, Mexico Objective: Previous epidemiological studies have demonstrated an increased prevalence of melanoma in patients with Parkinson’s Disease (PD). The pathological marker of PD is Lewy Bodies which are composed of a-synuclein protein. This study examines the extent of a-synuclein expression in variety of melanocytic neoplasms. Methodology: Using conventional immunohistochemistry with monoclonal antibodies against a-synuclein and tyrosinase, we processed skin-punch or shave biopsies containing Spitz nevus, intradermal nevus, dysplastic nevus, and melanoma. Control tissue included normal skin and acrochordons. Results: In all cases, between 70% to 85% of the melanocytic cells: benign, dysplastic and malignant, were found to express a-synuclein immunopositivity to various degrees. Conclusions: In the brain, a-synuclein participates releasing pre-synaptic vesicles from nerve terminals among other functions. Given its neural ectodermal origin, melanocytic cells may be using a-synuclein in a similar role to distribute melanin to keratinocytes. The typical aggregates of a-synuclein (Lewy Bodies) are not observed in cutaneous melanomas, thus it is unclear how a-synuclein plays a pathological role in the development of melanoma. Further research to delineate the role of a-synuclein may lead to a therapeutic agent or improved understanding of the pathophysiology of both melanoma and PD.
www.jidonline.org S117
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Phenformin targets the stem cell compartment in melanoma K Dallaglio5, A Romagnani1, T Petrachi7, G Grisendi2, M Dominici2, C Longo3, G Argenziano3, S Piana4, A Albini6 and A Ciarrocchi7 1 Oncology, ASMN-IRCCS, Reggio Emilia, Italy, 2 Division of Oncology, University of Modena and Reggio Emilia, Modena, Italy, 3 Skin Cancer Unit, ASMN-IRCCS, Reggio Emilia, Italy, 4 Pathology Unit, ASMN-IRCCS, Reggio Emilia, Italy, 5 Scientific Direction, Translational Research Lab., Arcispedale S. Maria Nuova ASMN-IRCCS, Reggio Emilia, Italy, 6 Scientific and Technology Pole, IRCCS MultiMedica, Milan, Milan, Italy and 7 Scientific Direction, Translational Research Lab., ASMN-IRCCS, Reggio Emilia, Italy Melanoma, the deadliest form of skin cancer, is highly resistant to conventional therapies due to the persistence in the patients of stem-like tumor cells, namely cancer stem cells (CSC). Therapies targeting CSCs are warranted. A promising therapeutic strategy is to target cell metabolism, as resistant cells display a specific metabolic asset. Phenformin (Phe) is an anti-diabetic drug perturbing cell metabolism and displaying anti-cancer activity. To date, the effect of Phe on melanoma CSC is unknown. We evaluated the ability of Phe to target melanoma CSC (FACS sorted ALDHhigh cells) and stem cell traits using melanoma cell lines, patient-derived primary cells in monolayer cell cultures and 3D spheroids. We show that Phe abrogates cell viability and growth in monolayer cell cultures and spheroids. In parallel, it reduces the levels of the pro-survival factor MITF in these cells. Phe blocks melanoma spheroids invasion in matrigel, independently from Phe-induced reduction of cell growth. Once sorted, ALDHhigh melanoma cells generate slightly bigger, more viable and less necrotic spheres as compared to ALDHlow cells. While paclitaxel kills mainly ALDHlow cells, Phe reduces ALDHhigh cell viability and the number of viable cells in ALDHhigh -derived spheroids. Treatment of melanoma cells with Phe significantly decreases the expression of the stemness markers ALDH1A and SOX2. Consistently, Phe reduces cell viability and growth in a model of A375 genetically modified to constitutively overexpress SOX2. Our results show that Phe decreases melanoma stem cell traits in melanoma and suggest its potential therapeutic use.
