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

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

Melanoma Patients with Positive Sentinel Nodes Who Did Not Undergo Completion Lymphadenectomy: A Multi-Institutional Study

Completion lymph node dissection (CLND) is considered the standard of care in melanoma patients found to have sentinel lymph node (SLN) metastasis. However, the therapeutic utility of CLND is not known. The natural history of patients with positive SLNs who do not undergo CLND is undefined. This multi-institutional study was undertaken to characterize patterns of failure and survival rates in these patients and to compare results with those of positive-SLN patients who underwent CLND.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45875/1/10434_2006_Article_10237.pd