u-0126 and Melanoma

The extracellular signal-regulated kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as melanoma. Melanoma remains incurable despite the use of conventional chemotherapy; consequently, development of new therapeutic agents for melanoma is highly desirable. Here, we carried out a chemical genetic screen using a fission yeast phenotypic assay and showed that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), which is a natural ginger compound, effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. ACA-28 more potently inhibited the growth of melanoma cells than did the parental compound ACA. Importantly, the growth of normal human epidermal melanocytes (NHEM) was less affected by ACA-28 at the same 50% inhibitory concentration. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells which were oncogenically transformed with human epidermal growth factor receptor-2 (HER2/ErbB2), but not in the parental cells. Notably, the ACA-28-induced apoptosis in melanoma and HER2-transformed cells was abrogated when ERK activation was blocked with a specific MEK inhibitor U0126. Consistently, ACA-28 more strongly stimulated ERK phosphorylation in melanoma cells, as compared in NHEM. ACA-28 might serve as a promising seed compound for melanoma treatment.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; Benzyl Alcohols; Butadienes; Cell Line, Tumor; Cell Transformation, Neoplastic; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Humans; MAP Kinase Kinase 1; Melanocytes; Melanoma; Mice; Nitriles; Phosphorylation; Signal Transduction

Cancer treatment often tends to involve direct targeting enzymes essential for the growth and proliferation of cancer cells. The aim of this study was the recognition of the possible role of selected protein kinases: PI3K, ERK1/2, and mTOR in cell proliferation and cell cycle in malignant melanoma. We investigated the role of protein kinase inhibitors: U0126 (ERK1/2), LY294002 (PI3K), rapamycin (mTOR), everolimus (mTOR), GDC-0879 (B-RAF), and CHIR-99021 (GSK3beta) in cell proliferation and expression of crucial regulatory cell cycle proteins in human melanoma cells: WM793 (VGP) and Lu1205 (metastatic). They were used either individually or in various combinations. The study on the effect of signaling kinases inhibitors on proliferation-BrdU ELISA test after 48-72 h. Their effect on the expression of cell cycle regulatory proteins: cyclin D1 and D3, cyclin-dependent kinase CDK4 and CDK6, and cell cycle inhibitors: p16, p21, and p27, was studied at the protein level (western blot). Treatment of melanoma cells with protein kinase inhibitors led to significantly decreased cell proliferation except the use of a GSK-3β kinase inhibitors-CHIR-99021. The significant decrease in the expression of selected cyclins and cyclin-dependent kinases (CDKs) with parallel increase in the expression of some of cyclin-dependent kinases inhibitors and in consequence meaningful reduction in melanoma cell proliferation by the combinations of inhibitors of signaling kinases clearly showed the crucial role of AKT, ERK 1/2, and mTOR signal transduction in melanoma progression. The results unanimously indicate those pathways as an important target for treatment of melanoma.

Topics: Antineoplastic Agents; Butadienes; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Cyclin D3; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Everolimus; Humans; Indenes; Melanoma; Morpholines; Nitriles; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Pyrimidines; Sirolimus

Sildenafil, an inhibitor of the cGMP-degrading phosphodiesterase 5 that is used to treat erectile dysfunction, has been linked to an increased risk of melanoma. Here, we have examined the potential connection between cGMP-dependent signaling cascades and melanoma growth. Using a combination of biochemical assays and real-time monitoring of melanoma cells, we report a cGMP-dependent growth-promoting pathway in murine and human melanoma cells. We document that C-type natriuretic peptide (CNP), a ligand of the membrane-bound guanylate cyclase B, enhances the activity of cGMP-dependent protein kinase I (cGKI) in melanoma cells by increasing the intracellular levels of cGMP. Activation of this cGMP pathway promotes melanoma cell growth and migration in a p44/42 MAPK-dependent manner. Sildenafil treatment further increases intracellular cGMP concentrations, potentiating activation of this pathway. Collectively, our data identify this cGMP-cGKI pathway as the link between sildenafil usage and increased melanoma risk.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic Nucleotide Phosphodiesterases, Type 5; Female; Humans; Melanoma; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Natriuretic Peptide, C-Type; Nitriles; Phosphodiesterase 5 Inhibitors; Protein Isoforms; Signal Transduction; Sildenafil Citrate; Transplantation, Homologous

Vemurafenib (PLX4032), an inhibitor of BRAF(V600E), has demonstrated significant clinical anti-melanoma effects. However, the majority of treated patients develop resistance, due to a variety of molecular mechanisms including MAPK reactivation through MEK. The induction of a cancer cell death modality associated with danger-signalling resulting in surface mobilization of crucial damage-associated-molecular-patterns (DAMPs), e.g. calreticulin (CRT) and heat shock protein-90 (HSP90), from dying cells, is emerging to be crucial for therapeutic success. Both cell death and danger-signalling are modulated by autophagy, a key adaptation mechanism stimulated during melanoma progression. However, whether melanoma cell death induced by MAPK inhibition is associated with danger-signalling, and the reliance of these mechanisms on autophagy, has not yet been scrutinized. Using a panel of isogenic PLX4032-sensitive and resistant melanoma cell lines we show that PLX4032-induced caspase-dependent cell death and DAMPs exposure in the drug-sensitive cells, but failed to do so in the drug-resistant cells, displaying heightened MEK activation. MEK inhibitor, U0126, treatment sensitized PLX4032-resistant cells to death and re-established their danger-signalling capacity. Only melanoma cells exposing death-induced danger-signals were phagocytosed and induced DC maturation. Although the PLX4032-resistant melanoma cells displayed higher basal and drug-induced autophagy, compromising autophagy, pharmacologically or by ATG5 knockdown, was insufficient to re-establish their PLX4032 sensitivity. Interestingly, autophagy abrogation was particularly efficacious in boosting cell death and ecto-CRT/ecto-HSP90 in PLX4032-resistant cells upon blockage of MEK hyper-activation by U0126. Thus combination of MEK inhibitors with autophagy blockers may represent a novel treatment regime to increase both cell death and danger-signalling in Vemurafenib-resistant metastatic melanoma.

