The EphB4 receptor promotes the growth of melanoma cells expressing the ephrin-B2 ligand

Cutaneous melanoma is the most aggressive form of skin cancer and several families of receptor tyrosine kinases have been implicated in its development and progression, including the Eph receptor family (Hess et al., 2007; Smalley et al., 2009). Among Eph receptors, EphA2 has been most extensively studied in melanoma and linked to increased malignancy (Hess et al., 2007; Margaryan et al., 2009).Fil: Yang, Nai Ying . University of California; Estados UnidosFil: Lopez Bergami, Pablo Roberto. Sanford-burnham Medical Research Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Goydos, James S.. Robert Wood Johnson Medical School; Estados UnidosFil: Yip, Dana . Robert Wood Johnson Medical School; Estados UnidosFil: Walker, Ameae . University of California; Estados UnidosFil: Pasquale, Elena B.. Sanford-burnham Medical Research Institute; Estados UnidosFil: Ethell, Iryna. University of California; Estados Unido

Gβγ subunits inhibit Epac-induced melanoma cell migration

<p>Abstract</p> <p>Background</p> <p>Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca ²⁺release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca ²⁺signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca ²⁺signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration.</p> <p>Methods</p> <p>SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca ²⁺was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers.</p> <p>Results</p> <p>The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca ²⁺response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca ²⁺signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca ²⁺elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca ²⁺elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca ²⁺elevation. Subsequently, the mechanism by which Gβγ inhibits Epac-induced Ca ²⁺elevation was explored. mSIRK activates Ca ²⁺influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca ²⁺elevation, and cell migration. These data suggest that, the mSIRK-induced Ca ²⁺from the extracellular space inhibits the Epac-induced Ca ²⁺release from the ER, resulting suppression of cell migration.</p> <p>Conclusion</p> <p>We found the cross talk of Ca ²⁺signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.</p

Occupational sunscreen use among US Hispanic outdoor workers

Background: Occupational ultraviolet radiation (UVR) exposure is a risk factor for skin cancer, and Hispanic individuals are over-represented in a number of outdoor occupations (e.g., farming, landscaping). This study examined predictors of occupational sunscreen use in a group of US Hispanic adults who work outdoors. Results: A population-based sample of outdoor workers (n = 149, 85 % male) completed survey measures regarding their demographics, melanoma risk, perceived skin cancer risk, skin cancer knowledge, and their occupational sunscreen use. Sixty-nine percent of the sample reported never or rarely wearing sunscreen while working outdoors. Being female (p = .02), having a higher level of education (p = .03), and residing at a higher latitude (p = .04) were associated with more frequent sunscreen use. Conclusions: This study highlights the importance of interventions to promote sun protection behaviors among US Hispanic outdoor workers, and identifies potential intervention targets.Ashley K. Day, Jerod L. Stapleton, Ana M. Natale‑Pereira, James S. Goydos and Elliot J. Coup

The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma.

Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma

The Role of Photodynamic Therapy for Hilar Cholangiocarcinoma

The prognosis for hilar cholangiocarcinoma is limited by tumor spread along the biliary tree leading to refractory obstructive cholestasis, cholangitis, and liver failure. Palliation with biliary endoprostheses results in median survival times of 4-6 months for advanced bile duct cancer. Photodynamic therapy (PDT) is a local photochemical tumor treatment consisting of a photosensitizing agent combined with laser irradiation of a distinct wavelength. Tumor ablation with PDT combined with biliary stenting reduces cholestasis and significantly improves median survival time. However, the treatment is not widely available, and the photosensitizers used for PDT cause prolonged photosensitivity. Optimum control of tumor spread along the bile ducts and control of cholestasis and cholangitis will prolong survival in one to two thirds of patients, and renders them suitable for other antitumor therapies

GLI2-Mediated Melanoma Invasion and Metastasis

Background The transforming growth factor-β (TGF-β) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-β signaling. Methods We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. Results Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm2, difference = 1.88 mm2, 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-β-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-β vs shCtrl + TGF-β, 31.0 vs 161.9, difference = −130.9, 95% CI = −96.2 to −165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors. Conclusion GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical stud

