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791 DNA methyltransferases play important but antagonistic roles in melanoma formation and growth
ABSTRACTS | Pigmentation and Melanoma 789
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Inhibition of isoprenylation synergizes with MAPK blockade to prevent growth in treatment-resistant melanoma N Theodosakis1, C Langdon2, G Micevic1, D Stern1 and M Bosenberg1 1 Yale School of Medicine, New Haven, CT and 2 Yale School of Medicine, Memphis, TN While originally approved for treatment of hypercholesterolemia, HMG-CoA reductase inhibitors, or statins, have also been postulated to have anti-cancer properties. Nevertheless, significant concern over their toxicity at required doses has limited their evaluation in the past. We demonstrate marked potentiation of statin-induced growth inhibition in BRAFmutant melanoma, as well as NRAS-mutant melanoma in combination with small molecule MAPK inhibitors. We also present evidence that this effect is mediated by impaired production of isoprenoid farnesyl and geranylgeranyl groups: products of the mevalonate pathway, with downstream effects on both PI3K/AKT and Hippo signaling. Mouse studies of dual therapy with statins and BRAF inhibitors in the setting of vemurafenib-resistant BRAFmutant melanoma provide additional in vivo evidence of efficacy, supporting a possible role for trials of statin and MAPK inhibitor combination therapy in human cancer patients.
Metabolic alterations in in vemurafenib-resistant melanoma: enolase a viable therapeutic target DN Syed1, A Singh2, SO Afolabi3, A Aljohani2 and H Mukhtar2 1 UW-Madison Wisconsin, Madison, WI, 2 UW-Madison, Madison, WI and 3 University of Wisconsin-Madison, Madison, WI Despite an increase in survival, resistance develops in most melanoma patients treated with BRAF-inhibitors. There is increasing evidence that melanoma like other cancers with acquired resistance to chemotherapeutic agents undergoes substantial metabolic reprogramming to sustain cell survival and proliferation. To study the metabolic alterations involved in acquired resistance, vemurafenib-resistant cell lines in the BRAFV600E mutated melanoma cell background were generated. Utilizing a quantitative proteomic strategy, we found enolase, triose-phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase differentially regulated in resistant cells. Flux studies validated suppressed glycolysis and reduced lactic acid levels in resistant cells. Treatment with vemurafenib abrogated phosphorylated ERK levels but had little effect on basal enolase levels and glucose uptake in sensitive cells. In contrast, markedly elevated phosphorylated ERK levels accompanied with suppressed glucose uptake indicated that metabolic alterations may be responsible for the observed decrease in enolase levels in resistant cells. Metabolomic studies revealed significantly decreased intracellular levels of arginine and lysine in vemurafenib-resistant cells. This was associated with reduced expression of synthesis enzyme ASS1 and suppressed uptake of arginine and lysine as assessed by media analysis studies. Vemurafenib-sensitive cells responded with enhanced glucose uptake and increased enolase levels to arginine/lysine deprivation. Notably, absence of a compensatory increase in enolase was associated with decreased viability of resistant cells. Our data are consistent with a model of prolonged treatment response to vemurafenib and shows that de-repression of ERK pathway upon emergence of resistance is associated with adaptive changes in cellular metabolism. Targeting enolase through modulation of arginine/lysine metabolism may be a viable option to harness tumor growth in vemurafenib-resistant melanomas.