Topics: Autophagy; Butadienes; Cell Death; Coculture Techniques; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Indoles; MAP Kinase Kinase Kinases; Melanoma; Nitriles; Signal Transduction; Sulfonamides; Vemurafenib

Metabolic reprogramming is increasingly being viewed as a hallmark of cancer. Accordingly, metabolic readouts can serve as biomarkers of response to therapy. The goal of this study was to investigate some of the MRS-detectable metabolic consequences of mitogen-activated protein kinase kinase (MEK) inhibition. We investigated PC3 prostate cancer, MCF-7 breast cancer and A375 melanoma cells, and determined that, consistent with previous studies, MRS-detectable levels of phosphocholine decreased significantly in all cell lines (to 63%, 50% and 18% of the control, respectively) following MEK inhibition with U0126. This effect was mediated by a decrease in the expression of choline kinase α, the enzyme that catalyzes the phosphorylation of choline. In contrast, the impact of MEK inhibition on glycolysis was cell line dependent. A375 cells, which express mutant BRAF, demonstrated significant decreases in glucose uptake (to 36% of control) and lactate production (to 42% of control) in line with positron emission tomography data. In contrast, in PC3 and MCF-7 cells, increases in glucose uptake (to 198% and 192% of control, respectively) and lactate production (to 177% and 212% of control, respectively) were observed, in line with a previous hyperpolarized (13) C MRS study. This effect is probably mediated by the activation of the phosphoinositide 3-kinase pathway and AMP-activated protein kinase. Our findings demonstrate the value of translatable non-invasive MRS methods for the provision of information on cellular metabolism as an indication of the activation of potential feedback loops following MEK inhibition.

Topics: AMP-Activated Protein Kinases; Breast Neoplasms; Butadienes; Cell Line, Tumor; Glycolysis; Humans; Magnetic Resonance Spectroscopy; Male; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Prostatic Neoplasms; Protein Kinase Inhibitors

Malignant melanoma has the highest risk of mortality among all types of skin cancer due to its highly metastatic potential. The ocular albinism type 1 (OA1) protein is a pigment cell‑specific glycoprotein, which shares significant structural and functional features with G protein‑coupled receptors. However, the role of OA1 in melanoma has yet to be elucidated. The present study aimed to investigate whether OA1 is involved in melanoma cell migration. OA1 was found to stimulate cell migration in a dose‑dependent manner in cultured human melanoma cells. Furthermore, knockdown of OA1 using small interfering RNA was observed to significantly inhibit melanoma cell migration. In addition, the mechanism underlying OA1‑induced melanoma cell migration was investigated. Stimulation of the RAS/RAF/mitogen activated protein kinase kinase (MEK)/extracellular signal‑regulated kinase (ERK) pathway using growth factors enhanced OA1 expression and melanoma cell migration, whereas inhibition of this pathway using U0126 was observed to markedly decrease OA1 expression and the number of migrated cells. These findings indicate that OA1 is involved in melanoma cell migration and that OA1‑induced melanoma cell migration is mediated through the RAS/RAF/MEK/ERK signaling pathway. Therefore, OA1 may serve as a novel therapeutic target for melanoma.

Topics: Butadienes; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Eye Proteins; Humans; Melanoma; Membrane Glycoproteins; Mitogen-Activated Protein Kinase Kinases; Nitriles; Platelet-Derived Growth Factor; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; ras Proteins; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction

The treatment of metastatic melanoma by conventional chemotherapeutic agents remains unsatisfactory. The present study was undertaken to reveal the role of co-inhibition of survival signaling pathways in apoptosis of melanoma cells.. A panel of human melanoma cell lines and fresh melanoma isolates was assessed for their sensitivity to the MEK inhibitor U0126 and/or AKT inhibitor LY294002. The proliferation and apoptosis of the cells were examined after treatment with the inhibitors.. Constitutive activation of ERK1/2 and AKT was closely related to concentrations of serum in the culture medium (extracellular signals). The sensitivity of melanoma cells to apoptosis induced by inhibition of MEK/ERK was not correlated with the active BRAF mutation (BRAF(V600E)). Inhibition of MEK/ERK predominantly induced apoptosis; whereas inhibition of PI3K/AKT primarily inhibited proliferation. Co-inhibition of MEK/ERK1/2 and PI3K/AKT synergistically induced apoptosis.. Co-targeting MEK/ERK and PI3K/AKT pathways may further improve treatment for melanoma.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Cell Line, Tumor; Chromones; Humans; MAP Kinase Signaling System; Melanoma; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Inhibition of RAF/MEK/ERK signaling is beneficial for many patients with BRAF(V600E)-mutated melanoma. However, primary and secondary resistances restrict long-lasting therapy success. Combination therapies are therefore urgently needed. Here, we evaluate the cellular effect of combining a MEK inhibitor with a genotoxic apoptosis inducer. Strikingly, we observed that an activated MAPK pathway promotes in several melanoma cell lines the pro-apoptotic response to genotoxic stress, and MEK inhibition reduces intrinsic apoptosis. This goes along with MEK inhibitor induced increased RAS and P-AKT levels. The protective effect of the MEK inhibitor depends on PI3K signaling, which prevents the induction of pro-apoptotic PUMA that mediates apoptosis after DNA damage. We could show that the MEK inhibitor dependent feedback loop is enabled by several factors, including EGF receptor and members of the SPRED family. The simultaneous knockdown of SPRED1 and SPRED2 mimicked the effects of MEK inhibitor such as PUMA repression and protection from apoptosis. Our data demonstrate that MEK inhibition of BRAF(V600E)-positive melanoma cells can protect from genotoxic stress, thereby achieving the opposite of the intended anti-tumorigenic effect of the combination of MEK inhibitor with inducers of intrinsic apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; Butadienes; Cell Line, Tumor; Cell Proliferation; Cisplatin; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Indazoles; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Melanoma; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation, Missense; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sulfonamides

Translocator protein TSPO and MAPK signaling partway are important regulators of numerous cell functions including carcinogenesis, cell proliferation and apoptosis. We have studied MAPK inhibitor UO126 and TSPO specific ligand PK11195 effects on TSPO expression level in melanoma cells. Using immunocytochemical staining, we have detected increased expression of TSPO after incubation with PK11195 and UO126 in all concentrations. These results were confirmed by real time PCR: the increase in mRNA TSPO expression level was detected after incubation with PK11195 in concentration 100 nmol/L, and with UO126 in concentration 10 micromol/L. In the case of a combination of treatments with PK11195 and UO126, we also observed an increase in the level of TSPO expression. So, we conclus de that TSPO ligand PK11195 and MAPK signaling partway inhibitor UO126 modulate TSPO expression. These data are crucial for indentifying of regulatory processes for TSPO expression. Pathogenetic interconnection between MAPK signaling partway and TSPO is important for novel therapeutic approaches in melanoma treatment.