UCHL1 inhibits melanogenesis via regulation of MITF protein level in primary human epidermal melanocyte S Jin1,2,3, E Seo1,2, Y Lee1,2,3, Y Kim1,2,3, D Lee1,2 and J Chung1,2,3 1 Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2 Institute of Human-Environment Interface Biology, Medical Research Center, Seoul National University, Seoul, Korea (the Republic of) and 3 Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Korea (the Republic of) MITF (microphthalmia-associated transcription factor) plays as a master regulator in skin pigmentation. Its protein level is controlled by post-transcriptional regulation, which is such as phosphorylation and ubiquitination. However, it is to be elucidated that which enzymes catalyze MITF ubiquitination. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is an important member of ubiquitin proteasome system and has an E3 ligase activity. Although UCHL1 is expressed in human melanocytes, there has been no reports of its functional role in skin pigmentation. In this study, we examined whether UCHL1 is involved in the regulation of melanogenesis in normal human epidermal melanocytes. Employing in vitro and in vivo studies, we found UCHL1 inhibits melanin synthesis, as well as tyrosinase and tyrosinaserelated protein (TRP) levels were down-regulated. These are results from reduced MITF protein levels which is mediated by overexpression of UCHL1. Taken together, we provide an evidence that MITF is a new targets for UCHL1 E3 ligase activity and UCHL1 may be a biological effector for the developments of hyperpigmented disorders.
662
663
Numb is induced by GSK3 inhibition and inhibits melanoma migration, invasion and metastasis D Hristova1, X Hua1, J Wang1, L Li1, M Beqiri1, A Watters1, A Vultur1, Z Wei2, M Herlyn1 and M Fukunaga-Kalabis1 1 Wistar Institute, Philadelphia, PA and 2 New Jersey Institute of Technology, Newark, NJ The up-regulation of adaptor protein Numb by the canonical Wnt pathway plays a role in triggering melanocytic differentiation from multipotent skin stem cells, which share many properties with aggressive melanoma cells. Numb acts as a tumor suppressor in various human cancer types, however little is known about its role in melanoma. Here, we investigated the role of Numb in melanoma progression and the mechanism of its regulation. An analysis of The Cancer Genome Atlas (TCGA) melanoma dataset revealed that high levels of Numb expression in melanoma tissues correlate with improved patient survival. In the majority of melanoma cell lines, Numb was not up-regulated by canonical Wnt ligand treatment, while the activation of Wnt pathway by a GSK3 inhibitor BIO was able to increase Numb expression, even in the Wnt ligand-refractory cells. BIO treatment reduced migration of melanoma cells in a 3D context in Numb-dependent manner. Numb-depleted melanoma cells significantly increased tumor metastasis nodules in lung tissues by tail vein injection compared to control cells. These results suggest that Numb acts as a metastatic suppressor in melanoma, and the targeted inhibition of GSK3 may have therapeutic effects through up-regulating Numb.
PIKFYVE regulates the maturation of the melanosome MC Liggins1, J Flesher1, S Sandhu2, R Boissy3 and A Ganesan1,2 1 Department of Biological Chemistry, University of California, Irvine, Irvine, CA, 2 Department of Dermatology, University of California, Irvine, Irvine, CA and 3 Department of Dermatology, University of Cincinnati, Cincinnati, OH Phosphoinositide lipids coat the surface of transport vesicles and interact with PI binding proteins on target membranes to direct vesicle fusion events. While specific phosphoinositides involved in autophagy and Golgi trafficking have been identified, it is not currently known what phosphoinositides and phosphoinositide binding proteins regulate melanosome biogenesis. VAC14, SAC3, and PIKFYVE form the only mammalian PtdIns5P kinase complex, which synthesizes PI(3,5)P2 which can then be converted to PI(5)P. Published studies have determined that VAC14 or SAC3 mouse mutants and knockouts have severe neurological defects and dilute coat color, implicating a role for the PIKFYVE complex in regulating melanosome maturation. In this study, we investigated the role of PIKFYVE, the only PtdIns5P kinase, in melanogenesis. Partial depletion of PIKfyve, VAC14, or SAC3 using shRNAs in melanocytes resulted in decreased tyrosinase protein and mRNA accumulation and decreased expression of the melanogenic regulator MITF-M but did not block melanosome maturation. These results suggest that the PIKFYVE complex can regulate the transcription of proteins involved in melanogenesis. More complete inhibition of PIKFYVE using pharmacologic agents inhibited melanin accumulation by blocking the formation of stage III melanosomes and the trafficking of proteins to the melanosome and lysosome, validating that PIKFYVE also controls vesicle maturation. Addition of PI(5)P but not PI(3,5)P2 to cultured cells could induce the expression of tyrosinase, indicating that PI(5)P, which is primarily derived from PI(3,5)P2, regulates melanogenesis. Melanocyte-specific PIKFYVE knockout mice exhibited a coat that contained numerous white hairs, indicating that PIKFYVE regulates pigmentation in vivo. Taken together, these results define a specific and novel role for the phosphoinositide PI(5)P in regulating melanogenesis.
