BACKGROUND. Skin cancer represents a significant health threat with over 1.3 million diagnoses, 8000 melanoma deaths, and more than $1 billion spent annually for skin cancer healthcare in the US. Despite findings from laboratory, case-control, and prospective studies that indicate a link between youthful indoor tanning (IT) and skin cancer, IT is increasing among US youth. Appearance-focused interventions represent a promising method to counteract these trends. METHODS. A total of 430 female indoor tanners were randomized into intervention or no intervention control conditions. Intervention participants received an appearance-focused booklet based on decision-theoretical models of health behavior. Outcome variables included self-reports of IT behavior and intentions, as well as measures of cognitive mediating variables. RESULTS. Normative increases in springtime IT rates were significantly lower (ie, over 35%) at 6-month follow-up in intervention versus control participants with similar reductions in future intentions. Mediation analyses revealed 6 cognitive variables (IT attitudes, fashion attitudes, perceived susceptibility to skin cancer and skin damage, subjective norms, and image norms) that significantly mediated change in IT behavior. CONCLUSIONS. The appearance-focused intervention demonstrated strong effects on IT behavior and intentions in young indoor tanners. Appearance-focused approaches to skin cancer prevention need to present alternative behaviors as well as alter IT attitudes. Mediational results provide guides for strengthening future appearance-focused interventions directed at behaviors that increase risk of skin cancer.

Genetic changes acquired in the pigment producing cells of the skin, called melanocytes, can lead to formation of the deadly cancer melanoma. Mutations or amplifications leading to the activation of the RAS/MAPK pathway occur in more than 90% of melanomas. Melanocyte development and survival requires the stimulation of this pathway by the receptor tyrosine kinase (RTK) KIT. In ~2% of melanomas, oncogenic KIT mutations drive tumor formation; however, the majority of melanomas lose wild-type KIT expression, suggesting that KIT could suppress melanoma formation. In human melanoma patients of The Cancer Genome Atlas (TCGA), we found an association between BRAFV600E mutations and low KIT mRNA expression, so we tested whether KIT loss would affect BRAFV600E-driven tumor onset by crossing a kit(lf) mutant allele into melanoma-prone Tg(mitfa:BRAFV600E); p53(lf) zebrafish. We observed that kit(lf)-mutant zebrafish experienced accelerated tumor onset and their tumors had increased RAS/MAPK pathway activation. In BRAFV600E-mutant melanoma cells, KIT activity reduced RAS/MAPK signaling by promoting activation of wild-type BRAF (BRAFWT). Furthermore, we found that overexpression of BRAFWT delayed tumor onset in Tg(mitfa:BRAFV600E); p53(lf); mitfa(lf) zebrafish, but had no effect in kit(lf); Tg(mitfa:BRAFV600E); p53(lf); mtifa(lf) zebrafish and a cohort of TCGA BRAFV600E-mutant melanoma patients with high KIT expression and high BRAFWT allele ratios experienced a reduced likelihood of metastasis and extended overall survival. These studies indicate that wild-type KIT acts to suppress melanoma formation through activation of BRAFWT, causing reduced signaling output of BRAFV600E-mutant cells.

Melanoma is among the most aggressive and treatment-resistant cancers. Currently, available therapies for melanoma are not satisfactory, and the prognosis for patients with metastatic melanoma is still poor. Vemurafenib, a BRAF kinase inhibitor (BRAFi), provides an approximately 50% response rate in patients with metastatic melanoma, but eventually, relapse occurs due to acquired resistance to the drug. Novel therapeutics and BRAFi adjuvants for treating melanoma are needed. Si-Jun-Zi-Tang (SJZT) is a traditional Chinese medicine formula used to treat chronic and debilitating diseases including melanoma. SJZT-based therapies alone or in combination with chemotherapies have achieved good clinical outcomes in melanoma management. However, the pharmacological basis of SJZT for its clinical use in melanoma treatment is not fully understood. c-Met is a receptor tyrosine kinase (RTK), and hepatocyte growth factor (HGF) is the only known ligand of c-Met. Abnormal activation of HGF/c-Met has been implicated in melanoma progression. HGF/c-Met has been proposed as a therapeutic target for melanoma. Some bioactive constituents in SJZT have been shown to inhibit c-Met signaling. In this study, we investigated the anti-melanoma effects and c-Met signaling-related action mechanisms of SJZT. Our in vivo study showed that SJZT-A, an ethanolic extract of SJZT, inhibited B16 tumor growth in mice without overt toxicity. Mechanistic investigations revealed that SJZT-A elevated miR-34b (a tumor-suppressing miRNA), and lowered c-Met (a miR-34b target gene) and β-catenin (a downstream molecule of c-Met signaling) expression levels in the B16 tumors. But SJZT-A failed to reduce the viability of B16 and A375 melanoma cells. Active component-enriched SJZT-B, which was prepared from SJZT-A using macroporous resin column chromatography, exhibited potent anti-proliferation effect and inhibited miR-34b/c-Met/β-catenin signaling in cultured melanoma cells. Overexpression of constitutively active β-catenin partially diminished the inhibitory effect of SJZT-B on cell proliferation. SJZT-B also exerted anti-proliferative effects, inhibited c-met signaling, and induced ER stress in vemurafenib resistance melanoma cells. In vivo experiments showed that intragastric administration of SJZT-B for consecutive 14 days overcame vemurafenib resistance in melanoma-bearing mice without observable toxicities. Overall, these results indicate that SJZT has anti-melanoma effects and is relatively safe. Also, we found that licochalcone A, a flavonoid presented in SJZT-B, overcame vemurafenib resistance both in vitro and in vivo, as well as inhibited c-Met signaling and induced ER stress in A375-VR cells, which is in line with the effects of SJZT-B in melanoma. The role of triggering ER stress in licochalcone A's effects in overcoming vemurafenib resistance effects has also been established. Overall, these results suggest that licochalcone A is one of the active compounds responsible for the anti-melanoma effects of SJZT-B. In conclusion, our results demonstrated that SJZT has anti-melanoma effects and is safe in cell and mouse melanoma models. Licochalcone A has been identified to be one of the active components responsible for the anti-melanoma effects of SJZT. This study provides a pharmacological and chemical basis for the traditional use of the formula SJZT in treating melanoma, and suggests that SJZT and SJZT-derived compounds have the potential to be developed as modern alternative and/or complementary agents for melanoma management.

Melanoma is among the most aggressive and treatment-resistant cancers. Currently, available therapies for melanoma are not satisfactory, and the prognosis for patients with metastatic melanoma is still poor. Vemurafenib, a BRAF kinase inhibitor (BRAFi), provides an approximately 50% response rate in patients with metastatic melanoma, but eventually, relapse occurs due to acquired resistance to the drug. Novel therapeutics and BRAFi adjuvants for treating melanoma are needed. Si-Jun-Zi-Tang (SJZT) is a traditional Chinese medicine formula used to treat chronic and debilitating diseases including melanoma. SJZT-based therapies alone or in combination with chemotherapies have achieved good clinical outcomes in melanoma management. However, the pharmacological basis of SJZT for its clinical use in melanoma treatment is not fully understood. c-Met is a receptor tyrosine kinase (RTK), and hepatocyte growth factor (HGF) is the only known ligand of c-Met. Abnormal activation of HGF/c-Met has been implicated in melanoma progression. HGF/c-Met has been proposed as a therapeutic target for melanoma. Some bioactive constituents in SJZT have been shown to inhibit c-Met signaling. In this study, we investigated the anti-melanoma effects and c-Met signaling-related action mechanisms of SJZT. Our in vivo study showed that SJZT-A, an ethanolic extract of SJZT, inhibited B16 tumor growth in mice without overt toxicity. Mechanistic investigations revealed that SJZT-A elevated miR-34b (a tumor-suppressing miRNA), and lowered c-Met (a miR-34b target gene) and β-catenin (a downstream molecule of c-Met signaling) expression levels in the B16 tumors. But SJZT-A failed to reduce the viability of B16 and A375 melanoma cells. Active component-enriched SJZT-B, which was prepared from SJZT-A using macroporous resin column chromatography, exhibited potent anti-proliferation effect and inhibited miR-34b/c-Met/β-catenin signaling in cultured melanoma cells. Overexpression of constitutively active β-catenin partially diminished the inhibitory effect of SJZT-B on cell proliferation. SJZT-B also exerted anti-proliferative effects, inhibited c-met signaling, and induced ER stress in vemurafenib resistance melanoma cells. In vivo experiments showed that intragastric administration of SJZT-B for consecutive 14 days overcame vemurafenib resistance in melanoma-bearing mice without observable toxicities. Overall, these results indicate that SJZT has anti-melanoma effects and is relatively safe. Also, we found that licochalcone A, a flavonoid presented in SJZT-B, overcame vemurafenib resistance both in vitro and in vivo, as well as inhibited c-Met signaling and induced ER stress in A375-VR cells, which is in line with the effects of SJZT-B in melanoma. The role of triggering ER stress in licochalcone A's effects in overcoming vemurafenib resistance effects has also been established. Overall, these results suggest that licochalcone A is one of the active compounds responsible for the anti-melanoma effects of SJZT-B. In conclusion, our results demonstrated that SJZT has anti-melanoma effects and is safe in cell and mouse melanoma models. Licochalcone A has been identified to be one of the active components responsible for the anti-melanoma effects of SJZT. This study provides a pharmacological and chemical basis for the traditional use of the formula SJZT in treating melanoma, and suggests that SJZT and SJZT-derived compounds have the potential to be developed as modern alternative and/or complementary agents for melanoma management.