Topics: Butadienes; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Isoquinolines; MAP Kinase Signaling System; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Receptors, GABA

Dendritic cells (DCs) can induce strong tumor-specific T-cell immune responses. Constitutive upregulation of the mitogen-activated protein kinase (MAPK) pathway by a BRAF(V600) mutation, which is present in about 50 % of metastatic melanomas, may be linked to compromised function of DCs in the tumor microenvironment. Targeting both MEK and BRAF has shown efficacy in BRAF(V600) mutant melanoma.. We co-cultured monocyte-derived human DCs with melanoma cell lines pretreated with the MEK inhibitor U0126 or the BRAF inhibitor vemurafenib. Cytokine production (IL-12 and TNF-α) and surface marker expression (CD80, CD83, and CD86) in DCs matured with the Toll-like receptor 3/Melanoma Differentiation-Associated protein 5 agonist polyI:C was examined. Additionally, DC function, viability, and T-cell priming capacity were assessed upon direct exposure to U0126 and vemurafenib.. Cytokine production and co-stimulation marker expression were suppressed in polyI:C-matured DCs exposed to melanoma cells in co-cultures. This suppression was reversed by MAPK blockade with U0126 and/or vemurafenib only in melanoma cell lines carrying a BRAF(V600E) mutation. Furthermore, when testing the effect of U0126 directly on DCs, marked inhibition of function, viability, and DC priming capacity was observed. In contrast, vemurafenib had no effect on DC function across a wide range of dose concentrations.. BRAF(V600E) mutant melanoma cells modulate DC through the MAPK pathway as its blockade can reverse suppression of DC function. MEK inhibition negatively impacts DC function and viability if applied directly. In contrast, vemurafenib does not have detrimental effects on important functions of DCs and may therefore be a superior candidate for combination immunotherapy approaches in melanoma patients.

Topics: Antigens, CD; B7-1 Antigen; Butadienes; CD40 Antigens; CD83 Antigen; Cell Line, Tumor; Coculture Techniques; Cytokines; Dendritic Cells; Humans; Immunoglobulins; Indoles; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Melanoma; Membrane Glycoproteins; Mutation; Nitriles; Poly I-C; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; T-Lymphocytes; Vemurafenib

Oncogenesis reflects an orchestrated interaction between misguided growth signals. Although much effort has been launched to pharmacologically disable activated oncogenes, one sidelined approach is the restoration of tumor suppressive signals. As TP53 is often structurally preserved, but functionally crippled, by CDKN2A/ARF loss in melanoma, rescue of p53 function represents an attractive point of vulnerability in melanoma. In this study, we showed that both p53 protein and activity levels in melanoma cells were strongly induced by nutlin-3, a canonical HDM2 antagonist. Among a test panel of 51 cell lines, there was a marked reduction in melanoma viability that was directly linked to TP53 status. Moreover, we also found that the melanoma growth suppression mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase inhibition was potentiated by HDM2 antagonism. These results provide fundamental insights into the intact p53 circuitry, which can be restored through small molecule inhibitors and potentially deployed for therapeutic gain.

Topics: Apoptosis; Butadienes; Cell Cycle Checkpoints; Cell Line, Tumor; Humans; Imidazoles; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Piperazines; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-mdm2; Skin Neoplasms; Tumor Suppressor Protein p53

Selection for targeted therapies in melanoma is currently based on the search for mutations in selected genes. We aimed at evaluating the interest of signalling and chemosensitivity studies in addition to genotyping for assessing the best suitable treatment in an individual patient. We extracted genomic DNA and melanoma cells from tumor tissue of a skin metastasis of a 17-year-old woman with stage IV melanoma progressing despite three successive lines of treatment. Despite the absence of mutation in BRAF, NRAS cKIT, the MAPK pathway was activated and a significant response to sorafenib, a mitogen-activated protein kinase (MAPK)/RAF inhibitor, was found in signalling and chemosensitivity assays. A treatment combining sorafenib and dacarbazine produced a partial response for 9 months, with marked necrosis in some lesions. Chemosensitivity assays and signalling pathway studies could be of great value in addition to genotyping for assessing the most appropriate treatment in melanoma.

Topics: Adolescent; Antineoplastic Agents; Benzenesulfonates; Butadienes; Cell Survival; Dacarbazine; Drug Therapy, Combination; Extracellular Signal-Regulated MAP Kinases; Fatal Outcome; Female; Genes, ras; Humans; Melanoma; Molecular Targeted Therapy; Mutation; Niacinamide; Nitriles; Nitrosourea Compounds; Organophosphorus Compounds; Phenylurea Compounds; Phosphorylation; Precision Medicine; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-kit; Pyridines; Signal Transduction; Sorafenib; Treatment Outcome

The serine/threonine kinase, B-RAF, is frequently mutated in melanoma and is required for cell proliferation. Proteasomal turnover of cyclins and cyclin-dependent kinase inhibitors via E3 ubiquitin ligases regulates cell cycle progression. We previously showed that B-RAF regulates Cks1, a co-factor for the F-box protein Skp2. Recently, a second F-box protein cofactor was identified, alphaB-crystallin, that binds Fbx4 and promotes cyclin D1 degradation. Here, we demonstrate that alphaB-crystallin is down-regulated in mutant B-RAF melanoma cells compared to melanocytes in a B-RAF and MEK-dependent manner. In a subset of lines, MEK inhibition was sufficient to up-regulate alphaB-crystallin protein levels; whereas in other lines combined MEK and proteasome inhibition was required. alphaB-crystallin knockdown partially stabilized cyclin D1 in melanocytes. Expression of alphaB-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. Together, these data show that alphaB-crystallin is highly expressed in melanocytes contributing, in part, to cyclin D1 turnover. Furthermore, alphaB-crystallin is down-regulated in a B-RAF-dependent manner in melanoma cells and its re-expression regulates cyclin D1 turnover after DNA damage.

Topics: alpha-Crystallin B Chain; Bleomycin; Butadienes; Cells, Cultured; Cyclin D1; Cycloheximide; DNA Damage; Etoposide; Humans; Leupeptins; Melanocytes; Melanoma; Mutant Proteins; Mutation; Nitriles; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Structure-Activity Relationship; Tetracycline

This study investigated the growth-regulating effects of progesterone (Prog) on nPR-negative malignant melanoma cells and the possible mechanisms. A375 and A875 cells were cultured and treated with Prog of different concentrations. For signal transduction pathway studies, the cells were pretreated with Prog receptor antagonist (RU486, 1 x 10(-7) mol/L) or MAPK inhibitor (U0126, 5 x 10(-6) mol/L) for 1 h and then co-incubated with prog (10(-9) mol/L) for another 24 h. Indirect immunofluorescence assay, MTT, flow cytometry and Western blotting were used for assessing the nPR expression, cell growth, cell apoptosis and ERK1/2 Phosphorylation, respectively. Our results showed that lower progesterone concentration promoted the proliferation of both A375 and A875 cells, but this growth-stimulatory effect decreased at progesterone concentration of 1 x 10(-7) mol/L or higher. The response could be abolished by MAPK inhibitor U0126, but could not be blocked by progesterone antagonist RU486. Flow cytometry exhibited that high concentration ([Symbol: see text]1 x 10(-7) mol/L) progesterone increased the apoptosis of the two cells in a dose-dependent manner. The level of ERK1/2 phosphorylation was increased by a lower progesterone concentration, but reduced by a higher concentration (1 x 10(-6) mol/L). These results suggest progesterone exerts growth-regulating effects on nPR-negative tumor cells through a non-genomic mechanism.

