[Show abstract][Hide abstract] ABSTRACT: While melanoma is believed to be a highly immunogenic tumor and recent developments in immunotherapies are promising. Interferon-γ (IFN-γ) produced by immune cells plays a crucial role in tumor immune surveillance; however, it has also been reported to be pro-tumorigenic. In the current study, we found that IFN-γ enhances the expression of CD74, which interacts with its ligand, macrophage migration inhibitory factor (MIF), and thereby activates the PI3K/AKT pathway in melanoma, promoting tumor survival. IFN-γ increased phosphorylation of AKT Ser473 and upregulated total and cell surface expression of CD74 in human melanoma cell lines tested. CD74 was highly expressed in melanoma tissues. Moreover, the expression of CD74 on tumor cells correlated with plasma IFN-γ levels in melanoma patient samples. In our analysis of melanoma cell lines, all produced MIF constitutively. Blockade of CD74-MIF interaction reduced AKT phosphorylation and expression of pro-tumorigenic molecules, including interleukin-6, interleukin-8 and BCL-2. Inhibition of CD74-MIF interaction significantly suppressed tumor growth in the presence of IFN-γ in our xenograft mouse model. Thus, we conclude that IFN-γ promotes melanoma cell survival by regulating CD74-MIF signaling, suggesting that targeting the CD74-MIF interaction under IFN-γ-stimulatory conditions would be an effective therapeutic approach for melanoma.Journal of Investigative Dermatology accepted article preview online, 03 June 2015. doi:10.1038/jid.2015.204.
Journal of Investigative Dermatology 06/2015; DOI:10.1038/jid.2015.204 · 7.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primary uveal melanoma tumors have been shown to harbor activating mutations in the heterotrimeric G-alpha protein subunits GNAQ or GNA11 genes in approximately 85% of samples.(Van Raamsdonk et al., 2009, Van Raamsdonk et al., 2010) Mutations in GNAQ or GNA11 are mutually exclusive, and result in amino acid changes at either the R183 or Q209 sites. GNAQ or GNA11 mutations have also been identified in uveal melanoma liver metastases suggesting that the metastatic cells in the liver are derived from primary tumor cell clones with these mutations.(Carvajal et al. 2014) In addition, the many uveal melanoma cell lines derived from metastatic sites also harbor GNAQ or GNA11 mutations.(Griewank et al., 2012)This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: c-Jun N-terminal kinase (JNK) known as the stress activated protein kinase, is a key mediator of a variety of stress stimuli, including proinflammatory cytokines, reactive oxygen species, and ultraviolet radiation (Bogoyevitch et al., 2010). By phosphorylating crucial molecules such as c-Jun, p53, and Bcl-2, active JNK regulates cell growth, differentiation, apoptosis, and autophagy (Bogoyevitch et al., 2010). Previous studies of JNK in cancer cells have produced paradoxical findings, with some supporting a pro-oncogenic function and others demonstrating its role as a tumor suppressor (Bogoyevitch et al., 2010; Tournier, 2013). To date, the role of JNK in melanoma remains ambiguous.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: An obstacle to effective gene-based cancer therapies is the limited number of cancer-specific growth suppressing and apoptosis-inducing genes. Using a differentiation induction subtraction hybridization (DISH) approach with human melanoma cells, melanoma differentiation associated (mda) genes were isolated that display elevated expression as a function of irreversible growth arrest, cancer reversion and terminal differentiation. This screening paradigm resulted in the cloning of mda-7 in the context of terminal differentiation of human melanoma cells. Based on its structure, chromosomal location, sequence homology and cytokine-like properties, mda-7 has now been renamed IL-24 and classified as a member of the expanding IL-10 cytokine gene family. Expression of mda-7/IL-24 inversely correlates with melanoma progression and administration of mda-7/IL-24 by means of a replication incompetent adenovirus, Ad.mda-7, results in growth suppression and apoptosis in melanoma cells as well as in a broad-spectrum of additional cancer cell types. In contrast, Ad.mda-7 does not elicit deleterious effects in normal cells, including those of epithelial, fibroblast, astrocyte, melanocyte or endothelial origin. Based on these distinctive properties and anti-tumor and anti-angiogenic activities in human tumor xenograft animal models, mda-7/IL-24 has now entered the clinical arena. A Phase I/II clinical trial in patients with advanced carcinomas involving intratumoral administration of mda-7/IL-24 [using a replication incompetent adenovirus; ING241 (Ad.mda-7)] has documented that this gene is safe and well tolerated by patients and a single virus injection elicits apoptosis in a majority of the tumor. Current data suggests that mda-7/IL-24 may function as a dual-acting cytokine in which its normal physiological functions may be related to specific aspects of the immune system and over-expression culminates in cancer-specific apoptosis. This review will provide a prospectus of our current understanding of mda-7/IL-24.
