WM266-4 Cells Research Articles

Significant and Various Effects of ML329-Induced MITF Suppression in the Melanoma Cell Line.

To study the inhibitory effects on microphthalmia-associated transcription factor (MITF)-related biological aspects in malignant melanomas (MMs) in the presence or absence of the low-molecular MITF specific inhibitor ML329, cell viability, cellular metabolic functions, and three-dimensional (3D) spheroid formation efficacy were compared among MM cell lines including SK-mel-24, A375, dabrafenib- and trametinib-resistant A375 (A375DT), and WM266-4. Upon exposure to 2 or 10 μM of ML329, cell viability was significantly decreased in WM266-4, SK-mel-24, and A375DT cells, but not A375 cells, in a dose-dependent manner, and these toxic effects of ML329 were most evident in WM266-4 cells. Extracellular flux assays conducted using a Seahorse bioanalyzer revealed that treatment with ML329 increased basal respiration, ATP-linked respiration, proton leakage, and non-mitochondrial respiration in WM266-4 cells and decreased glycolytic function in SK-mel-24 cells, whereas there were no marked effects of ML329 on A375 and A375DT cells. A glycolytic stress assay under conditions of high glucose concentrations also demonstrated that the inhibitory effect of ML329 on the glycolytic function of WM266-4 cells was dose-dependent. In addition, ML329 significantly decreased 3D-spheroid-forming ability, though the effects of ML329 were variable among the MM cell lines. Furthermore, the mRNA expression levels of selected genes, including STAT3 as a possible regulator of 3D spheroid formation, KRAS and SOX2 as oncogenic-signaling-related factors, PCG1a as the main regulator of mitochondrial biogenesis, and HIF1a as a major hypoxia transcriptional regulator, fluctuated among the MM cell lines, possibly supporting the diverse ML329 effects mentioned above. The findings of diverse ML329 effects on various MM cell lines suggest that MITF-associated biological activities are different among various types of MM.

IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration.

Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca2+-dependent, IK-like, clotrimazole- and senicapoc-sensitive currents, which could not be detected in Panc-1 cells. Neither clotrimazole nor senicapoc altered the intracellular Ca2+ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.

Hypoxia-Induced Changes in L-Cysteine Metabolism and Antioxidative Processes in Melanoma Cells.

This study was performed on human primary (WM115) and metastatic (WM266-4) melanoma cell lines developed from the same individual. The expression of proteins involved in L-cysteine metabolism (sulfurtransferases, and cystathionine β-synthase) and antioxidative processes (thioredoxin, thioredoxin reductase-1, glutathione peroxidase, superoxide dismutase 1) as well as the level of sufane sulfur, and cell proliferation under hypoxic conditions were investigated. Hypoxia in WM115 and WM266-4 cells was confirmed by induced expression of carbonic anhydrase IX and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 by the RT-PCR and Western blot methods. It was shown that, under hypoxic conditions the inhibition of WM115 and WM266-4 melanoma cell proliferation was associated with decreased expression of thioredoxin reductase-1 and cystathionine β-synthase. These two enzymes may be important therapeutic targets in the treatment of melanoma. Interestingly, it was also found that in normoxia the expression and activity of 3-mercaptopyruvate sulfurtransferase in metastatic WM266-4 melanoma cells was significantly higher than in primary melanoma WM115 cells.