Topics: Butadienes; Cell Line, Tumor; Cell Proliferation; Humans; Melanoma; Mifepristone; Mitogen-Activated Protein Kinase Kinases; Nitriles; Progesterone; Receptors, Progesterone; Signal Transduction; Skin Neoplasms

Targeted therapy against the BRAF/mitogen-activated protein kinase (MAPK) pathway is a promising new therapeutic approach for the treatment of melanoma. Treatment with selective BRAF inhibitors results in a high initial response rate but limited duration of response. To counter this, investigators propose combining this therapy with other targeted agents, addressing the issue of redundancy and signaling through different oncogenic pathways. An alternative approach is combining BRAF/MAPK-targeted agents with immunotherapy. Preliminary evidence suggests that oncogenic BRAF (BRAF(V600E)) contributes to immune escape and that blocking its activity via MAPK pathway inhibition leads to increased expression of melanocyte differentiation antigens (MDA). Recognition of MDAs is a critical component of the immunologic response to melanoma, and several forms of immunotherapy capitalize on this recognition. Among the various approaches to inhibiting BRAF/MAPK, broad MAPK pathway inhibition may have deleterious effects on T lymphocyte function. Here, we corroborate the role of oncogenic BRAF in immune evasion by melanoma cells through suppression of MDAs. We show that inhibition of the MAPK pathway with MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors or a specific inhibitor of BRAF(V600E) in melanoma cell lines and tumor digests results in increased levels of MDAs, which is associated with improved recognition by antigen-specific T lymphocytes. However, treatment with MEK inhibitors impairs T lymphocyte function, whereas T-cell function is preserved after treatment with a specific inhibitor of BRAF(V600E). These findings suggest that immune evasion of melanomas mediated by oncogenic BRAF may be reversed by targeted BRAF inhibition without compromising T-cell function. These findings have important implications for combined kinase-targeted therapy plus immunotherapy for melanoma.

Topics: Antigens, Neoplasm; Benzamides; Butadienes; Cell Line, Tumor; Combined Modality Therapy; Diphenylamine; Epitopes, T-Lymphocyte; Extracellular Signal-Regulated MAP Kinases; gp100 Melanoma Antigen; Humans; Intramolecular Oxidoreductases; MAP Kinase Signaling System; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Nitriles; Oxidoreductases; Proto-Oncogene Proteins B-raf; T-Lymphocytes

Melanoma responds poorly to standard chemotherapy due to its intrinsic chemoresistance. Multiple genetic and molecular defects, including an activating mutation in the BRaf kinase gene, are associated with melanoma, and the resulting alterations in signal transduction pathways regulating proliferation and apoptosis are thought to contribute to its chemoresistance. Sorafenib, a multikinase inhibitor that targets BRaf kinase, is Food and Drug Administration approved for use in advanced renal cell and hepatocellular carcinomas. Although sorafenib has shown little promise as a single agent in melanoma patients, recent clinical trials suggest that, when combined with chemotherapy, it may have more benefit. We evaluated the ability of sorafenib to augment the cytotoxic effects of melphalan, a regional chemotherapeutic agent, and temozolomide, used in systemic and regional treatment of melanoma, on a panel of 24 human melanoma-derived cell lines and in an animal model of melanoma. Marked differences in response to 10 micromol/L sorafenib alone were observed in vitro across cell lines. Response to sorafenib significantly correlated with extracellular signal-regulated kinase (ERK) downregulation and loss of Mcl-1 expression (P < 0.05). Experiments with the mitogen-activated protein kinase/ERK kinase inhibitor U0126 suggest a unique role for ERK downregulation in the observed effects. Sorafenib in combination with melphalan or temozolomide led to significantly improved responses in vitro (P < 0.05). In the animal model of melanoma, sorafenib in combination with regional melphalan or regional temozolomide was more effective than either treatment alone in slowing tumor growth. These results show that sorafenib in combination with chemotherapy provides a novel approach to enhance chemotherapeutic efficacy in the regional treatment of in-transit melanoma.

Topics: Animals; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzenesulfonates; Blotting, Western; Butadienes; Cell Line, Tumor; Cell Survival; Dacarbazine; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Melanoma; Melphalan; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Niacinamide; Nitriles; Phenylurea Compounds; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-bcl-2; Pyridines; Rats; Rats, Nude; Sorafenib; Temozolomide; Tumor Burden; Xenograft Model Antitumor Assays

In this study, the processes of differentiation and melanogenesis induced by 5,7-dimethoxycoumarin in murine (B16) and human (A375) melanoma cells were investigated. Taking into account the previously demonstrated antiproliferative and differentiation activities of this compound, we examined Ras/Raf/Mek/Erk mitogen-activated protein kinase activity following treatment; inhibition of Mek 1/2 kinase activity and subsequent reduction in Erk 1/2 activation were detected in both cell types. We observed melanogenesis induction associated to an increase in cAMP-response element-binding protein (CREB) and microphthalmia-associated transcription factor (Mitf) expression, both involved in its regulation. Mitf is fundamental for development, survival and differentiation of melanocyte and melanoma, since it regulates transcription of genes encoding for proteins involved in cell cycle progression or in melanogenesis, such as the enzyme tyrosinase. A significant increase of tyrosinase activity was revealed following treatment in B16 but not in A375 cells, although a strong synthesis of melanin was induced by 5,7-dimethoxycoumarin in both cell lines. The treatment induced protoporphyrine IX accumulation involved in melanogenesis since it promotes stability of cAMP. Finally, the Mek 1/2 inhibitor U0126 significantly potentiated growth inhibition of B16 cells triggered by 5,7-dimethoxycoumarin, suggesting that down-regulation of Raf/Mek/Erk pathway sensitizes melanoma cells to 5,7-dimethoxycoumarin treatment.

Topics: Animals; Blotting, Western; Butadienes; Cell Differentiation; Coumarins; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanins; Melanoma; Mice; Microphthalmia-Associated Transcription Factor; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; raf Kinases; ras Proteins; Signal Transduction; Tumor Cells, Cultured

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system.