The T1799A point mutation is present in posterior uveal melanoma

An activating mutation in exon 15 of the BRAF gene is present in a high proportion of cutaneous pigmented lesions. Until recently this mutation had however only been identified in one case of posterior uveal melanoma. Despite this apparent lack of the BRAF mutation, inappropriate downstream activation of the Ras/Raf/MAPK pathway has been described in posterior uveal melanoma. Based on the already recognised morphological and cytogenetic heterogeneity in uveal melanoma, we hypothesised that the BRAF mutation may be present in uveal melanoma but only in some of the tumour cells. In this study, we analysed 20 ciliary body and 30 choroidal melanomas using a nested PCR-based technique resulting in the amplification of a nested product only if the mutation was present. This sensitive technique can identify mutated DNA in the presence of wild-type DNA. The mutation was identified in 4 of 20 (20%) ciliary body and 11 of 30 (40%) choroidal melanomas. Further analysis of separate areas within the same choroidal melanoma demonstrated that the mutation was not present in the entire tumour. In conclusion, the T1799A BRAF mutation is present in a proportion of posterior uveal melanomas but within these tumours the distribution of the mutation is heterogeneous

Targeting the IL-6 Dependent Phenotype Can Identify Novel Therapies for Cholangiocarcinoma

The need for new therapies for cholangiocarcinoma is highlighted by their poor prognosis and refractoriness to chemotherapy. Increased production of Interleukin-6 promotes cholangiocarcinoma growth and contributes to chemoresistance by activating cell survival mechanisms. We sought to identify biologically active compounds capable of ameliorating the phenotypic effects of IL-6 expression and to explore their potential therapeutic use for cholangiocarcinoma.A genomic signature associated with Interleukin-6 expression in Mz-ChA-1 human malignant cholangiocytes was derived. Computational bioinformatics analysis was performed to identify compounds that induced inverse gene changes to the signature. The effect of these compounds on cholangiocarcinoma growth was then experimentally verified in vitro and in vivo. Interactions with other therapeutic agents were evaluated using median effects analysis.A group of structurally related compounds, nitrendipine, nifedipine and felodipine was identified. All three compounds were cytotoxic to Mz-ChA-1 cells with an IC50 for felodipine of 26 µM, nitrendipine, 44 µM and nifedipine, 15 µM. Similar results were observed in KMCH-1, CC-LP-1 and TFK-1 cholangiocarcinoma cell lines. At a fractional effect of 0.5, all three agents were synergistic with either camptothecin or gemcitabine in Mz-ChA-1 cells in vitro. Co-administration of felodipine and gemcitabine decreased the growth of Mz-ChA-1 cell xenografts in nude athymic mice.Computational bioinformatics analysis of phenotype-based genomic expression can be used to identify therapeutic agents. Using this drug discovery approach based on targeting a defined tumor associated phenotype, we identified compounds with the potential for therapeutic use in cholangiocarcinoma

A novel AKT3 mutation in melanoma tumours and cell lines

Recently, a rare activating mutation of AKT1 (E17K) has been reported in breast, ovarian, and colorectal cancers. However, analogous activating mutations in AKT2 or AKT3 have not been identified in any cancer lineage. To determine the prevalence of AKT E17K mutations in melanoma, the most aggressive form of skin cancer, we analysed 137 human melanoma specimens and 65 human melanoma cell lines for the previously described activating mutation of AKT1, and for analogous mutations in AKT2 and AKT3. We identified a single AKT1 E17K mutation. Remarkably, a previously unidentified AKT3 E17K mutation was detected in two melanomas (from one patient) as well as two cell lines. The AKT3 E17K mutation results in activation of AKT when expressed in human melanoma cells. This represents the first report of AKT mutations in melanoma, and the initial identification of an AKT3 mutation in any human cancer lineage. We have also identified the first known human cell lines with naturally occurring AKT E17K mutations