3D melanoma spheroid model for the development of positronium biomarkers

It was recently demonstrated that newly invented positronium imaging may be used for improving cancer diagnostics by providing additional information about tissue pathology with respect to the standardized uptake value currently available in positron emission tomography (PET). Positronium imaging utilizes the properties of positronium atoms, which are built from the electrons and positrons produced in the body during PET examinations. We hypothesized that positronium imaging would be sensitive to the in vitro discrimination of tumor-like three-dimensional structures (spheroids) built of melanoma cell lines with different cancer activities and biological properties. The lifetime of ortho-positronium (o-Ps) was evaluated in melanoma spheroids from two cell lines (WM266-4 and WM115) differing in the stage of malignancy. Additionally, we considered parameters such as the cell number, spheroid size and melanoma malignancy to evaluate their relationship with the o-Ps lifetime. We demonstrate pilot results for o-Ps lifetime measurement in extracellular matrix-free spheroids. With the statistical significance of two standard deviations, we demonstrated that the higher the degree of malignancy and the rate of proliferation of neoplastic cells, the shorter the lifetime of ortho-positronium. In particular, we observed the following indications encouraging further research: (i) WM266-4 spheroids characterized by a higher proliferation rate and malignancy showed a shorter o-Ps lifetime than WM115 spheroids characterized by a lower growth rate. (ii) Both cell lines showed a decrease in the lifetime of o-Ps after spheroid generation on day 8 compared to day 4 in culture, and the mean o-Ps lifetime was longer for spheroids formed from WM115 cells than for those formed from WM266-4 cells, regardless of spheroid age. The results of this study revealed that positronium is a promising biomarker that may be applied in PET diagnostics for the assessment of the degree of cancer malignancy.

Quantifying Cell Heterogeneity and Subpopulations Using Single Cell Metabolomics.

Mass spectrometry (MS) has become an indispensable tool for metabolomics studies. However, due to the lack of applicable experimental platforms, suitable algorithm, software, and quantitative analyses of cell heterogeneity and subpopulations, investigating global metabolomics profiling at the single cell level remains challenging. We combined the Single-probe single cell MS (SCMS) experimental technique with a bioinformatics software package, SinCHet-MS (Single Cell Heterogeneity for Mass Spectrometry), to characterize changes of tumor heterogeneity, quantify cell subpopulations, and prioritize the metabolite biomarkers of each subpopulation. As proof of principle studies, two melanoma cancer cell lines, the primary (WM115; with a lower drug resistance) and the metastatic (WM266-4; with a higher drug resistance), were used as models. Our results indicate that after the treatment of the anticancer drug vemurafenib, a new subpopulation emerged in WM115 cells, while the proportion of the existing subpopulations was changed in the WM266-4 cells. In addition, metabolites for each subpopulation can be prioritized. Combining the SCMS experimental technique with a bioinformatics tool, our label-free approach can be applied to quantitatively study cell heterogeneity, prioritize markers for further investigation, and improve the understanding of cell metabolism in human diseases and response to therapy.

Adriamycin-loaded exosome with anti-CD20 aptamers selectively suppresses human CD20+ melanoma stem cells.

Targeting CD20+ melanoma cancer stem cells (CSCs) subset is essential for treating melanoma. Anti-CD20 aptamer-modified exosomes (ACEXO) loaded with Adriamycin could be a therapeutic strategy for targeting CSCs. Exosomes loaded with Adriamycin were modified with anti-CD20 aptamer and characterized by size and molecular markers using transmission electron microscope and dynamic light scattering. The uptake of ACEXO into CD20+ cells was checked, and its cytotoxicities in CD20+ melanoma cells, HEK 293T, and 3T3 cells were evaluated. At the same time, the in vivo distribution of ACEXO in the tumor-bearing mice model was determined. The particle size of the exosome is about 80-100nm. Western blot analysis showed that they expressed the characteristic exosome markers: CD9 and CD63. Quantitative analysis of the mean fluorescence intensity after 4h incubation showed that ACEXO significantly improved Adriamycin uptake. Notably, the ACEXO killed only CD20+ melanoma cells. In addition, they exhibited good biocompatibility with both 293T and 3T3 cells at all doses. After intravenous injection, exosome distribution data showed that ACEXO's accumulation in the tumor is higher than anti-CD20-modified exosomes (AEXO)'s at all time points, and the accumulation increased as time prolonged. Addition of ACEXO reduces the number of tumorspheres in A375 or WM266-4 cells compared to untreated controls or AEXO-treated group. More important, while treating melanoma tumor-bearing mice, ACEXO-treated group showed the lowest tumor weight without body weight loss. ACEXO loaded with Adriamycin could suppress tumor cell growth in vitro and in vivo, probably by targeting CD20+ melanoma CSCs.

From Proteomic Mapping to Invasion-Metastasis-Cascade Systemic Biomarkering and Targeted Drugging of Mutant BRAF-Dependent Human Cutaneous Melanomagenesis.