Topics: Apoptosis; Butadienes; Cell Line, Tumor; Cycloheximide; Down-Regulation; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 1; Melanoma; Mitogen-Activated Protein Kinases; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitriles; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Thermodynamics; Up-Regulation

The RAS-RAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways are activated through multiple mechanisms and appear to play a major role in melanoma progression. Herein, we examined whether targeting the RAS-RAF-MEK-ERK pathway with the RAF inhibitor sorafenib and/or the PI3K-AKT-mTOR pathway with the mTOR inhibitor rapamycin has therapeutic effects against melanoma. A combination of sorafenib (4 microM) with rapamycin (10 nM) potentiated growth inhibition in all six metastatic melanoma cell lines tested. The absolute enhancement of growth inhibition rates ranged from 13.0-27.8% in different cell lines (P<0.05, combination treatment vs monotreatment). Similar results were obtained with combinations of the MEK inhibitors U0126 (30 microM) or PD98059 (50 microM) with rapamycin (10 nM). The combined treatment of melanoma cells with sorafenib and rapamycin led to an approximately twofold increase of cell death compared with sorafenib monotreatment (P<0.05) as assessed by propidium iodide staining and cell death detection ELISA. Moreover, sorafenib in combination with rapamycin completely suppressed invasive melanoma growth in organotypic culture mimicking the physiological context. These effects were associated with complete downregulation of the antiapoptotic proteins Bcl-2 and Mcl-1. Sorafenib combined with rapamycin appears to be a promising strategy for the effective treatment of melanoma and merits clinical investigation.

Topics: Androstadienes; Apoptosis; Benzenesulfonates; Butadienes; Cell Line, Tumor; Cell Proliferation; Chromones; Down-Regulation; Flavonoids; Humans; Mechanistic Target of Rapamycin Complex 1; Melanoma; Mitogen-Activated Protein Kinase Kinases; Morpholines; Multiprotein Complexes; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Invasiveness; Niacinamide; Nitriles; Phenylurea Compounds; Protein Kinase Inhibitors; Proteins; Proto-Oncogene Proteins c-bcl-2; Pyridines; Signal Transduction; Sirolimus; Skin Neoplasms; Sorafenib; TOR Serine-Threonine Kinases; Transcription Factors; Wortmannin

Dysfunctional dendritic cells (DC) are common in cancer patients; however, the underlying molecular targets are poorly understood. Nevertheless, adoptive-transfer and in situ vaccination protocols continue, largely without addressing immune-suppression. Understanding tumour-mediated DC suppression would assist rational design of next generation immunotherapy. This study used a tumour-lysate loaded DC model of adoptive immunotherapy and also necrotic cells common in many tumours. Patient-derived and healthy-donor monocyte-derived DC were examined for disruptions in mitogen-activated protein kinase (MAPK) signalling pathways associated with their capacity to generate Type-1 helper-T cell populations (Th1). Melanoma-lysate markedly suppressed TLR4-dependent IL-12p40 and p70 production. Suppression of IL-12p70 occurred independently of maturation markers (e.g., CD40, CD80, CD83) and correlated with depressed p35 and p40 transcription. Decreased IL-12 secretion was not associated with IL-10, TGFbeta, VEGF, PGE(2) or ganglioside in tumour lysates or attributable to endogenous PGE(2) release from DC. In contrast to HUVEC lysate, melanoma-lysate-treated DC were less able to generate Th1-responses from naïve T-cells. The p44/42 MAPK was hyper-activated by melanoma lysate, but not that of HUVEC. Blockade of MEK1/2, the upstream kinase for p44/42, with U0126 prevented p44/42 activation, restored IL-12p70, and permitted effective generation of Th1-responses. Therefore the p44/42 MAPK is a target for tumour-mediated immune suppression of DC resulting in transcriptional down-regulation of IL-12 p35 and p40 genes, reduced IL-12 secretion and suppressed Th1-responses. Pharmacological intervention in the MEK-p44/42 axis may be applicable to render DC resistant to the suppressive effects of tumour lysates and may form part of a combination immunotherapy.

Topics: Blotting, Western; Butadienes; Cell Line, Tumor; Dendritic Cells; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy, Adoptive; Interleukin-12; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Necrosis; Nitriles; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Th1 Cells; Transcription, Genetic

The loss of tumour phospho-extracellular responsive kinase (pERK) positivity is the major treatment biomarker for mitogen-activated protein kinase/extracellular responsive kinase (MEK) inhibitors. Here, we demonstrate that there is a poor correlation between pERK inhibition and the anti-proliferative effects of MEK inhibitors in melanoma cells. We suggest that Ki67 is a better biomarker for future clinical studies.

Topics: Biomarkers, Tumor; Butadienes; Cell Line, Tumor; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; G1 Phase; Humans; Ki-67 Antigen; Melanoma; Mitogen-Activated Protein Kinase Kinases; Mutation; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins B-raf

Past studies have shown that activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK is a common cause for resistance of melanoma cells to death receptor-mediated or mitochondria-mediated apoptosis. We report in this study that inhibition of the MEK/ERK pathway also sensitizes melanoma cells to endoplasmic reticulum (ER) stress-induced apoptosis, and this is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperon glucose-regulated protein 78 (GRP78) expression. Treatment with the ER stress inducer tunicamycin or thapsigargin did not induce significant apoptosis in the majority of melanoma cell lines, but resistance to these agents was reversed by the MEK inhibitor U0126 or MEK1 small interfering RNA (siRNA). Induction of apoptosis by ER stress when MEK was inhibited was caspase dependent with caspase-4, caspase-9, and caspase-3 being involved. Caspase-4 seemed to be the apical caspase in that caspase-4 activation occurred before activation of caspase-9 and caspase-3 and that inhibition of caspase-4 by a specific inhibitor or siRNA blocked activation of caspase-9 and caspase-3, whereas inhibition of caspase-9 or caspase-3 did not inhibit caspase-4 activation. Moreover, overexpression of Bcl-2 inhibited activation of caspase-9 and caspase-3 but had minimal effect on caspase-4 activation. Inhibition of MEK/ERK also resulted in down-regulation of GRP78, which was physically associated with caspase-4, before and after treatment with tunicamycin or thapsigargin. In addition, siRNA knockdown of GRP78 increased ER stress-induced caspase-4 activation and apoptosis. Taken together, these results seem to have important implications for new treatment strategies in melanoma by combinations of agents that induce ER stress and inhibitors of the MEK/ERK pathway.