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DNA methyltransferases play important but antagonistic roles in melanoma formation and growth G Micevic and M Bosenberg Yale School of Medicine, New Haven, CT The purpose of this study is to determine the requirement of DNA methyltransferases for melanoma formation, and the effect of their inactivation on tumor growth. The requirement of individual DNMT enzymes for melanoma formation is currently incompletely understood, despite important roles DNMT enzymes play in many cancers, including melanoma. The role of DNMT3B in melanoma was previously investigated, and was found to be a therapeutic target, but the requirement for DNMT1/DNMT3A is currently unknown. In this study, we examined the effect of individual genetic inactivation of DNMT1 and DNMT3A in a genetically engineered mouse model of melanoma. We confirmed our key findings in a panel of melanoma and melanocyte cell lines. We examined in vivo tumor formation and proliferation, DNA damage response, and requirement of DNMT enzymes for melanoma cell and melanocyte viability. We uncovered that inactivation of DNMT1 resulted in a significant and surprising acceleration of melanoma formation in vivo (p < 0.001), which was associated with increased activation of DNA damage pathways (p < 0.001). Inactivation of DNMT3A did not significantly affect melanoma formation. To test the effect on melanocyte and melanoma cell viability, we performed knockout experiments and uncovered that DNMT1 and DNMT3A are dispensable for melanocyte viability, while DNMT1 loss significantly affected melanoma cell proliferation (p < 0.01). Furthermore, loss of DNMT1 or DNMT3A did not significantly affect the global level of DNA methylation. This study examined the requirement of DNMT1 and DNMT3A for melanoma formation and uncovered that they are not essential for tumor formation, and that inactivation of DNMT1 surprisingly accelerates melanoma formation by a non-methylation based mechanism. These findings address an unresolved question in the field, and suggest that unlike DNMT3B, which may be a therapeutic target in melanoma, DNMT1 may plays anti-tumorigenic roles, and its inhibition using non-specific hypomethylating agents in clinic should be approached with caution in melanoma.
CK1a ablation in keratinocytes induces p53-dependent, sunburn-protective, skin hyperpigmentation C Chang1, T Ito2, T Stiewe3, K Wakamatsu4 and Y Ben-Neriah5 1 Skin Institute, China Medical University Hospital; Department of Dermatology, China Medical University, Taichung, Taichung, Taiwan, 2 Skin Institute, China Medical University Hospital, Taichung, Taichung, Taiwan, 3 Philipps-University Marburg, Molecular Oncology and Genomics Core Facility, Center for Tumor and Immunobiology, Marberg, Germany, 4 Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan and 5 The Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel Casein kinase 1a (CK1a), a component of the b-catenin-destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1a (encoded by Csnk1a1) in skin physiology, we generated mice in which tamoxifen induces the deletion of Csnk1a1 exclusively in keratinocytes (SKOKer). CK1a loss was accompanied by b-catenin and p53 stabilization, Wnt and p53 target gene activation, yet phenotypically, most striking was hyperpigmentation. To clarify the role of p53 in hyperpigmentation, we established K14eCreeERT2 CK1a/p53 double-knockout (DKOKer) mice and found coablation failed to induce hyperpigmentation, demonstrating that it was p53dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit and KitL. SKOKer treatment with ACK2 (a Kit neutralizing antibody) or imatinib (a c-Kit inhibitor) abrogated the CK1a-ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway, which is in contrast to the p53-dependent UV tanning mechanisms. Nevertheless, acute sunburn effects were successfully prevented. CK1a inhibition induces skin protective eumelanin but no carcinogenic pheomelanin, and may therefore constitute an effective new strategy for safely increasing skin pigmentation via UV-independent pathways, protecting against acute sunburns.
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Clusterin negatively regulates melanogenesis via TGFb receptor-Smad pathway M Kim, J Lee, T Park and H Kang Ajou University, Suwon, Kyonggi-do, Republic of Korea Clusterin has been known as a biomarker of aging and is highly induced in stressed and senescent cells. In this study, RNA sequencing analysis showed transcripts for clusterin were highly expressed in UV irradiated fibroblasts. A considerable increase of clusterin was also detected at the protein level. We found that the clusterin is overexpressed in the acutely UVirradiated skin and fibroblasts. To investigate the effect of clusterin on melanogenesis, normal human melanocytes were treated with conditioned media of fibroblasts infected with clusterin-lentivirus or sh-clusterin. It was found that clusterin inhibits melanogenesis through MITF/tyrosinase downregulation viaTGFb signaling. The findings suggest that clusterin inhibits melanogenesis and that it plays a role in controlling UV-induced pigmentary changes.