664
665
Vasoactive intestinal peptide (VIP) regulates human melanocyte biology and hair follicle pigmentation M Bertolini1,2, M Ba¨hr3, M Sulk1, L Ponce1,2, JA Hardman4, T Bı´ro´5, D Tobin6 and R Paus1,2,4 1 University of Mu¨nster, Mu¨nster, Germany, 2 Monasterium Laboratory, Mu¨nster, Germany, 3 University of Lu¨beck, Lu¨beck, Germany, 4 University of Manchester, Manchester, United Kingdom, 5 University of Debrecen, Debrecen, Hungary and 6 University of Bradford, Bradford, United Kingdom Exploratory pilot experiments in our lab(s) had raised the possibility that vasoactive intestinal peptide (VIP), a key immunoinhibitory neuropeptide released by perifollicular sensory nerve fibers, may be a novel modulator of human HF pigmentation. This hypothesis was followedup in the current study. After showing that the VIP receptors, VPAC1 and VPAC2, are expressed at gene and protein level in human anagen hair bulbs, we investigated whether VIP (10-7M) modulates human HF pigmentation in situ and in isolated human HF melanocytes (HFiMc). VIP significantly increased both the number of intraepithelial c-Kit+ HF melanocytes (HFMc) in situ and intrafollicular c-Kit transcription. By contrast, VIP down-regulated SCF gene and protein expression in organ-cultured human HFs. Interestingly, c-Kit/gp100 double- immunostaining indicated that most of the VIP-induced c-Kit+ HFMc represent immature/amelanotic HFMc. Intriguingly, VIP up-regulated the total number of gp100+, MITF+, or p-MITF+ HFMc and promoted intrafollicular melanin production in situ. VIP also significantly stimulated melanogenesis in isolated human HFiMc, suggesting that it impacts directly on HFMc, but also slightly inhibited HFMc/HFiMc proliferation in situ and in vitro. Preliminary results obtained with specific antagonists suggested that VPAC1 and VPAC2 mediated the VIP effects on HFMc. Since microarray and qRT-PCR analyses showed an up-regulation of phosphodiesterase 4D7 transcription in VIP-treated HFs, VIP may regulate the cAMP level in HFMc, possibly by utilizing downstream pathways shared with a-MSH signaling. In summary, our results reveal VIP as a novel, complex and clinically-relevant neuroendocrine regulator of human HF pigmentation.
Expression of a-Synuclein in melanocytic cells W Eng1, S Saikaly1, M Jimenez-Capdeville2, I Rodriguez-Leyva2 and RA Norman1 1 Pathology, University of Central Florida Medical School, Lake Nona, FL and 2 Neurology, University of St Luis, St Luis- Potsi, Mexico Objective: Previous epidemiological studies have demonstrated an increased prevalence of melanoma in patients with Parkinson’s Disease (PD). The pathological marker of PD is Lewy Bodies which are composed of a-synuclein protein. This study examines the extent of a-synuclein expression in variety of melanocytic neoplasms. Methodology: Using conventional immunohistochemistry with monoclonal antibodies against a-synuclein and tyrosinase, we processed skin-punch or shave biopsies containing Spitz nevus, intradermal nevus, dysplastic nevus, and melanoma. Control tissue included normal skin and acrochordons. Results: In all cases, between 70% to 85% of the melanocytic cells: benign, dysplastic and malignant, were found to express a-synuclein immunopositivity to various degrees. Conclusions: In the brain, a-synuclein participates releasing pre-synaptic vesicles from nerve terminals among other functions. Given its neural ectodermal origin, melanocytic cells may be using a-synuclein in a similar role to distribute melanin to keratinocytes. The typical aggregates of a-synuclein (Lewy Bodies) are not observed in cutaneous melanomas, thus it is unclear how a-synuclein plays a pathological role in the development of melanoma. Further research to delineate the role of a-synuclein may lead to a therapeutic agent or improved understanding of the pathophysiology of both melanoma and PD.
www.jidonline.org S117