Topics: Apoptosis; Butadienes; Caspase Inhibitors; Caspases; Cell Line, Tumor; Down-Regulation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Activation; Enzyme Inhibitors; Heat-Shock Proteins; Humans; Isoenzymes; MAP Kinase Kinase Kinases; Melanoma; Molecular Chaperones; Nitriles; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Thapsigargin; Transfection; Tunicamycin

MEK1/2 inhibitors like U0126 can potentiate or antagonize the antitumor activity of cytotoxic agents such as cisplatin, paclitaxel or vinblastine, depending on the drug or the target cells. We now investigated whether U0126, differentially regulates melanoma signaling in response to UV radiation or betulinic acid, a drug lethal against melanoma. This report shows that U0126 inhibits early response (ERK) kinase activation and cyclin A expression in wt p53 C8161 melanoma exposed to either UV radiation or betulinic acid. However, U0126 does not protect from UV damage, but counteracts betulinic acid-mediated apoptosis in the same cells. Protection from the latter drug by joint treatment with U0126 was also evident in wt p53 MelJuso melanoma and mutant p53 WM164 melanoma. The latter cells were the most responsive to betulinic acid, showing a selective decline in the cdk4 protein, without a comparable change in other key cell cycle proteins like cdc2, cdk2, cdk7 or cyclin A, prior to apoptosis-associated PARP fragmentation. Laser scanning cytometry also showed that betulinic acid induced a significant increase in chromatin condensation in WM164 melanoma irrespective of whether they were in adherent form or as multicellular spheroids. All these betulinic acid-induced changes were counteracted by U0126. Our data show for the first time that (a) cdk4 protein is an early target of betulinic acid-induced apoptosis and (b) unrestricted ERK signaling favours betulinic acid-induced apoptosis, but this is counteracted by U0126, partly through counteracting chromatin condensation and restoring Akt activation decreased by betulinic acid treatment.

Topics: Apoptosis; Betulinic Acid; Butadienes; Cell Adhesion; Cell Line, Tumor; Chromatin; Cyclin A; Cyclin-Dependent Kinase 4; DNA Damage; Down-Regulation; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Pentacyclic Triterpenes; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Triterpenes; Tumor Suppressor Protein p53

Although >66% of melanomas harbor activating mutations in BRAF and exhibit constitutive activity in the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase signaling pathway, it is unclear how effective MEK inhibition will be as a sole therapeutic strategy for melanoma. We investigated the anticancer activity of MEK inhibition in a panel of cell lines derived from radial growth phase (WM35) and vertical growth phase (WM793) of primary melanomas and metastatic melanomas (1205Lu, 451Lu, WM164, and C8161) in a three-dimensional spheroid model and found that the metastatic lines were completely resistant to MEK inhibition (U0126 and PD98059) but the earlier stage cell lines were not. Similarly, these same metastatic melanoma lines were also resistant to inhibitors of the phosphatidylinositol 3-kinase/Akt pathway (LY294002 and wortmannin). Under adherent culture conditions, the MEK inhibitors blocked growth through the induction of cell cycle arrest and up-regulation of p27, but this was readily reversible following inhibitor washout. However, when the phosphatidylinositol 3-kinase and MEK inhibitors were combined, the growth and invasion of the metastatic melanoma three-dimensional spheroids were blocked. Taken together, these results suggest that the most aggressive melanomas are resistant to strategies targeting one signaling pathway and that multiple signaling pathways may need to be targeted for maximal therapeutic efficacy. It is further suggested that BRAF mutational status is not predictive of response to MEK inhibition under three-dimensional culture conditions.

Topics: Butadienes; Cell Line, Tumor; Chromones; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Humans; MAP Kinase Kinase Kinases; Melanoma; Morpholines; Neoplasm Metastasis; Nitriles; Phosphatidylinositol 3-Kinases; Protein Subunits; Signal Transduction; Spheroids, Cellular

Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by (V600E)B-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)-mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant (V600E)B-Raf signaling cascade to inhibit melanoma metastases.

Topics: Amino Acid Substitution; Animals; Butadienes; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Metastasis; Nitriles; Proto-Oncogene Proteins B-raf; RNA, Neoplasm; RNA, Small Interfering; Signal Transduction

Heterogeneous expression of melanocytic antigens occurs frequently in melanomas and represents a potent barrier to immunotherapy. We previously showed that coordinated losses of several melanocytic antigens are generally attributable to down-regulation of antigen gene expression rather than irreversible mutation. Treatment of melanoma cells with mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors blocks ERK activation and increases steady-state levels of mRNAs and corresponding protein expression for the melanocytic antigens Melan-A/MART-1, gp100, and tyrosinase. Although the degree of MEK inhibitor enhancement of antigen expression varied among different cell lines irrespective of their antigen expression status, all showed detectable responses. Notably, the antigen-enhancing effects of the MEK inhibitors could not be attributed to the master melanocytic regulator MITF-M. Because MAPK pathway activation via constitutively active mutant forms of BRAF is common in melanomas, correlation between BRAF function and antigen expression was investigated. No simple correlation of endogenous BRAF mutational status and antigen levels was observed, but transient overexpression of V600E BRAF increased ERK activation and reduced Melan-A/MART-1 levels in antigen-positive cell lines. These data indicate that whereas multiple factors may regulate antigen expression in melanomas, enhancement of MAPK signaling can act as a negative influence. Blocking such signaling with MEK inhibitors accordingly augments antigen levels, thereby enhancing Melan-A/MART-1-specific cytotoxic T-cell responses to antigen-negative cells following MEK inhibition treatment. Consequently, MAPK inhibition may assist targeting of melanomas for immunotherapy.

Topics: Antigens, Neoplasm; Butadienes; Cell Line, Tumor; Enzyme Inhibitors; Humans; MAP Kinase Signaling System; MART-1 Antigen; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nitriles; Organic Chemicals; Polymorphism, Restriction Fragment Length; Proto-Oncogene Proteins B-raf; T-Lymphocytes, Cytotoxic; Transcription, Genetic; Transfection

The RAS/BRAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is emerging as a key modulator of melanoma initiation and progression. However, a variety of clinical studies indicate that inhibiting the MAPK pathway is insufficient per se to effectively kill melanoma cells. Here, we report on a genetic and pharmacologic approach to identify survival factors responsible for the resistance of melanoma cells to MEK/ERK antagonists. In addition, we describe a new tumor cell-selective means to bypass this resistance in vitro and in vivo. By generating a panel of isogenic cell lines with specific defects in the apoptotic machinery, we found that the ability of melanoma cells to survive in the absence of functional MEK relies on an ERK-independent expression of the antiapoptotic factor Mcl-1 (and to a lesser extent, Bcl-x(L) and Bcl-2). Using computer-based modeling, we developed a novel Bcl-2 homology domain 3 (BH3) mimetic. This compound, named TW-37, is the first rationally designed small molecule with high affinity for Mcl-1, Bcl-x(L), and Bcl-2. Mechanistic analyses of the mode of action of TW-37 showed a synergistic tumor cell killing in the presence of MEK inhibitors. Importantly, TW-37 unveiled an unexpected role of the MAPK pathway in the control of reactive oxygen species (ROS). This function was critical to prevent the activation of proapoptotic functions of p53 in melanoma cells, but surprisingly, it was dispensable for normal melanocytes. Our results suggest that this MAPK-dependent ROS/p53 feedback loop is a point of vulnerability of melanoma cells that can be exploited for rational drug design.

