Melanoma incidence is increasing faster than any other cancer in the world today. Disease detected early can be cured by surgery, but once melanoma progresses to the metastatic stage it is lethal, with an overall median survival of less than one year. The poor prognosis for late stage melanoma patients is attributed to the intrinsic resistance of melanoma to all Federal Drug Administration approved melanoma therapies. Therefore, there is a critical need for novel treatment approaches that circumvent melanoma therapy resistance. Emerging evidence suggests that differences in melanoma metabolism relative to non-malignant cells represents a potential target for therapeutic intervention. The research presented here demonstrates the potential for using triphenylphosphonium-based compounds as a new therapeutic platform for metastatic melanoma that is designed to take advantage of these metabolic differences. In vitro experiments demonstrate that triphenylphosphonium-based compounds modified with an aliphatic side chain target melanoma cell mitochondria and promote melanoma cell death via mitochondria metabolism inhibition and subsequent reactive oxygen species production. Increased reactive oxygen species production results in decreased glutathione levels and an oxidized cellular state. There is also a structure-activity relationship between side chain length, metabolic disruption, and melanoma cell cytotoxicity. Further, results demonstrate that traditional in vivo triphenylphosphonium drug administration routes such as oral gavage, intraperitoneal injection, and intravenous injection do not result in significant tumor accumulation of triphenylphosphonium drugs. However, the use of a thermosensitive hydrogel delivery system localizes triphenylphosphonium drugs directly at the melanoma tumor site and decreases melanoma tumor growth rate. These results suggest that a hydrogel-based triphenlyphosphonium delivery system could potentially be a therapeutic strategy that circumvents melanoma resistance mechanisms in order to provide durable therapy for an increasing number of metastatic melanoma patients worldwide.

Philosophiae Doctor - PhD / Dyspigmentation is one of the commonest dermatological presenting complaints from patients, particularly hyperpigmentation. Hyperpigmentation can cause dangerous psychological and emotional impact on self-perception and health quality of lives of people affected. However, of all the diseases encountered globally, epidemiological data has shown that skin diseases account for almost 34% of all the diseases and these dermatological disorders have gotten worse over time. The gold standard for treatment of hyperpigmentation is hydroquinone. Despite its efficacy, hydroquinone and other current modalities of treatments are associated with some side effects. There are a number of natural products derived from medicinal plants that have proven to be an abundant source of biologically active compounds and a lot of these have served as the basis for the development of new lead chemicals for pharmaceutical. The present study focused on screening of selected South African plants (Maclura pomifera, Otholobium fruticans, Phyylica ericoides, Psoralea aphylla, Rhynchosia villosa, and Serruria furcellata) for their antimelanogenic potentials. Methanol and ethyl acetate were used for the extraction of plant materials. Standard methods were employed for evaluation of cytotoxicity of the methanolic leaf extracts (MLE), ethyl acetate leaf extracts (ELE) and melanin synthesis potentials on B16 melanoma cells. To elucidate mechanisms of melanin reduction action, intracellular tyrosinase activity was determined by measuring the rate of L-DOPA oxidation. Tyrosinase activity was assessed further with dihydroxyphenylalanine (DOPA) staining. The mode of action was further determined by evaluating reactive oxygen species (ROS) and expressions of melanogenesis gene using qPCR. The results showed that O. fruticans and S. furcellata reduced melanin synthesis without cytotoxicity. O. fruticans inhibited tyrosinase activity, increased ROS and suppressed the expression of TYR, TRP-1, TRP-2/Dopachrome tautomerase, MITF, MC1R but upregulated β-Catenin. S. furcellata stimulated tyrosinase activity and did not increase ROS. It upregulated the expression of TYR, TRP-1, TRP-2, and MC1R while MITF and β-Catenin were suppressed. The results showed that O. fruticans reduced melanin synthesis via cAMP pathway while S. furcellata reduced the synthesis via possibly degradation of melanin pigment. The present study on O. fruticans and S. furcellata has shown that leaves of these plants are candidate anti-melanogenic agents. However, more work still needs to be done to elucidate other possible mechanisms that are relevant to antimelanogenic effects of these two plants. / 2020-08-31