S136 Journal of Investigative Dermatology (2017), Volume 137
Effect of a new lipophilic pro-vitamin C, tetra-isopalmitoyl ascorbic acid (VCIP), on senile lentigo (aging spot) via controlling of melanocytes-keratinocytes interaction M Yokota1 and S Yahagi2 1 NIKKOL GROUP Cosmos Technical Center Co.,Ltd., Tokyo, Japan and 2 NIKKOL GROUP Cosmos Technical Center Co.,Ltd., Tokyo, Japan Senile lentigo or aging spots are hyperpigmented macules of skin that occur in irregular shapes, appearing most commonly in the sun-exposed areas of the skin such as on the face and back of the hands, and are a common component of photoaged skin. Vitamin C is well known to play an important role in maintaining skin physiology, especially as a skin whitening and blighting agent. We previously demonstrated that a new stable lipophilic provitamin C derivative, tetra-isopalmitoyl ascorbic acid (VC-IP), showed significant suppressive effect for UVB-induced skin pigmentation by conversion into vitamin C in skin tissue. In addition, we reported that VC-IP showed multiple physiological activities represented by scavenging of ROS and reduction of inflammatory cytokines, IL-1a and PGE2, in keratinocytes. In this study, we evaluated that effectiveness of VC-IP on senile lentigo (aging spot) by conducting clinical test approved by the institutional ethical committee. VC-IP significantly improved brightness of aging spot and skin tone to evenness by mechanical measurements. Moreover, self-assessment study demonstrated that subjects were satisfied with the following parameters; appearance of aged spot, skin brightness, fading of dullness or yellowing and zero adverse reaction. Furthermore, we newly identified that VC-IP strongly suppressed the melanin delivery by inhibition of dendrite elongation stimulated by endothelin-1 in melanocytes-keratinocytes co-culture system. There are still many discussions on how senile lentigo represents in skin, however, these results suggested possibility that VC-IP is effective for improving hyperpigmented spot based on controlling of melanocytes-keratinocytes interaction without any adverse reaction.
790
Inhibition of isoprenylation synergizes with MAPK blockade to prevent growth in treatment-resistant melanoma N Theodosakis1, C Langdon2, G Micevic1, D Stern1 and M Bosenberg1 1 Yale School of Medicine, New Haven, CT and 2 Yale School of Medicine, Memphis, TN While originally approved for treatment of hypercholesterolemia, HMG-CoA reductase inhibitors, or statins, have also been postulated to have anti-cancer properties. Nevertheless, significant concern over their toxicity at required doses has limited their evaluation in the past. We demonstrate marked potentiation of statin-induced growth inhibition in BRAFmutant melanoma, as well as NRAS-mutant melanoma in combination with small molecule MAPK inhibitors. We also present evidence that this effect is mediated by impaired production of isoprenoid farnesyl and geranylgeranyl groups: products of the mevalonate pathway, with downstream effects on both PI3K/AKT and Hippo signaling. Mouse studies of dual therapy with statins and BRAF inhibitors in the setting of vemurafenib-resistant BRAFmutant melanoma provide additional in vivo evidence of efficacy, supporting a possible role for trials of statin and MAPK inhibitor combination therapy in human cancer patients.