Topics: Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Benzamides; Butadienes; Cell Line, Tumor; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Drug Synergism; Humans; MAP Kinase Signaling System; Melanoma; Mice; Mice, Nude; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Molecular Structure; Nitriles; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA Interference; Sulfones; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

A number of human melanomas show hyperactivation of the Ras pathway due to mutations of the molecule or alteration of upstream or downstream effectors. In this study, we evaluated the effect of blocking the two Ras downstream pathways phosphatidylinositol-3-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase on melanoma development and regression in the TPRas mouse model. The inhibition of these two signaling cascades by topically applied Ly294002 and U0126 significantly delayed melanoma development and significantly decreased the tumor incidence, particularly when the drugs were applied in combination. Treatment with the inhibitors of established melanomas resulted in complete remission in 33% of mice and partial regression in 46% of mice when drugs were delivered in combination. These responses correlated with increased apoptosis and decreased proliferation both in vitro and in vivo and reduced tumor angiogenesis. In conclusion, this study strongly supports the role of the phosphatidylinositol-3-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways in the development and maintenance of Ras-dependent melanomas and supports the notion that specific inhibition of these effectors may represent a very promising avenue for the treatment and prevention of the disease.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Butadienes; Chromones; Enzyme Inhibitors; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Melanoma; Mice; Mice, Transgenic; Morpholines; Neovascularization, Pathologic; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; raf Kinases

The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.

Topics: Autocrine Communication; Butadienes; Cell Line, Tumor; DNA-Binding Proteins; Early Growth Response Protein 1; Extracellular Signal-Regulated MAP Kinases; Fibronectins; Genes, Reporter; Hepatocyte Growth Factor; Humans; Immediate-Early Proteins; Luciferases; MAP Kinase Signaling System; Melanoma; Nitriles; Phosphorylation; Promoter Regions, Genetic; ras Proteins; Signal Transduction; Skin Neoplasms; Transcription Factors; Up-Regulation

The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotides against PKC-alpha, or the PKC inhibitor Gö6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.

Topics: Anoikis; Apoptosis; Butadienes; Carbazoles; Cell Adhesion; Cell Culture Techniques; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Progression; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Humans; Immunoblotting; Indoles; Melanocytes; Melanoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Nitriles; Oligonucleotides, Antisense; Protein Kinase C; Protein Kinase C-alpha; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors; Transfection

The Ras-mitogen-activated protein (Ras-MAP) kinase pathway regulates various cellular processes, including gene expression, cell proliferation, and survival. Ribosomal S6 kinase (RSK), a key player in this pathway, modulates the activities of several cytoplasmic and nuclear proteins via phosphorylation. Here we report the characterization of the cytoskeletal protein filamin A (FLNa) as a membrane-associated RSK target. We show that the N-terminal kinase domain of RSK phosphorylates FLNa on Ser(2152) in response to mitogens. Inhibition of MAP kinase signaling with UO126 or mutation of Ser(2152) to Ala on FLNa prevents epidermal growth factor (EGF)-stimulated phosphorylation of FLNa in vivo. Furthermore, phosphorylation of FLNa on Ser(2152) is significantly enhanced by the expression of wild-type RSK and antagonized by kinase-inactive RSK or specific reduction of endogenous RSK. Strikingly, EGF-induced, FLNa-dependent migration of human melanoma cells is significantly reduced by UO126 treatment. Together, these data provide substantial evidence that RSK phosphorylates FLNa on Ser(2152) in vivo. Given that phosphorylation of FLNa on Ser(2152) is required for Pak1-mediated membrane ruffling, our results suggest a novel role for RSK in the regulation of the actin cytoskeleton.

Topics: Amino Acid Sequence; Animals; Butadienes; Cell Line, Tumor; Cell Movement; Colforsin; Contractile Proteins; Enzyme Inhibitors; Epidermal Growth Factor; Filamins; Humans; MAP Kinase Signaling System; Melanoma; Microfilament Proteins; Molecular Sequence Data; Nitriles; Phosphorylation; Protein Isoforms; Protein Structure, Tertiary; Ribosomal Protein S6 Kinases; Sequence Alignment; Serine

We have previously shown that Smac/DIABLO release from mitochondria appears to be the principal pathway by which TRAIL induces apoptosis of human melanoma. We report that TRAIL-induced release of Smac/DIABLO appears to be downregulated by concomitant signaling through the MEK Erk1/2 kinase pathway and that this inhibits TRAIL-induced apoptosis. Inhibition of Erk1/2 signaling by either the MEK inhibitor U0126 or a dominant-negative mutant of MKK1 markedly sensitized melanoma cells to TRAIL-induced apoptosis. The site in the apoptotic pathway acted on by U0126 appeared to be downstream of caspase-8 and Bid but upstream of caspase-3 in that the levels of proteolytic cleavage of caspase-8 and Bid by TRAIL were similar in cells with or without exposure to U0126. Caspase-3 activation and cleavage of its substrates, PARP, ICAD and XIAP, were however increased by cotreatment with U0126. This was associated with a rapid reduction in mitochondrial transmembrane potential (MMP) and increased release of Smac/DIABLO into the cytosol. Exploration of events leading to the changes in MMP revealed an increased translocation of Bax from the cytosol to mitochondria in the presence of U0126. There was also a delayed decrease in the levels of expression of Mcl-1. Bcl-2 and Bcl-X(L). Over expression of Bcl-2 blocked TRAIL-induced apoptosis in the presence of U0126. Cytochrome c appeared not to play a major role in sensitization of melanoma to TRAIL in that caspase-9 activation was not detected in most of the cell lines. These results suggest that Erk1/2 signaling may protect melanoma cells against TRAIL-induced apoptosis by inhibiting the relocation of Bax from the cytosol to mitochondria and that this may reduce TRAIL-mediated release of Smac/DIABLO and induction of apoptosis.