Melanoma incidence is increasing faster than any other cancer worldwide.1 Early detection is often curative, but metastatic melanoma is lethal (5-year survival <20%) due to the development of resistance to all approved drugs.1 However, emerging evidence suggests that differences in melanoma metabolism relative to non-malignant cells may provide a target to improve treatment.2-14 Specifically, melanoma cells have increased mitochondrial electron transport chain (ETC) activity, elevated levels of reactive oxygen species, and a simultaneous hyperpolarized mitochondrial membrane potential relative to non-malignant cells.4, 8, 11, 15-17 Furthermore, melanoma cells have upregulated glucose consumption and concurrent increased levels of glucose transporters (GLUTs) relative to non-malignant cells; the products of glycolysis (pyruvate and NADPH) aid in the detoxification reactive oxygen species (ROS), while the intermediates are utilized in energy production via increased oxidative metabolism.15, 18 Collectively, melanoma cells exhibit alterations in metabolic, mitochondrial, and cell-surface targets that can be potentially exploited for therapeutic strategies for selective cancer cell killing relative to non-malignant cells. The research presented here demonstrates the therapeutic potential for a new class of mitochondrial-targeted fluorescent lipophilic-cations: pyridinium derivatives (UIRF 17023.186PV1 U.S. Provisional Patent Application No. 62/268,980 Patent Pending). Importantly, the pyridinium derivatives presented in this study have not been previously investigated as a mitochondrial-targeted therapy.19-21 Furthermore, the research presented outlines the feasibility of improving melanoma cellular accumulation of these pyridinium derivatives by including a GLUT targeting moiety in the form of a hexosamine. The addition of a hexosamine molecule to pyridinium derivatives has the potential to increase melanoma cell accumulation by targeting upregulation of GLUT expression in melanoma cells relative to normal cells. Thus, the results of this study identified: (1) a triphenylvinylpyridine (TPVP) lipophilic cation derivative that increased melanoma oxidative metabolism and decreased melanoma cell viability; and (2) the targeting potential for GLUT-mediated melanoma cell specific delivery of glucosamine-modified TPVP derivatives. These findings support the hypothesis that TPVP-based therapies can be developed to exploit fundamental differences in glucose and mitochondrial metabolism to selectively kill melanoma cells relative to non-malignant cells.

Doctor en Ciencias Biomédicas / Melanoma es la neoplasia cutánea más agresiva, con una incidencia ascendente a pesar de numerosos esfuerzos de prevención. En la actualidad existen diversas estrategias de tratamiento, sin embargo, no todos los pacientes responden a ellas. En nuestro laboratorio se ha ensayado una inmunoterapia basada en células dendríticas (DCs) maduradas con un lisado de líneas celulares de melanoma (TRIMEL), que ha incrementado hasta 3 veces la sobrevida en pacientes que desarrollan una respuesta inmunológica de hipersensibilidad retardada (DTH+). Hasta la fecha no se han desarrollado avances en el estudio de la fracción no peptídica de este lisado ni de su capacidad de activar células iNKT, capaces de reconocer antígenos glicolipídicas en contexto CD1d. Hipótesis: Células iNKT infiltran tumores de pacientes con melanoma avanzado y son capaces de ser activadas in vitro por células dendríticas utilizadas en inmunoterapia antitumoral. Objetivo General: Determinar el rol en pacientes con melanoma de células iNKT infiltrantes de tumor y activadas por células dendríticas maduradas ex vivo utilizadas en inmunoterapia antimelanoma. Métodos: Se determinaron células iNKT infiltrantes de tumor mediante inmunofluorescencia y se correlacionó su presencia en lesiones de melanoma primario en todos los estadios según Breslow con la sobrevida de la enfermedad. Se evaluó la capacidad de las fracciones glicolipídica y proteica de TRIMEL para inducir maduración en células dendríticas. Para este fin, las fracciones se obtuvieron mediante gradiente de densidad con MTBE (Metil-terbutil-éter) y se usaron para estimular DCs generadas en base a rhIL-4 y GM-CSF. En estas condiciones se evaluó la presencia de diversas moléculas de superficie y la producción de citoquinas. Para evaluar la capacidad de estas DCs de