Metabolic alterations in in vemurafenib-resistant melanoma: enolase a viable therapeutic target DN Syed1, A Singh2, SO Afolabi3, A Aljohani2 and H Mukhtar2 1 UW-Madison Wisconsin, Madison, WI, 2 UW-Madison, Madison, WI and 3 University of Wisconsin-Madison, Madison, WI Despite an increase in survival, resistance develops in most melanoma patients treated with BRAF-inhibitors. There is increasing evidence that melanoma like other cancers with acquired resistance to chemotherapeutic agents undergoes substantial metabolic reprogramming to sustain cell survival and proliferation. To study the metabolic alterations involved in acquired resistance, vemurafenib-resistant cell lines in the BRAFV600E mutated melanoma cell background were generated. Utilizing a quantitative proteomic strategy, we found enolase, triose-phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase differentially regulated in resistant cells. Flux studies validated suppressed glycolysis and reduced lactic acid levels in resistant cells. Treatment with vemurafenib abrogated phosphorylated ERK levels but had little effect on basal enolase levels and glucose uptake in sensitive cells. In contrast, markedly elevated phosphorylated ERK levels accompanied with suppressed glucose uptake indicated that metabolic alterations may be responsible for the observed decrease in enolase levels in resistant cells. Metabolomic studies revealed significantly decreased intracellular levels of arginine and lysine in vemurafenib-resistant cells. This was associated with reduced expression of synthesis enzyme ASS1 and suppressed uptake of arginine and lysine as assessed by media analysis studies. Vemurafenib-sensitive cells responded with enhanced glucose uptake and increased enolase levels to arginine/lysine deprivation. Notably, absence of a compensatory increase in enolase was associated with decreased viability of resistant cells. Our data are consistent with a model of prolonged treatment response to vemurafenib and shows that de-repression of ERK pathway upon emergence of resistance is associated with adaptive changes in cellular metabolism. Targeting enolase through modulation of arginine/lysine metabolism may be a viable option to harness tumor growth in vemurafenib-resistant melanomas.
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792
DNA methyltransferases play important but antagonistic roles in melanoma formation and growth G Micevic and M Bosenberg Yale School of Medicine, New Haven, CT The purpose of this study is to determine the requirement of DNA methyltransferases for melanoma formation, and the effect of their inactivation on tumor growth. The requirement of individual DNMT enzymes for melanoma formation is currently incompletely understood, despite important roles DNMT enzymes play in many cancers, including melanoma. The role of DNMT3B in melanoma was previously investigated, and was found to be a therapeutic target, but the requirement for DNMT1/DNMT3A is currently unknown. In this study, we examined the effect of individual genetic inactivation of DNMT1 and DNMT3A in a genetically engineered mouse model of melanoma. We confirmed our key findings in a panel of melanoma and melanocyte cell lines. We examined in vivo tumor formation and proliferation, DNA damage response, and requirement of DNMT enzymes for melanoma cell and melanocyte viability. We uncovered that inactivation of DNMT1 resulted in a significant and surprising acceleration of melanoma formation in vivo (p < 0.001), which was associated with increased activation of DNA damage pathways (p < 0.001). Inactivation of DNMT3A did not significantly affect melanoma formation. To test the effect on melanocyte and melanoma cell viability, we performed knockout experiments and uncovered that DNMT1 and DNMT3A are dispensable for melanocyte viability, while DNMT1 loss significantly affected melanoma cell proliferation (p < 0.01). Furthermore, loss of DNMT1 or DNMT3A did not significantly affect the global level of DNA methylation. This study examined the requirement of DNMT1 and DNMT3A for melanoma formation and uncovered that they are not essential for tumor formation, and that inactivation of DNMT1 surprisingly accelerates melanoma formation by a non-methylation based mechanism. These findings address an unresolved question in the field, and suggest that unlike DNMT3B, which may be a therapeutic target in melanoma, DNMT1 may plays anti-tumorigenic roles, and its inhibition using non-specific hypomethylating agents in clinic should be approached with caution in melanoma.