Simple SummaryDespite the recent advances in human malignancy therapy, metastasis and chemoresistance remain the principal causes of cancer-derived deaths. Given the fatal forms of cutaneous metastatic melanoma, we herein employed primary (WM115) and metastatic (WM266-4) melanoma cells, both obtained from the same patient, to identify novel biomarkers and therapeutic agents. Through state-of-the-art technologies including deep proteome landscaping, immunofluorescence phenotyping, and drug toxicity screening, we were able to describe new molecular programs, oncogenic drivers, and drug regimens, controlling the invasion-metastasis cascade during BRAFV600D-dependent melanomagenesis. It proved that proteomic navigation could foster the development of systemic biomarkering and targeted drugging for successful treatment of advanced disease.Melanoma is classified among the most notoriously aggressive human cancers. Despite the recent progress, due to its propensity for metastasis and resistance to therapy, novel biomarkers and oncogenic molecular drivers need to be promptly identified for metastatic melanoma. Hence, by employing nano liquid chromatography-tandem mass spectrometry deep proteomics technology, advanced bioinformatics algorithms, immunofluorescence, western blotting, wound healing protocols, molecular modeling programs, and MTT assays, we comparatively examined the respective proteomic contents of WM115 primary (n = 3955 proteins) and WM266-4 metastatic (n = 6681 proteins) melanoma cells. It proved that WM115 and WM266-4 cells have engaged hybrid epithelial-to-mesenchymal transition/mesenchymal-to-epithelial transition states, with TGF-β controlling their motility in vitro. They are characterized by different signatures of SOX-dependent neural crest-like stemness and distinct architectures of the cytoskeleton network. Multiple signaling pathways have already been activated from the primary melanoma stage, whereas HIF1α, the major hypoxia-inducible factor, can be exclusively observed in metastatic melanoma cells. Invasion-metastasis cascade-specific sub-routines of activated Caspase-3-triggered apoptosis and LC3B-II-dependent constitutive autophagy were also unveiled. Importantly, WM115 and WM266-4 cells exhibited diverse drug response profiles, with epirubicin holding considerable promise as a beneficial drug for metastatic melanoma clinical management. It is the proteome navigation that enables systemic biomarkering and targeted drugging to open new therapeutic windows for advanced disease.

Contribution of sialic acids to integrin α5β1 functioning in melanoma cells

PurposeTo establish the relationship between sialylation of integrin α5β1 and possible alteration in the function of α5β1 receptor in melanoma cells. Materials and methodsIntegrin α5β1 was isolated from primary WM115 (RGP/VGP-like phenotype) and metastatic WM266-4 (lymph node metastasis) cells via affinity chromatography. Integrin α5β1 sialylation and the shift in relative masses of the enzymatically desialylated subunits were confirmed by confocal microscopy and SDS-PAGE, respectively. The ELISA assay was performed to evaluate sialic acid (SA) influence on integrin α5β1 binding to fibronectin (FN). Cell invasion was investigated by the Transwell invasion assay. The effect of neuraminidases treatment on melanoma cells was assessed by flow cytometry using Maackia amurensis and Sambucus nigra lectins. ResultsBoth subunits of integrin α5β1 were found to be more abundantly sialylated in primary than in metastatic cells. The removal of SA had no effect on the purified integrin α5β1 binding to FN. Although metastatic cells underwent more pronounced desialylation than primary cells, invasion of primary WM115 cells was more dependent on the presence of α2-3 linked SA than it was in the case of metastatic WM266-4 cells. In both melanoma cell lines not only integrin α5β1 was involved in invasion, however simultaneous desialylation and usage of anti-integrin α5β1 antibodies resulted in lower invasion abilities of primary WM115 cells. ConclusionsOur data suggest that in primary melanoma cells integrin α5β1 action is more likely dependent on its glycosylation profile, i.e. the presence of SA residues, which influence (decreased) their invasion properties and may facilitate malignant melanoma progression.