[Show abstract][Hide abstract] ABSTRACT: The role of inflammation in cancer has been reported in various adult malignant neoplasms. By contrast, its role in pediatric tumors has not been as well studied. In this study, we have identified and characterized the infiltration of various inflammatory immune cells as well as inflammatory markers in Wilms tumor (WT), the most common renal malignancy in children. Formalin-fixed paraffin-embedded blocks from tumors and autologous normal kidneys were immunostained for inflammatory immune cells (T cells, B cells, macrophages, neutrophils, and mast cells) and inflammatory markers such as cyclooxygenase-2 (COX-2), hypoxia-inducible factor 1α, phosphorylated STAT3, phosphorylated extracellular signal–related kinases 1 and 2, inducible nitric oxide synthase, nitrotyrosine, and vascular endothelial growth factor expression. Overall, we found that there was predominant infiltration of tumor-associated macrophages in the tumor stroma where COX-2 was robustly expressed. The other tumor-associated inflammatory markers were also mostly localized to tumor stroma. Hence, we speculate that COX-2–mediated inflammatory microenvironment may be important in WT growth and potential therapies targeting this pathway may be beneficial and should be tested in clinical settings for the treatment of WTs in children.
[Show abstract][Hide abstract] ABSTRACT: Mutagen sensitivity assay, which measures the enhanced cellular response to DNA damage induced in vitro by mutagens/carcinogens, has been used in the study of cancer susceptibility. 4-Nitroquinoline-1-oxide (4-NQO), an ultraviolet (UV) radiation-mimetic chemical, can produce chromosomal breaks in mammalian cells and induce cancer. Given the potential role of 4-NQO as the experimental mutagen substituting for UV as the etiological carcinogen of cutaneous melanoma (CM), we tested the hypothesis that cellular sensitivity to 4-NQO is associated with the risk of developing CM in a case-control study of 133 patients with primary CM and 176 cancer-free controls. Short-term blood cultures were treated with 4-NQO at a final concentration of 10 μmol/l for 24 h and scored chromatid breaks in 50 well-spread metaphases. Multivariate logistic regression was used to calculate odds ratios and 95% confidence intervals. We found that the log-transformed frequency of chromatid breaks was significantly higher in 133 patients than in 176 controls (P=0.004) and was associated with an increased risk for CM (adjusted odds ratio=1.78, 95% confidence interval: 1.12-2.84) after adjustment for age and sex. Moreover, as the chromatid break values increased, the risk for CM increased in a dose-dependent manner (Ptrend=0.003). Further analysis explored a multiplicative interaction between the sensitivity to 4-NQO and a family history of skin cancer (Pinteraction=0.004) on the risk of CM. Therefore, our findings suggest that sensitivity to 4-NQO may be a risk factor for the risk of CM, which is more sensitive than UV-induced chromotid breaks.
Melanoma Research 06/2014; Publish Ahead of Print. DOI:10.1097/CMR.0000000000000106 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cutaneous melanoma is one of the most aggressive forms of skin cancer. In the United States, its incidence rate increased on average 2.7% annually between 1985 and 2010. Targeted molecular chemotherapy drugs, such as BRAF and MEK inhibitors, significantly improve survival and quality of life among late-stage patients; but oftentimes de novo or acquired resistance render these therapies ineffective. Therefore, ongoing search for novel therapies for advanced melanoma is imperative. In laboratory studies, we found that Zyflamend (New Chapter, Inc., Brattleboro, VT), a promising anticancer multi-herbal extract, inhibited melanoma proliferation by regulating the autophagy-apoptosis switch. Based on our preliminary findings, we speculated that a combination of Zyflamend and vemurafenib, a mutant BRAF inhibitor, may synergistically inhibit melanoma proliferation. To test this hypothesis, we prepared 3 concentrations of Zyflamend (low [LZ], 0.001 µL/mL; medium [MZ], 0.025 µL/mL; high [HZ], 0.050 µL/mL) and vemurafenib (low [LV], 0.1 µM; medium [MV], 1.0 µM; high [HV], 5.0 µM) each, and 4 combinations—LZLV, LZMV, MZMV, MZLV. HZ and HV concentrations were not used in combination treatments due to their individual extensive toxicity. A375 (BRAF mutant) and MeWo (BRAF wild type) melanoma cells and normal BJ fibroblasts were diluted to 50,000 cells/mL, seeded in 24-well plates, and incubated with the treatments for 48 hours. Our controls (50,000 cells/mL each) included media with DMSO (HV concentration), media with DMSO and olive oil (HZ concentration), and media only in each experiment. All media solutions were prepared with Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum. Trypan blue and Cellometer Auto T4 (Nexcelom Bioscience, Lawrence, MA) were used for cell counting. Each preparation was done in duplicate, and each experiment was conducted in duplicate at separate times. We observed that the MZLV combination had the greatest synergistic effect against our melanoma cell lines: this combination resulted in a 54% reduction in A375 cells compared with a predicted reduction of 22%, while having less than 20% reduction in BJ fibroblasts. The proliferation of BJ fibroblasts incubated with MZ (70%) was lower than expectation; but surprisingly, vemurafenib (LV) appeared to rescue partially this inhibition (MZLV, 86%). We also observed similar phenomenon with MeWo Cells. These findings with MeWo cells are in agreement with BRAF wild-type studies showing BRAF inhibitor resistance via MEK and ERK signaling. In our study, we found that a combination of Zyflamend and vemurafenib may be a novel therapy for late-stage cutaneous melanoma. Further studies should focus on elucidating the mechanism behind this dosage-dependent synergistic effect on BRAF-mutant cells and the partial rescue of Zyflamend’s toxic effect on normal human fibroblasts.