Topics: Apoptosis; Apoptosis Regulatory Proteins; BH3 Interacting Domain Death Agonist Protein; Butadienes; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line; Humans; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Melanoma; Membrane Glycoproteins; Mitochondria; Mitochondrial Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

A combination of paclitaxel (Taxol) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK/Erk) inhibitor represents a rational new approach to chemotherapy. We performed Affymetrix microarray analysis to understand the global effects of this combination in lung carcinoma. Genes involved in cell cycle control, apoptosis, adhesion, proliferation, invasion, and metastasis were modulated. We observed similar patterns of gene modulation in ovarian and melanoma cell lines, indicating the general applicability of these findings. Functional genomic analysis identified two genes as new targets of drug-induced tumor apoptosis. The MGSA/Gro1 gene, important in melanoma growth, was induced by paclitaxel and reduced by MEK inhibition. Blockage of paclitaxel-induced melanoma growth stimulatory activity significantly reduced melanoma growth. Additionally, the expression of topoisomerase III beta, which exhibited a clear pattern of gene reduction by a combination of the two drugs, was significantly increased (5.7-fold) in primary lung cancers but not adjacent tissues. These findings provide potential new biomarkers and gene targets for the development of improved cancer treatment.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Butadienes; Chemokine CXCL1; Chemokines; Chemokines, CXC; Chemotactic Factors; Dactinomycin; Enzyme Inhibitors; Female; Gene Expression Profiling; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Paclitaxel

The teleost Xiphophorus provides a genetically well-described model system to study the molecular processes underlying melanoma formation. As transcriptional deregulation is a widespread phenomenon in many tumors, we have studied the regulation of melanoma-specific gene expression in this fish. A central regulator of melanocyte specific gene expression, which is also a marker for melanomas, is the transcription factor microphthalmia-associated transcription factor (MITF). One of its targets, the tyrosinase gene, codes for a key enzyme in the melanin synthesis pathway. We could show that the promoter of the medaka tyrosinase gene is highly active in the Xiphophorus melanoma cell line PSM (platyfish-swordtail melanoma) but not in non-melanoma cells. Functional dissection of the promoter revealed that three E-boxes are essential for its pigment cell-specific activity. These binding sites for basic helix-loop-helix transcription factors are recognized by a nuclear protein from the melanoma cell line PSM, most likely MITF, as its exogenous delivery could activate the promoter in non-melanoma cells. The use of specific signalling inhibitors demonstrated that the activity of the tyrosinase promoter is negatively regulated by the melanoma-inducing receptor tyrosine kinase Xmrk in PSM cells. This repression is mediated by MAPkinase and dependent on E-box integrity, again implicating the involvement of MITF. The cumulative evidence indicates that in Xiphophorus, Xmrk suppresses differentiation signals relayed by MITF as part of the transformation process finally resulting in melanoma formation.

Topics: Alternative Splicing; Animals; Base Sequence; Binding Sites; Butadienes; Cell Line; Cell Line, Tumor; Cyprinodontiformes; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Fish Proteins; Humans; Luciferases; Melanoma; Microphthalmia-Associated Transcription Factor; Mitogen-Activated Protein Kinases; Monophenol Monooxygenase; Nitriles; Nuclear Proteins; Oryzias; Promoter Regions, Genetic; Protein Binding; Protein Isoforms; Receptor Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Signal Transduction; Transcription Factors; Transcriptional Activation; Transfection; Tyrphostins

Tumor necrosis factor alpha (TNF-alpha) modulates various events through several different pathways. Many tumor cells are resistant to this cytokine. Pretreatment of these cells with actinomycin D enhances TNF-alpha-induced apoptosis. In the present study, we investigated the mechanism of this enhancement and whether or not the apoptosis of TNF-alpha-resistant cancer cells can be induced by the inhibition of Protein kinase C (PKC). When TNF-alpha was added after inhibition of PKC by H7, apoptosis was observed, and companied with the activation of nuclear factor kappa B (NF-kappaB). After the inhibition of protein kinase B (Akt) by LY294002 or p38 mitogen-activated protein kinase (p38MAPK) by SB203580, the addition of TNF-alpha did not cause apoptosis. However, after the inhibition of MAPK/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) with U0126, apoptosis was observed when TNF-alpha was added. In the Western blotting analysis, phosphorylation of MEK1/2 occurred at 60 minutes after the addition of TNF-alpha. However, it was noted that after pretreatment with H7, a significant decrease in phosphorylated MEK1/2 was observed. The present findings suggest that MEK1/2 plays an important role in TNF-alpha-resistance in TNF-alpha-resistant B16 melanoma BL6 cells. Furthermore, it was found that MEK1/2 is more important than NF-kappaB, Akt, and p38MAPK in anti-apoptotic PKC signaling and that TNF-alpha-resistance can be overcome by inhibiting MEK1/2. These results suggest the possibility of development of a new anticancer drug treatment.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antineoplastic Agents; Apoptosis; Butadienes; Caspase 3; Caspases; Cell Division; Cell Line, Tumor; Cell Survival; Dactinomycin; Dose-Response Relationship, Drug; Drug Combinations; Drug Resistance, Neoplasm; Enzyme Inhibitors; Melanoma; Mice; Nitriles; Phosphorylation; Protein Kinase C; Tumor Necrosis Factor-alpha

The anti-invasive ability of the mitogen-activated protein kinase (MAPK) kinase inhibitor, U0126, was examined in human A375 melanoma cells in vitro. The effect was compared to that of PD98059, another commonly used MEK (MAPK kinase) inhibitor. U0126 or PD98059 showed a dose-dependent inhibition of A375 cell invasion through growth factor-reduced Matrigel. U0126 was more potent than PD98059 in suppressing tumor cell invasion. Both compounds significantly decreased urokinase plasminogen activator (uPA) and matrix metalloproteinases-9 (MMP-9) concentrations in conditioned media. At 5 microM, U0126 inhibited phosphorylation of the MEK 1/2 to a non-detectable level within 24 h. The phosphorylation of extracellular signal-related kinase 1/2 was also dramatically suppressed by the treatment with 10 microM U0126 or 40 microM PD98059. Both compounds suppressed the protein expression of c-Jun, but not c-Fos. The expression of uPA and MMP-9 was also inhibited. Our data suggest that U0126 is an effective agent in inhibiting human A375 melanoma cell invasion and that the effect is partially due to the decreased production of uPA and MMP-9.

Topics: Butadienes; Enzyme Inhibitors; Flavonoids; Humans; Matrix Metalloproteinase 9; Melanoma; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Nitriles; Phosphorylation; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator