phosphothreonine and Melanoma

ErbB4 receptor tyrosine kinase has four different isoforms that are classified based on variants in the extracellular juxtamembrane domain (JM-a and JM-b) and the C-terminal region (CYT-1 and CYT-2). Here, we used the JM-b/CYT-1 isoform to investigate the roles of serine/threonine phosphorylation in MEK-ERK-dependent feedback inhibition. TPA as an activator of the ERK pathway markedly induced ErbB4 phosphorylation at Thr-674, the conserved common feedback site in the intracellular JM domain, which resulted in the downregulation of tyrosine autophosphorylation. We also identified Ser-1026 as an ErbB4-specific ERK target site in the CYT-1 region. Moreover, double mutations (Thr-674/Ser-1026 to Ala) significantly upregulated ErbB4 activation, indicating that Thr-674 and Ser-1026 are cooperatively involved in negative feedback regulation. Given the fact that ErbB4 mutation is one of the most common genetic alterations in melanoma cells, we demonstrated that a typical oncogenic ErbB4 mutant was resistant to the negative feedback regulation to maintain a highly active status of tyrosine kinase activity. Together, these findings indicate that feedback mechanisms are key switches determining oncogenic potentials of ErbB receptor kinases.

Topics: Amino Acid Sequence; Extracellular Signal-Regulated MAP Kinases; Feedback, Physiological; HEK293 Cells; Humans; Isoenzymes; Melanoma; Mutation; Phosphorylation; Phosphoserine; Phosphothreonine; Receptor, ErbB-4

A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines.. The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot.. Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways.. The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

Topics: Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Culture Media; DNA Mutational Analysis; Drug Synergism; Flow Cytometry; Glucose; Humans; Indoles; Melanoma; Metformin; Mutation; Phosphorylation; Phosphothreonine; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Signal Transduction; Sulfonamides; Time Factors; Vemurafenib

MicroRNAs-221 and -222 are highly upregulated in several solid tumors, including melanomas. We demonstrate that the proto-oncogene ETS-1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR-222 by direct binding to its promoter region. Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS-1 represses miR-222 transcription, in metastatic melanoma the constitutively Thr-38 phosphorylated fraction of ETS-1 induces miR-222. Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS-1 relies on its RAS/RAF/ERK-dependent phosphorylation status more than on its total amount. To close the loop, we demonstrate ETS-1 as a direct target of miR-222, but not miR-221, showing the novel option of their uncoupled functions. In addition, a spatial redistribution of ETS-1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells. Finally, in vivo studies confirmed the contribution of miR-222 to the increased invasive potential obtained by ETS- silencing.

Topics: Animals; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Melanocytes; Melanoma; Mice; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Phosphorylation; Phosphothreonine; Proto-Oncogene Mas; Proto-Oncogene Protein c-ets-1; Signal Transduction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transcription, Genetic