Association for Genetic Technologists, Louisville, KY; 06/2014
[Show abstract][Hide abstract] ABSTRACT: Nearly all primary uveal melanoma (UM) that metastasize involve the liver. Hepatocyte growth factor (HGF) is proposed to be an important microenvironmental element in attracting/supporting UM metastasis through activation of MET. The majority (>85%) of UM express mutations in the G-alpha proteins, that drive the MEK-ERK1/2 pathway. Thus, we proposed that the combination of MET and MEK inhibition would inhibit the growth and migration of G-alpha protein mutant versus non-mutant UM cells.
Western-blots demonstrated the relative protein levels of ERK1/2 and MET in UM cells. Cells were treated with the small molecule inhibitors AZD6244 (MEKi) and/or MK-8033 (METi) and downstream markers evaluated. Further studies determined the effect of combination MEKi and METi treatment on cell growth, apoptosis and migration.
All G-alpha protein mutant UM cell lines express MET mRNA and protein. The level of mRNA expression correlates with protein expression. MEKi, but not METi treatment results in markedly reduced ERK1/2 phosphorylation. Either MEKi or METi treatment alone results in reduced cell proliferation, but only modest induction of apoptosis. The combination MEKi+METi results in significant reduction of proliferation in G-alpha protein mutant cells. UM cell migration was blocked by METi, but not MEKi treatment.
MET protein expression showed no correlation with G-alpha protein mutation status. Combining MEKi with METi treatment has added benefit to either treatment alone in reducing G-alpha protein mutant UM cell growth. Combining METi with MEKi treatment adds the effect of limiting uveal melanoma cell migration.
PLoS ONE 02/2014; 9(2):e83957. DOI:10.1371/journal.pone.0083957 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Melanoma is one of the cancers of fastest-rising incidence in the world. iNOS is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K/AKT/mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p-P70S6K, p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of TSC2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Rheb, a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of mTOR pathway members. Exogenously-supplied NO was also sufficient to reverse mTOR pathway inhibition by the B-Raf inhibitor Vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers.
Cancer Research 01/2014; 74(4). DOI:10.1158/0008-5472.CAN-13-0588 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oral melanoma (OM) in dogs is an aggressive malignancy, with clinical behavior resembling cutaneous melanomas in humans. Melanoma in humans is promoted by an inflammatory environment that is contributed to by leptin and inducible nitric oxide synthase (iNOS).
To determine if the patterns of leptin and iNOS expression are similar in OM in dogs and cutaneous melanomas in humans.
Twenty client-owned dogs.
Retrospective case study. Immunostaining of the OM tumors from each dog was scored for percentage and intensity of leptin and iNOS expression. Mitotic index was used as an indicator of tumor aggression.
Leptin was detected in ≥75% of the tumor cells in specimens from 11 dogs. One tumor expressed leptin in ≤25% of the cells. The intensity of leptin expression was variable with 6, 9, and 5 cases exhibiting low-, moderate-, and high-intensity staining, respectively. OM with the lowest percentage of iNOS positive cells displayed the highest mitotic indices (P = .006, ANOVA).
The expression of leptin is a common finding in melanomas in dogs. These data suggest that the possibility of future clinical applications, such as measuring the concentrations of plasma leptin as a screening tool or leptin as a target for therapy. The relevance of iNOS is not as clear in dogs with OM, for which other directed therapeutics might be more appropriate.
Journal of Veterinary Internal Medicine 09/2013; 27(5):1278-82. DOI:10.1111/jvim.12169 · 1.88 Impact Factor