Adaptability of single melanoma cells to surfaces with distinct hydrophobicity and roughness

Cell-substrate interaction is one of the crucial phenomenon that determines cell behavior and fate. Here, we present the results on the effect of physical properties (i.e., hydrophobicity/hydrophilicity and roughness) of polyethylene oxide (PEO) and 1,4-polyisoprene (PI) thin films on adhesion of two melanoma cell lines, originating from primary tumor site from vertical growth phase (VGP WM115 cells) and from its metastasis to skin (WM266-4 cells). Our findings show that cell–substrate interaction is strongly dependent on the stage of melanoma progression. For both studied cell types, the dependence of cellular adhesive properties (quantified from single cell force spectroscopy) on surface free energy, particularly, on its polar component, was observed. Adhesive forces of VGP WM115 cells to thin polymer films (TPF) with various hydrophobicity and roughness are larger as compared to those observed in the case of metastatic WM266-4 cells. The proliferation of the latter cells was higher as for VGP WM115 cells, what agrees with the invasive phenotype of these melanoma cells. In summary, the metastatic WM266-4 cells adapt better to different substrates properties as compared to VGP WM115 ones.

Piceatannol induced apoptosis through up-regulation of microRNA-181a in melanoma cells

BackgroundMelanoma took top position among the lethal cancers and, despite there have been some great attempts made to increase the natural life of patients with metastatic disease, long-lasting and complete remissions are few. Piceatannol, owns the similar function as resveratrol, has been defined as an anti-cancer agent playing important role in inhibition of proliferation, migration and metastasis in various cancer. Thus, we aim to investigate the anti-cancer effect and mechanisms of piceatannol in melanoma cells.MethodsMelanoma cell lines WM266-4 and A2058 were treated either with or without piceatannol. Cell viability and cell apoptosis were assessed by using MTT and Annexin V/PI assay, respectively. Cells were transfected with specific miRNA using Lipfectamine 2000. miRNA bingding ability to 3'-UTR region within specific gene was assed by firefly luciferase analysis. Gene and protein expression was eveluated by qRT-PCR and western blot analysis, respectively.ResultsOur study showed that piceatannol inhibited WM266-4 and A2058 cells growth and induced apoptosis. Totally, 16 differentially expressed miRNAs were screened out including 8 up-regulated and 8 down-regulated miRNAs. Expression level of miR-181a is significantly higher in piceatannol-treated cells than normal control and is lower in melanoma cancer tissues than its adjacent normal tissues. Bcl-2 is a target gene of miR-181a. Moreover, silencing of miR-181a reverses the decrease of cell viability induced by piceatannol in WM266-4 and A2058 cells. Taken together, present study uncovered the ability of piceatannol to repress melanoma cell growth and clarified the contribution of miR-181a in the anticancer role of piceatannol.ConclusionThe present study proposes that piceatannol can be taken into account to be a hopeful anticancer agent for melanoma.

Cuprous oxide nanoparticle-inhibited melanoma progress by targeting melanoma stem cells.

Recent studies have shown that metal and metal oxide have a potential function in antitumor therapy. Our previous studies demonstrated that cuprous oxide nanoparticles (CONPs) not only selectively induce apoptosis of tumor cells in vitro but also inhibit the growth and metastasis of melanoma by targeting mitochondria with little hepatic and renal toxicities in mice. As a further study, our current research revealed that CONPs induced apoptosis of human melanoma stem cells (CD271+/high cells) in A375 and WM266-4 melanoma cell lines and could significantly suppress the expression of MITF, SOX10 and CD271 involved in the stemness maintenance and tumorigenesis of melanoma stem cells. CD271+/high cells could accumulate more CONPs than CD271−/low through clathrin-mediated endocytosis. In addition, lower dosage of CONPs exhibited good anti-melanoma effect by decreasing the cell viability, stemness and tumorigenesis of A375 and WM266-4 cells through reducing the expression of SOX10, MITF, CD271 and genes in MAPK pathway involved in tumor progression. Finally, CONPs obviously suppressed the growth of human melanoma in tumor-bearing nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice, accompanied with tumors structural necrosis and fibrosis remarkably and decreased expression of CD271, SOX10 and MITF. These results above proved the effectiveness of CONPs in inhibiting melanoma progress through multiple pathways, especially through targeting melanoma stem cells.

Towards understanding the role of sialylation in melanoma progression.