activar células iNKT, desde PBMC se aisló la población CD3+ mediante cell-sorting y cultivadas con distintas DCs generadas. Se analizó la proliferación celular utilizando el ensayo CFSE. También se evaluó la presencia de células iNKT mediante inmunofluorescencia en tacos de parafina de las DTH realizadas a pacientes de inmunoterapia. Resultados: Encontramos infiltración por células iNKT en todos los estadíos de la enfermedad, siendo mayor en Breslow avanzados, sin una correlación con la sobrevida global. Al evaluar la maduración de DCs, esta fue mayor en el grupo estimulada con la fracción peptídica de TRIMEL. DCs estimuladas con la fracción glicolipídica de TRIMEL no mostraron diferencias con la condición no tratada. Al evaluar mediante ensayo de proliferación realizado con CFSE, se evidenció mayor proliferación en la condición tratada con la fracción peptídica. Finalmente, sólo en 1 de 3 pacientes en los que se analizó la presencia de iNKT en las DTH se encontraron estas células. Conclusiones: unificando todos los hallazgos discutidos previamente, células iNKT infiltran tumores de melanoma, las que podrían ser inducidas a proliferar por DCs estimuladas con la fracción peptídica de TRIMEL y de esa forma migrar a sitios inflamados como representan las lesiones DTH. / Melanoma is the most aggressive skin tumor, with a growing incidence worldwide, despite of prevention strategies. Nowadays, there are several new therapies to treat it, however the overall survival continues to be low. Our lab has been studying a Dendritic Cell (DC) based immunotherapy maturated with a melanoma cell lines lysate (TRIMEL), which has increased survival up to 3 times in patients that developed a delayed type hypersensitivity response (DTH+). To this date no studies have been made regarding the non peptidic fraction of this lysate or its capability to activate a subpopulation of T Cells called iNKT cells, characterized by their ability to recognize glycolipids in a CD1d manner. Hypothesis: iNKT Cells infiltrate melanoma patient’s tumors and are capable to be activated in vitro by Dendritic cells Main Objective: To determinate the role of tumor infiltrating iNKT cells activated by matured Dendritic Cells ex vivo used in antimelanoma immunotherapy in melanoma patients Methods: The presence of tumor infiltrating iNKT Cells in primary melanoma in all of Breslow stages was evaluated by expression of TCR Vα24Jα18 by Immunofluorescence microscopy. To evaluate the capability of glycolipid and peptidic TRIMEL fraction to induce DCs maturation, they were isolated from TRIMEL using a MTBE (Metil-terbutil-ether) density gradient. Afterwards, these fractions were used to stimulate DCs generated with a rhIL-4 and GM-CSF protocol and surface molecules expression were assessed, as well as cytokine secretion. To evaluate DCs effectiveness to activate iNKT Cells’ proliferation, CD3+ population was isolated from Buffy Coats using cell-sorting technology and they were cultured with DCs. Proliferation was accessed with CFSE dye assay. Finally, DTH infiltrating iNKT cells were asessed in paraffined embedded samples by immunofluorescence microscopy. Results: We found that iNKT cells infiltrated melanoma tumors in every Breslow stage, with higher infiltration on advanced stages,with no correlation with overall survival. The peptidic fraction of TRIMEL induced higher expression of maturation related surface molecules in addition to cytokine secretion, whereas glycolipidic fraction showed no difference with the untreated condition. iNKT cell had a higher proliferation rate when cultured with DCs treated with the peptidic fraction of TRIMEL. And finally, we found the infiltration of iNKT in 1 of the 3 patients DTH. Conclusions: Taking together all the previous results, iNKT cells infiltrate melanoma Tumors, and they could be induced to proliferate by DCs stimulated with the peptidic fraction of TRIMEL and as well supporting their migration to inflamed tissues like the DTH samples. / 2021