Le mélanome constitue un problème majeur de santé publique. En 2018, on estimait à 15 513 nouveaux cas en France et 1975 décès dûs à ce cancer. S’il ne représente qu’environ 10% des cancers cutanés, il est le plus agressif en raison de son très fort potentiel métastatique. Le développement de ces métastases met en jeu différentes étapes qui nécessitent d’être coordonnées par un ensemble de gènes. Dans les mélanomes, le facteur de transcription MITF connu pour son importance dans la différenciation et la survie des mélanocytes, joue ainsi un rôle majeur dans la progression tumorale. Plusieurs altérations génétiques telles que des mutations ou amplifications du gène ont été décrites et montrent son caractère oncogène, sans que les mécanismes expliquant les conséquences de ces modifications soient encore bien compris. Les altérations génétiques de MITF ne sont pas les seuls événements impliqués dans la mélanomagenèse. Les variations des niveaux d’expression de MITF sont également déterminantes dans l’évolution du mélanome. En effet, si son expression stimule la prolifération, son inhibition est nécessaire à l'émergence des métastases. Il est donc essentiel de comprendre les mécanismes en aval de ce facteur de transcription impliqués dans la progression tumorale.Mes travaux de thèse s’articulent autour de deux axes qui visent à mieux comprendre l’influence de MITF dans l’invasion. Le premier consiste à étudier les effets d’une mutation de MITF dans la progression tumorale du mélanome. Une analyse comparative de plusieurs lignées de mélanomes exprimant la forme sauvage ou mutée de MITF a ainsi permis d’identifier S100A4 comme étant différentiellement régulé dans les mélanomes mutés pour MITF. Notre étude montre que MITF réprime l’expression de S100A4 et que cette répression est levée lorsque MITF est muté, ce qui a pour conséquence de stimuler le potentiel d’invasion des mélanomes. Ces résultats sont confirmés par les données cliniques qui montrent que les patients atteints de mélanomes et présentant une amplification de S100A4 ont une espérance de vie plus faible que les patients où l'expression de S100A4 n'est pas altérée.Le deuxième axe de mon projet s’intéresse au rôle d’une cible en aval de MITF, le récepteur à l’IGF2 (IGF2R), dont l’expression est nécessaire à l’invasion des mélanomes. L'analyse des différentes fonctions de ce récepteur ont révélé que son rôle dans l’adressage des hydrolases vers les lysosomes est requis pour que ces cellules soient invasives. Ce transport nécessite le marquage des hydrolases au mannose-6-phosphate notamment catalysé par l’enzyme codée par GNPTAB dans l’appareil de Golgi et la perte d’expression de ce gène entraîne le même effet que la déplétion d’IGF2R, à savoir une inhibition forte de l’invasion. De manière intéressante, la déplétion d’IGF2R et de GNPTAB induit également une augmentation du nombre de lysosomes, associée à une augmentation de l’expression de TFEB, un régulateur connu de la biogenèse des lysosomes. Nos résultats montrent que ce facteur de transcription appartenant à la même famille que MITF est également impliqué dans la régulation de l’invasion tumorale des mélanomes. / Melanoma constitutes a major public health issue. In 2018, we estimated 15 513 new melanoma cases in France and 1975 deaths caused by this cancer. If it only represents 10% of all cutaneous cancers, it is the most aggressive one due to its high potential to form metastasis. Metastasis cascade is a multi-step process requiring the coordination of specific genes. In melanoma, the microphthalmia-associated transcription factor MITF, the master regulator of melanocyte differentiation and survival has also a crucial role during melanoma tumor progression. Some genetic alterations have been described, mutations and gene amplification, and showed its oncogenic properties, even if mechanisms associated to these genetic modifications in tumor progression remained unknown. Genetic alterations of MITF are not the only events involved in melanomagenesis. In fact, MITF expression levels are determining for melanoma evolution. MITF expression activates proliferation and its inhibition is necessary for metastasis. It is therefore crucial to understand what are the mechanisms downstream of MITF, involved in tumor progression.My PhD project is indeed organized in two different axis, in order to better understand the importance of MITF in melanoma invasion. The first one consisted to study the impact of a single-point MITF mutation in melanoma tumor progression involving direct regulation of S100A4 gene. A comparative analysis using melanoma cell lines expressing the wild-type or mutated form of MITF led us to identify S100A4 as one of the most up-regulated genes in MITF-mutant melanoma. Our findings showed us that the MITFE87R mutant form increases S100A4 transcription, whereas the wild-type form of MITF has an opposite effect. This regulation is important in melanoma tumor progression because it can stimulate invasive potential of non-invasive melanoma. These results are confirmed by clinical data showing that harboring S100A4 amplification is associated to a poor prognosis in melanoma patients, compared to patients presenting no S100A4 alteration.The second axis was interested in the role of IGF2 receptor (IGF2R), a MITF target gene known to regulate melanoma invasion. Among all the different functions attributed to this receptor, our data indicates that only its role in hydrolase trafficking to lysosomes is required for invasion. This transport requires mannose-6-phosphate binding to these hydrolases by the enzyme encoding by GNPTAB gene in Golgi apparatus; loss of GNPTAB expression leads to the same effect as IGF2R depletion, a high decrease of melanoma invasion. Interestingly, IGF2R and GNPTAB depletion also induce an increase in lysosome number, associated to an increase of TFEB expression, a well-known regulator in lysosome biogenesis. Our data showed that this member of the same MiT-TFE family as MITF is also involved in this melanoma invasion regulation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20254号 / 医博第4213号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 川上 浩司, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Ca2+ is a ubiquitous and dynamic second messenger molecule that is induced by many factors including receptor activation, environmental factors, and voltage, leading to pleiotropic effects on cell function including changes in migration, metabolism and transcription. As such, it is not surprising that aberrant regulation of Ca2+ signals can lead to pathological phenotypes, including cancer progression. However, given the highly context-specific nature of Ca2+-dependent changes in cell function, delineation of its role in cancer has been a challenge. Hence, the role of store-operated Ca2+ entry (SOCE) in melanoma metastasis is still not fully elucidated. To address this, we examined UV-dependent metastasis, revealing a critical role for SOCE suppression. As previous literature demonstrated a role for cholesterol (CHL) in melanoma progression, our investigations corroborate this revealing UV-induced CHL biosynthesis as a critical mediator for UV-induced SOCE suppression and subsequent metastasis. However, SOCE suppression alone was both necessary and sufficient for metastasis to occur. Further, SOCE suppression facilitated UV-dependent differences in gene expression associated with increased invasion through altered glucose utilization. Functional analyses further establish that increased glucose uptake leads to a metabolic shift towards biosynthetic pathways critical for melanoma metastasis. Finally, examination of fresh surgically isolated human melanoma explants revealed CHL dependent low SOCE. Invasiveness could be reversed with either CHL biosynthesis inhibitors, pharmacological inhibition of terminal glycosylation enzyme, OGT, or pharmacological SOCE potentiation. In parallel, we demonstrate that Geranylgeranylpyrophosphate (GGPP) can function as a novel SOCE inhibitor either by saturation or prevention of transfer to membrane proteins; both of which lead to GGPP accumulation in the cytosol. Collectively, we provide evidence that Ca2+ signals can block invasive behavior, and suppression of these signals promotes invasion and metastasis. / Biomedical Sciences

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