Aberrant expression of sialic acids or altered linkage types is closely associated with malignant phenotype and metastatic potential, and can have prognostic significance in human cancer. The present study was undertaken to evaluate whether expression of sialylated derivatives on melanoma cell surface is associated with tumour progression. Four cell lines (WM1552C, WM115, IGR-39 and WM266-4) were used in the study. Cell surface expression of sialic acids was evaluated by flow cytometry with the use of Maackia amurensis and Sambucus nigra lectins. Moreover, adhesion and migration potential of melanoma cells and involvement of sialic acids in these processes were analysed. We have demonstrated that WM266-4 cells have a significantly higher level of α2,3-linked sialic acid residues than other cells, whereas IGR-39 cells had lower expression of α2,6-linked sialic acids. The adhesion efficiencies of WM1552C and WM115 cells were significantly lower than that of IGR-39 and WM266-4 cells. In contrast, WM266-4 cells repaired scratch wounds at least twice as fast as other cells. Melanoma cell adhesion to fibronectin in the presence of Sambucus nigra agglutinin (SNA) was reduced only in IGR-39 and WM266-4 cells, whereas the impact of Maackia amurensis agglutinin (MAA) on this process was much more important. Migration efficiency of melanoma cells was reduced more strongly in the presence of MAA than SNA. In conclusion, our results show that melanoma progression is associated with the increased expression of α2,3-linked sialic acids on the cell surface and these residues could promote melanoma cell interaction with fibronectin.

Abstract 1146: Enhancer of zeste homolog 2 (EZH2) inhibition in metastatic melanoma promotes tumor regression in vivo

Abstract Melanoma is a deadly variety of skin cancer, which is responsible for three-fourths of skin cancer-related deaths, but constitutes less than 5% of all skin cancer cases. It has been estimated that in 2015, about 73,870 new cases of melanoma will be diagnosed in United States alone, of which 9,940 are expected to die. According to the ACS, melanoma is also the fastest growing cancer, which makes it essential to investigate the pathogenesis involved in the advanced stage of melanoma, and design effective therapeutics for its treatment. Drugs targeting epigenetic modifiers like EZH2 are becoming increasingly popular because of the fact that most epigenetic modifications and their effects can be reversed using these drugs. There has been no study till date investigating the role of EZH2 in melanoma. Our laboratory is investigating the role of EZH2 in the pathogenesis involved in the advanced stage of melanoma, to enable the design of effective therapeutic strategies for the treatment of melanoma. Here we investigate the relationship between EZH2 and tumor suppressors E-cadherin and RUNX3 in melanoma using matched melanoma cell lines (WM115 and WM266-4), formalin-fixed-paraffin-embedded (FFPE) patient tissue samples and in vivo xenograft model. The studies performed in cell lines and FFPE tissue samples suggested EZH2-mediated silencing of E-cadherin and RUNX3 by promoting H3K27-trimethylation of the E-CADHERIN and RUNX3 promoters in WM266-4 cells (metastatic; high EZH2; low E-cadherin) relative to WM115 cells (primary; low EZH2; high E-cadherin). Furthermore, we observed that EZH2 inhibition restores E-cadherin and RUNX3 expression in metastatic melanoma cells by reducing EZH2 and H3K27me3 occupancy at E-CADHERIN and RUNX3 promoters respectively. In addition, we found that inhibition of EZH2 besides inducing apoptosis and inhibiting cellular proliferation, also adversely affected invasion of metastatic melanoma. E-cadherin depletion had an opposite effect, suggesting that EZH2 mediated E-cadherin silencing promotes invasion in metastatic melanoma. In vivo, treatment of WM266-4 xenografts with EZH2 inhibitor resulted in significant reduction of tumor growth. In combination with Dabrafenib, this effect was highly synergistic. Analysis of harvested tumors indicated increased E-cadherin and RUNX3 levels in EZH2 inhibited group compared to vehicle, due to reduced H3K27me3 enrichment by EZH2 at E-cadherin and RUNX3 promoters, validating our in vitro finding. Tumor regression by combination therapy was also accompanied by significant increased pro-apoptotic proteins like Bak, Bax and decreased anti-apoptotic proteins Bcl-xL and Bcl-2. Hence, our findings suggest strong potential of EZH2 inhibitors as promising melanoma drugs either by itself (monotherapy), or in conjunction with other chemotherapeutics (combination therapy). Citation Format: Deepanwita Sengupta, Nathan Avaritt, Sara C. Shalin, Alan J. Tackett. Enhancer of zeste homolog 2 (EZH2) inhibition in metastatic melanoma promotes tumor regression in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1146.

β1,6-branched complex-type N-glycans affect FAK signaling in metastatic melanoma cells

ABSTRACTIntegrin-dependent binding of the cell to extracellular matrix (ECM) is a key activator of the focal adhesion kinase (FAK) signaling pathway. N-glycosylation of integrins affects their interactions with ECM proteins. Using WM266-4 cells with overexpression of β1,6-acetylglucosaminyltransferase V, we showed that β1,6-branched N-glycans increased tyrosine phosphorylation of FAK in metastatic melanoma cells, resulting in enhanced migration on vitronectin (VN). The co-localization of αvβ3 integrin and FAK in focal adhesions of melanoma cells growing on VN indicates their interaction in signal transduction. Melanoma cell migration on VN was mediated by αvβ3 caring overexpressed β1,6-branched structures, important for FAK upregulation.

Comparing surface properties of melanoma cells using time of flight secondary ions mass spectrometry.

Various techniques have been already reported to differentiate between normal (non-malignant) and cancerous cells based on their physico-chemical properties. This is relatively simple when studied cancerous cells originate from distant stages of cancer progression. Here, studies on chemical properties of two closely related human melanoma cell lines are presented: WM115 melanoma cells were taken from the vertical growth phase while WM266-4 from the skin metastatic site of the same patient. Their chemical properties were studied by two techniques, namely time-of-flight secondary ion mass spectra (ToF SIMS) and photothermal microspectroscopy (PTMS), used to record mass and photothermal spectra of cells, respectively. In our approach, independently of the spectra type, its full range, i.e. masses and wavenumbers within the range 0-500 kDa and 500-4000 cm-1, underwent a similar methodology for principal component analysis (PCA). PCA outcome shows results groupped depending on the sample type (either WM115 or WM266-4 cells). The results are independent of the method applied to study chemical properties of melanoma cells, indicating that cancer-related changes are large enough to be identified with these techniques and to differentiate between cells originating from vertical growth phase and skin metastatis.

CD20 Antibody-Conjugated Immunoliposomes for Targeted Chemotherapy of Melanoma Cancer Initiating Cells.

Cancer initiating cells (CIC) are tumorigenic cancer cells that have properties similar to normal stem cells. CD20 is a phenotype of melanoma CIC that is responsible for melanoma drug resistance. Vincristine (VCR) is commonly used in melanoma therapy; however, it has been found ineffective against CIC. To target CD20+ melanoma CIC, we prepared VCR-containing immunoliposomes that were conjugated to CD20 antibodies (VCR-Lip-CD20). The drug release profile and the antibody-mediated targeting of the immunoliposomes were optimized to target CD20+ melanoma CIC. The immunoliposomes had desirable particle size (163 nm), drug encapsulation efficiency (91.8%), and drug release profile. We demonstrated that these immunoliposomes could successfully target more than 55% of CD20+ Chinese Hamster Ovary cells (CHO-CD20) even when the CHO-CD20 cells accounted for only 0.1% of a mixed population of CHO-CD20 and CHO cells. After treating WM266-4 melanoma mammospheres for 96 h, the ICo values of the drug delivered in VCR-Lip-CD20, VCR-Lip (VCR liposomes), and VCR were found to be 53.42, 98.99, and 99.09 μg/mL, respectively, suggesting that VCR-Lip-CD20 was 1.85 times more effective than VCR-Lip and VCR. VCR-Lip-CD20 could almost completely remove the tumorigenic ability of WM266-4 mammospheres in vivo, and showed the best therapeutic effect in WM266-4 melanoma xenograft mice. Significantly, VCR-Lip-CD20 could selectively kill CD20+ melanoma CIC in populations of WM266-4 cells both in vitro and in vivo. We demonstrated that VCR-Lip-CD20 has the potential to efficiently target and kill CD20+ melanoma CIC.

Human melanocytes form a PAX3-expressing melanocyte cluster on Matrigel by the cell migration process

BackgroundThe interactions between human epidermal melanocytes and their cellular microenvironment are important in the regulation of human melanocyte functions or in their malignant transformation into melanoma. Although the basement membrane extracellular matrix (BM-ECM) is one of major melanocyte microenvironments, the effects of BM-ECM on the human melanocyte functions are not fully explained at a molecular level. ObjectiveThis study was aimed to characterize the molecular and cellular interactions between normal human melanocytes (NHMs) and BM-ECM. MethodsWe investigated cell culture models of normal human melanocytes or melanoma cells on three-dimensional (3D) Matrigel to understand the roles of the basement membrane microenvironment in human melanocyte functions. Melanogenesis and melanobast biomarker expression in both primary human melanocytes and melanoma cells on 3D Matrigel were evaluated. ResultsWe found that NHMs migrated and formed reversible paired box 3 (PAX3) expressing cell clusters on three-dimensional (3D) Matrigel. The melanogenesis was significantly decreased in the PAX3 expressing cell cluster. The expression profile of PAX3, SOX10, and MITF in the melanocyte cluster on 3D Matrigel was similar to that of melanoblasts. Interestingly, PAX3 and SOX10 showed an inverse expression profile in NHMs, whereas the inverse expression pattern of PAX3 and SOX10 was disrupted in melanoma MNT1 and WM266-4 cells. ConclusionThe human melanocyte culture on 3D Matrigel provides an alternative model system to study functions of human melanoblasts. In addition, this system will contribute to the elucidation of PAX3-related tumorigenic mechanisms to understand human melanoma.

Common Glycoproteins Expressing Polylactosamine-Type Glycans on Matched Patient Primary and Metastatic Melanoma Cells Show Different Glycan Profiles

Recently, we reported comparative analysis of glycoproteins which express cancer-specific N-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type N-glycans that are abundantly present in malignant cells [ Mitsui et al., J. Pharm. Biomed. Anal. 2012 , 70 , 718 - 726 ]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type N-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these N-glycans, glycopeptides having polyLacNAc-type N-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing N-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells.

Abstract C232: B-raf/Mek/Erk Pathway inhibition induces GPNMB expression and sensitizes melanoma cells to the antibody-drug conjugate glembatumumab vedotin.

Abstract Background: Recent advances with B-raf and Mek targeted therapies have transformed the therapeutic landscape for metastatic melanoma and improved survival times for patients. Despite this progress, resistance to targeted therapies remains a universal problem that limits treatment efficacy. Combination therapy strategies represent a viable option to overcome the problem of therapeutic resistance. Glycoprotein Non-Metastatic B (GPNMB) is a cell surface protein that is highly expressed in a wide variety of cancer types, and regulates: tumor growth, cancer cell migration, invasion and metastasis. Glembatumumab vedotin (CDX-011) is a GPNMB-targeted antibody-drug conjugate that is in clinical trials for the treatment of melanoma and breast cancer. Interestingly, a wide spectrum of kinase inhibitors (KI) has been reported to induce GPNMB expression in a variety of cell types. Here we investigate the mechanism(s) responsible for elevated GPNMB levels in melanoma cells treated with clinically relevant KI, determine whether KI-mediated induction of GPNMB expression synergizes with CDX-011 in the treatment of melanoma. Methods: B-raf mutant (A375, WM-2664) and N-ras mutant (SkMel2) melanoma cells were exposed to inhibitors of B-raf (vemurafenib, dabrafenib), or Mek (trametinib, selumetinib). Immunoblot analysis or fluorescence-activated cell sorting was used to assess GPNMB expression. B-raf mutant cells were passaged in the presence of vemurafenib over the course of several weeks to months to generate vemurafenib-resistant cells. Transient siRNA knockdown studies were used to assess the requirement for the transcription factor, MiTF, in KI-mediated induction of GPNMB. To assess the interaction between individual KI and CDX011, dose response curves for the KI and CDX-011 alone or in combination in melanoma cells in vitro were generated. Melanoma cells were either 1) pretreated or 2) pre and co-treated with KI for 48 hours prior to treatment with CDX011 for 96 hours. Cell viability was assessed by XTT assay. Results: B-raf inhibition led to an induction of total and cell surface GPNMB protein in B-raf mutant, but not in B-raf WT melanoma cells. Mek inhibition induced GPNMB expression in all three melanoma cell lines. In addition to acute inhibitor treatment, Vemurafenib-resistant cells also expressed higher levels of GPNMB when compared to parental cells (inhibitor sensitive). Knock-down of the MiTF transcription factor abrogated the vemurafenib-mediated induction of GPNMB. Pretreatment of melanoma cells with Mek inhibitors led to a reduction in cell viability, compared to untreated cells, that was further exacerbated by exposure to CDX-011, in a dose-dependent manner. Interestingly, sub-optimal doses with B-raf inhibitors did not significantly affect cell viability of WM-2664 cells when used in isolation, but did enhance the sensitivity of melanoma cells to CDX-011. Conclusions: Induction of GPNMB in response to B-raf/Mek inhibition is a MiTF-dependent process that occurs in both B-raf mutant and WT cells. In this context, induction of GPNMB expression sensitizes cells to CDX-011 mediated cell killing. As such, the combination of B-raf/Mek inihibitors with CDX-011 represents an intriguing new combination therapy strategy for the treatment of melanoma, and warrants further validation in in vivo mouse models. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C232. Citation Format: April A.N. Rose, Peter M. Siegel. B-raf/Mek/Erk Pathway inhibition induces GPNMB expression and sensitizes melanoma cells to the antibody-drug conjugate glembatumumab vedotin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C232.

Abstract 3401: Vemurafenib-induced RhoB expression leads to melanoma cell resistance.

Abstract Most of melanoma harbour mutations leading to the constitutive activation of the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway (i.e: activating mutations of BRAF, NRAS, c-KIT). Addiction of melanoma to the MAPK pathways led to the development of inhibitors targeting either mutant BRAF, such as Vemurafenib (PLX4032), or MEK. Melanoma treatment recently met a breakthrough with the clinical evaluation of Vemurafenib that demonstrated an unprecedented 80% response rate among patients with BRAF V600E melanomas treated with Vemurafenib, but rapidly limited by drug resistance, which still requires to be elucidated. The MAPK pathway regulates the expression of many genes which can mediate therapeutic response but also resistance, such as RhoGTPases. We analysed the mRNA expression of the RhoGTPases in two BRAF mutated melanoma cell lines, WM266-4 and A375, in response to inhibition of the MAPK pathway by PLX4032. We observed that the mRNA of RhoB was increased by PLX4032 treatment. This result was confirmed in many other BRAF mutated melanoma cell lines by inhibition of the MAPK pathway with BRAF inhibitors (PLX4032 or SB-590885) as well as MEK inhibitors (AZD6244 or AS703026). MEK inhibitors (AZD6244 or AS703026) upregulate RhoB in BRAF wild-type melanoma cells harbouring N-Ras, K-Ras or c-KIT mutation suggesting that inhibiting MAPK pathway is critical for RhoB expression modulation. It is noteworthy that this modulation is cell type dependent, while it was not found in BRAF mutated non-melanoma cells as colon carcinoma cells (Colo205 and RKO). To investigate the role of RhoB induction in cellular responses to BRAF and MEK inhibitors, the RhoB induction was suppressed using RNA interference. The cells treated with siRNA targeting RhoB, WM266-4 and A375 cells, were more sensitive to PLX4032 and AZD6244. Moreover the blockade of RhoB induction led to a significant increase of PLX4032-induced apoptosis as shown by the increase of the subG1 population and PARP cleavage. Our findings show that RhoB upregulation is involved in PLX4032 response and both inhibitions of MAPK pathway and RhoB expression trigger cell death and should prompt a search for agents. Indeed co-targeting MAPK and RhoB pathways, could lead to a synthetic lethality and prevent resistance to either BRAF or MEK inhibitors. Ongoing work focuses on confirming this role in tumour-xenografted mouse models. Citation Format: Audrey Delmas, Julia Cherier, Claire Medale-Giamarchi, Anne Pradines, Gilles Favre. Vemurafenib-induced RhoB expression leads to melanoma cell resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3401. doi:10.1158/1538-7445.AM2013-3401