Very few cancer patients are cured through drug therapy alone, with the majority exhibiting acquired resistance. To date, most studies of therapeutic escape have focused upon tumor-intrinsic mechanisms of drug resistance with little attention paid to the role of normal host cells in preventing complete tumor eradication. In the present study we implicate co-operative bi-directional signaling between melanoma cells and fibroblasts in the generation of a pro-survival niche that mediates drug resistance. Mass-spectrometry based phosphoproteomics was used to show that BRAF inhibition and chemotherapy drugs enhanced the survival of both melanoma cells and fibroblasts through the induction of fibronectin (FN)/integrin α5β1 signaling. Immunohistochemical staining confirmed the induction of FN in mouse xenografts and human melanoma specimens following BRAF inhibitor treatment. Adhesion to FN amplified the adaptive EGFR, HER3 and c-MET receptor tyrosine kinase (RTK) signals required for PI3K/AKT/Mcl-1-mediated melanoma cell survival when BRAF was inhibited. At the same time, BRAF inhibition led, directly and indirectly, to the paracrine release of HGF and neuregulin from fibroblasts, with TGF-β release from the melanoma cells increasing both fibroblast differentiation and survival. Although dual inhibition of RTKs and BRAF did not reverse host-mediated resistance, therapeutic escape was overcome through combined BRAF/PI3K inhibition, suggesting the PI3K/AKT pathway to be a common signaling vulnerability in microenvironment-mediated drug resistance. Our work suggests that durable responses to targeted therapies will only be achieved through dual targeting of the tumor and the adaptive host responses to therapy. These findings are especially important for a cancer such as melanoma, where as few as one cell can repopulate the entire tumor in vivo.

Although melanoma accounts for only 4% of skin cancers, it is responsible for 80% of deaths from skin cancers and its incidence in Caucasians has been increasing steadily during the last 30 years. So far, no treatment - except surgery at the earliest stages of the disease - has been shown to significantly improve survival. New treatments are thus clearly needed. The interest of immunologists for melanoma is based on particular features of this tumor. Rare spontaneous regressions have been described, which are possibly mediated by immune responses. Moreover, melanoma cell lines are relatively easy to obtain, providing essential tools for laboratory studies. The first melanoma vaccines involved inoculations of patients with autologous or allogeneic melanoma cells, as well as a variety of immunological adjuvants. Since the beginning of the nineties, the identification of antigens recognized on human tumors by autologous T lymphocytes has opened the way for new vaccination strategies involving molecularly defined tumor antigens. An important group of antigens recognized by T lymphocytes is encoded by “cancer-germ line genes”, which are expressed in tumors of various histological types, but are silent in normal tissues, with the exception of testis germinal cells and placental trophoblast. Since the latter do not express the HLA molecules required to present these antigens to the T lymphocytes, cancer-germ line genes encoded antigens are only present on tumors, which should limit the risk of generating autoimmune diseases as a consequence of vaccination. Therefore, these widely shared tumor specific antigens should represent good targets for the development of cancer vaccines. Our clinical research program of therapeutic vaccinations focuses on antigens encoded by the MAGE family of cancer-germ line genes. Most of the patients included in our phase I/II immunization trials had measurable metastatic melanoma. Several MAGE peptides as well as a recombinant MAGE-3 protein have been tested, while several additional trials are ongoing, including immunizations with a recombinant poxvirus coding for 2 MAGE epitopes. No major toxicity was reported. Tumor regressions have been observed in a minority of patients, mainly those who had regional or distant metastases without visceral involvement. Some of these regressions have been complete and long lasting. Although the rate of objective tumor response observed is low, it is clearly higher than the rate of spontaneous tumor regression observed in melanoma. Other immunization modes against T-cell defined epitopes are currently being explored by several groups in human clinical trials. Vaccines include peptides presented by class I or class II HLA molecules, proteins given alone or mixed with immunological adjuvants or cytokines, recombinant viral or bacterial vectors, dendritic cells and DNA encoding the antigen. Adoptive transfer of T lymphocytes selected for their capacity to recognize defined epitopes presented by the tumor represents another type of approach aiming at the destruction of the tumor by the immune system. It is difficult to predict whether and when therapeutic vaccination against cancer will reach an efficacy that will be sufficient for a standard cancer treatment. Provided their low toxicity, these vaccines should be tested in an adjuvant setting, at earlier stages of the disease.

A model with human melanoma xenografts transplanted to athymic mice has been adopted for in vivo studies of 5-S-cysteinyldopa (an intermediate pigment metabolite), glutathione, and cysteine. L-Dopa is an intermediate metabolite in pigment formation and is also important in the treatment of Parkinson's disease, and therefore 1 have also studied the pharmacokinetics of this compound. We were first to describe in vivo microdialysis in melanoma tissue and showed that dialysis membranes of cuprophane or polyamide are suitable for studies of interstitial 5-S-cysteinyldopa and selected thiols. Analytical procedures were also improved for quantitation of 5-S-cysteinyldopa, L-dopa, glutathione, cysteine, and N-acetylcysteine (NAC). In the melanoma xenografts the interstitial concentration of 5-S-cysteinyldopa reflected the high intracellular production of this intermediate metabolite. For in vivo manipulation of glutathione in the melanoma tissue we gave intraperitoneal injection of buthionine sulphoximine to the animals and thus reduced the glutathione concentrations substantially. We showed that restitution of glutathione in melanoma tissue occurs spontaneously and is not much improved by treatment with the cysteine deliverers NAC and L-2-oxothiazolidine-4-carboxylate (OTC). 5-S-Cysteinyldopa was not substantially affected by great variations in glutathione concentrations. Transport of NAC from intraperitoneal injection to melanoma tissue occurred rapidly and deacetylation to cysteine in vivo could be detected soon after NAC injection. In vivo formation of cysteine was slower from OTC than from NAC. Pharmacokinetic studies of L-dopa in human subjects indicated a slight to moderate protein binding. Plasma free L-dopa had similar elimination T½ as interstitial L-dopa, but in some cases the elimination of total L-dopa was slower. Difficulties in intestinal absorption of L-dopa were revealed by microdialysis in blood and subcutaneous tissue. Studies showed that this was due to delayed emptying of the stomach. L-Dopa intake increased 5-S-cysteinyldopa concentrations in blood within 30 min in patients with Parkinson's disease and a history of melanoma. No melanoma activation occurred during long-term treatment with L-dopa. Microdialysis is thus a safe and easily applied method for in vivo studies of both pigment metabolites from human melanoma tissue transplanted to nude mice and for pharmacokinetic studies of L-dopa. / On the day of the public defence the status of the articles IV, V and VI was: Submitted.

Cutaneous malignant melanoma (CMM) is one of the most rapidly increasing cancers in the Swedish population. The aetiology of melanoma is a complex interplay between genetics, host characteristics and environmental factors. The host characteristic with the strongest association with CMM is a phenotype with high numbers of common naevi and with dysplastic naevi. The principal environmental factor is sun exposure. Melanoma risk assessment (paper I) In a multi-national study including 986 subjects from Sweden, Denmark, the UK, Germany and the Netherlands, the ability of primary care physicians and nurses to identify individuals at increased melanoma risk was assessed. The atypical mole syndrome (AMS) scoring system for melanoma risk was used. The AMS scoring system consists of a five point check list incorporating total body naevus counts, clinically dysplastic naevi and body distribution of naevi. After brief training, the overall agreement in diagnosis between the trained personnel and experienced dermatologists was 94.5% (kappa value 0.70, p&lt;0.05). The study showed that the scoring system successfully can be taught to personnel in primary care. The naevus phenotype in a population in northern Sweden (paper II) The naevus phenotype was investigated in a population living in the inland of northern Sweden with a low melanoma incidence. Two hundred and one participants from the community of Storuman were included. The median naevus count was15 common naevi/individual, and the prevalence of dysplastic naevi was 11%. The median naevus count and prevalence of dysplastic naevi were significantly lower than previously described in populations with higher melanoma incidence and higher ambient ultraviolet exposure in southern Sweden. This geographical variation in naevus phenotype might be explained by differences in levels of sun exposure and in genotype. Cutaneous malignant melanoma in children and adolescents (papers III–V) During the years 1973 to 2002, 250 cases of primary CMM in individuals