CK1a ablation in keratinocytes induces p53-dependent, sunburn-protective, skin hyperpigmentation C Chang1, T Ito2, T Stiewe3, K Wakamatsu4 and Y Ben-Neriah5 1 Skin Institute, China Medical University Hospital; Department of Dermatology, China Medical University, Taichung, Taichung, Taiwan, 2 Skin Institute, China Medical University Hospital, Taichung, Taichung, Taiwan, 3 Philipps-University Marburg, Molecular Oncology and Genomics Core Facility, Center for Tumor and Immunobiology, Marberg, Germany, 4 Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan and 5 The Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel Casein kinase 1a (CK1a), a component of the b-catenin-destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1a (encoded by Csnk1a1) in skin physiology, we generated mice in which tamoxifen induces the deletion of Csnk1a1 exclusively in keratinocytes (SKOKer). CK1a loss was accompanied by b-catenin and p53 stabilization, Wnt and p53 target gene activation, yet phenotypically, most striking was hyperpigmentation. To clarify the role of p53 in hyperpigmentation, we established K14eCreeERT2 CK1a/p53 double-knockout (DKOKer) mice and found coablation failed to induce hyperpigmentation, demonstrating that it was p53dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit and KitL. SKOKer treatment with ACK2 (a Kit neutralizing antibody) or imatinib (a c-Kit inhibitor) abrogated the CK1a-ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway, which is in contrast to the p53-dependent UV tanning mechanisms. Nevertheless, acute sunburn effects were successfully prevented. CK1a inhibition induces skin protective eumelanin but no carcinogenic pheomelanin, and may therefore constitute an effective new strategy for safely increasing skin pigmentation via UV-independent pathways, protecting against acute sunburns.
793
794
Clusterin negatively regulates melanogenesis via TGFb receptor-Smad pathway M Kim, J Lee, T Park and H Kang Ajou University, Suwon, Kyonggi-do, Republic of Korea Clusterin has been known as a biomarker of aging and is highly induced in stressed and senescent cells. In this study, RNA sequencing analysis showed transcripts for clusterin were highly expressed in UV irradiated fibroblasts. A considerable increase of clusterin was also detected at the protein level. We found that the clusterin is overexpressed in the acutely UVirradiated skin and fibroblasts. To investigate the effect of clusterin on melanogenesis, normal human melanocytes were treated with conditioned media of fibroblasts infected with clusterin-lentivirus or sh-clusterin. It was found that clusterin inhibits melanogenesis through MITF/tyrosinase downregulation viaTGFb signaling. The findings suggest that clusterin inhibits melanogenesis and that it plays a role in controlling UV-induced pigmentary changes.
S136 Journal of Investigative Dermatology (2017), Volume 137
Effect of a new lipophilic pro-vitamin C, tetra-isopalmitoyl ascorbic acid (VCIP), on senile lentigo (aging spot) via controlling of melanocytes-keratinocytes interaction M Yokota1 and S Yahagi2 1 NIKKOL GROUP Cosmos Technical Center Co.,Ltd., Tokyo, Japan and 2 NIKKOL GROUP Cosmos Technical Center Co.,Ltd., Tokyo, Japan Senile lentigo or aging spots are hyperpigmented macules of skin that occur in irregular shapes, appearing most commonly in the sun-exposed areas of the skin such as on the face and back of the hands, and are a common component of photoaged skin. Vitamin C is well known to play an important role in maintaining skin physiology, especially as a skin whitening and blighting agent. We previously demonstrated that a new stable lipophilic provitamin C derivative, tetra-isopalmitoyl ascorbic acid (VC-IP), showed significant suppressive effect for UVB-induced skin pigmentation by conversion into vitamin C in skin tissue. In addition, we reported that VC-IP showed multiple physiological activities represented by scavenging of ROS and reduction of inflammatory cytokines, IL-1a and PGE2, in keratinocytes. In this study, we evaluated that effectiveness of VC-IP on senile lentigo (aging spot) by conducting clinical test approved by the institutional ethical committee. VC-IP significantly improved brightness of aging spot and skin tone to evenness by mechanical measurements. Moreover, self-assessment study demonstrated that subjects were satisfied with the following parameters; appearance of aged spot, skin brightness, fading of dullness or yellowing and zero adverse reaction. Furthermore, we newly identified that VC-IP strongly suppressed the melanin delivery by inhibition of dendrite elongation stimulated by endothelin-1 in melanocytes-keratinocytes co-culture system. There are still many discussions on how senile lentigo represents in skin, however, these results suggested possibility that VC-IP is effective for improving hyperpigmented spot based on controlling of melanocytes-keratinocytes interaction without any adverse reaction.