aged 0-19 years were reported to the Swedish Cancer Registry. Histological material was available for review in 87% of the cases registered during the two first decades (1973–1992). The diagnostic accuracy in the reviewed material was 88%. The melanoma incidence doubled in teenagers between the first decade (1973–1982) and the second (1983–1992). During the third decade (1993–2002) the increasing trend was broken. A decrease in incidence was noted in boys during 1993–1997, and in girls during 1998–2002. In younger children the incidence remained extremely low, only 4 cases in children aged 0–9 years were reported during the studied 30-year period. The trunk was the most common melanoma site in boys, and legs and trunk were the most common sites in girls. Superficial spreading melanoma was the most frequent subtype, followed by nodular melanoma. During the two first decades (1973–1992), the median melanoma thickness decreased from 1.5 to 0.9 mm. The melanoma-specific 5-year survival rate was 93%. The most important prognostic factor was melanoma thickness. The prognosis for thin lesions was excellent, during a median follow up time of 12 years, no tumour less than 0.8 mm was lethal according to the Registry. The results indicate that CMM in teenagers has many features in common with adult onset melanoma. The tudy also underlines the importance of not neglecting lesions suspected for malignant change in children and adolescents, as early detection and removal is crucial for the prognosis also in this young age group. / The electronic version of the thesis is a corrected version of the printed version.

<p>The chaperone protein GRP78 is primarily expressed in the endoplasmic reticulum, but it is also aberrantly expressed on the surface of cells under pathological conditions. One the cell membrane, GRP78 acts as a signaling molecule with unique properties. The amino-terminal domain acts as a growth factor receptor-like protein, while the carboxyl-terminal domain functions as a death-signaling receptor-like protein to extrinsically induce apoptosis. Autoantibodies that react with cell-surface GRP78 on many tumor cell lines occur in the sera of patients with prostate cancer, melanoma, and ovarian cancer. These autoantibodies are a negative prognostic factor in prostate cancer and melanoma, and when purified, stimulate tumor cell proliferation in vitro. It is unclear, however, whether these IgGs are merely a biomarker, or if they actually promote tumor growth in vivo. We immunized C57Bl/6 mice with recombinant GRP78 and then implanted the B16F1 murine melanoma cell line as flank tumors. We employed the antisera from these mice for in vitro cell signaling and proliferation assays. The immunodominant epitope in human cancer patients was well represented in the antibody repertoire of these immunized mice. We observed significantly accelerated tumor growth, as well as shortened survival in GRP78-immunized mice as compared to controls. Furthermore, antisera from these mice, as well as purified anti-GRP78 IgG from similarly immunized mice, stimulate Akt phosphorylation and proliferation in B16F1 and human DM6 melanoma cells in culture. These studies demonstrate a causal link between a humoral response to GRP78 and the progression of cancer in a murine melanoma model. They support the hypothesis that such autoantibodies are involved in the progression of human cancers and are not simply a biomarker. Because GRP78 is present on the surface of many types of cancer cells, this hypothesis has broad clinical and therapeutic implications.</p><p>We generated and characterized a panel of monoclonal murine antibodies (mAbs) against GRP78 with the goal of identifying therapeutic candidate IgGs. We developed three stable hybridomas that produce interesting antibodies. The N88 IgG reacts with the NH2-terminal domain and is an agonist. The C38 IgG reacts with the COOH-terminal domain and is an antagonist of NH2-terminal signaling. The C107 IgG binds the COOH-terminal domain and induces apoptosis. </p><p>We examined the effect of these three mAbs on the growth of B16 flank tumors. N88 accelerated and C107 slowed tumor growth, while C38 had no net effect. We are currently developing these antibodies and derivatives thereof as therapeutics for melanoma as well as for cancers of the brain, breast, ovary, and prostate. In fact, any tumor cell that over-expresses GRP78 on its surface is a potential therapeutic target for our future studies.</p> / Dissertation

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.