hes1-protein--human and Melanoma

The role of Notch signaling in melanoma drug resistance is not well understood. In this study, we show that although NOTCH proteins are upregulated in metastatic melanoma cell lines, Notch signaling inhibition had no effect on cell survival, growth, migration or the sensitivity of BRAFV600E-melanoma cells to MAPK inhibition (MAPKi). We found that NOTCH1 is downregulated in melanoma cell lines with intrinsic and acquired resistance to MAPKi. Forced expression of NICD1, the active form of Notch1, caused apoptosis of the NOTCH

Topics: Apoptosis; Cell Death; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Endothelin-1; Gene Expression Regulation, Neoplastic; Humans; Ligands; Melanoma; Mitogen-Activated Protein Kinases; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-jun; Receptors, Notch; Signal Transduction; Transcription Factor HES-1; Transcriptome; Up-Regulation

Uveal melanoma (UM) is the most common primary intraocular tumour in adults and has a high incidence. Nearly 50% of patients with UM develop metastases after diagnosis. Long noncoding RNAs (lncRNAs) are involved in both oncogenic and tumour suppression pathways. We show that lncRNA PAUPAR is present at low levels in UM tissues and cell lines and modulates the tumourigenesis of UM in vitro and in vivo. The ectopic expression of PAUPAR in UM cells revealed that PAUPAR acts as a necessary UM suppressor and induces the silencing of HES1 expression, which significantly reduces tumour metastasis. Mechanistically, PAUPAR modulates HES1 expression by inhibiting histone H3K4 methylation. These data support a role of this lncRNA as a novel therapeutic target in cancer prevention and treatment.

Topics: Animals; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Histones; Humans; Male; Melanoma; Methylation; Mice; Mice, Nude; Neoplasm Proteins; RNA, Long Noncoding; RNA, Neoplasm; Transcription Factor HES-1; Uveal Neoplasms

Melanoma is an aggressive disease with limited therapeutic options. Here, we determined the effects of honokiol (HNK), a biphenolic natural compound on melanoma cells and stemness. HNK significantly inhibited melanoma cell proliferation, viability, clonogenicity and induced autophagy. In addition, HNK significantly inhibited melanosphere formation in a dose dependent manner. Western blot analyses also demonstrated reduction in stem cell markers CD271, CD166, Jarid1b, and ABCB5. We next examined the effect of HNK on Notch signaling, a pathway involved in stem cell self-renewal. Four different Notch receptors exist in cells, which when cleaved by a series of enzymatic reactions catalyzed by Tumor Necrosis Factor-α-Converting Enzyme (TACE) and γ-secretase protein complex, results in the release of the Notch intracellular domain (NICD), which then translocates to the nucleus and induces target gene expression. Western blot analyses demonstrated that in HNK treated cells there is a significant reduction in the expression of cleaved Notch-2. In addition, there was a reduction in the expression of downstream target proteins, Hes-1 and cyclin D1. Moreover, HNK treatment suppressed the expression of TACE and γ-secretase complex proteins in melanoma cells. To confirm that suppression of Notch-2 activation is critical for HNK activity, we overexpressed NICD1, NICD2, and performed HNK treatment. NICD2, but not NICD1, partially restored the expression of Hes-1 and cyclin D1, and increased melanosphere formation. Taken together, these data suggest that HNK is a potent inhibitor of melanoma cells, in part, through the targeting of melanoma stem cells by suppressing Notch-2 signaling.

Topics: ADAM Proteins; ADAM17 Protein; Amyloid Precursor Protein Secretases; Autophagy; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Homeodomain Proteins; Humans; Lignans; Melanoma; Neoplastic Stem Cells; Receptor, Notch2; Receptors, Notch; Signal Transduction; Transcription Factor HES-1

Controlling the spread of uveal melanoma is key to improving survival of patients with this common intraocular malignancy. The Notch ligand Jag2 has been shown to be upregulated in primary tumors that metastasize, and we therefore investigated its role in promoting invasion and clonogenic growth of uveal melanoma cells.. mRNA and protein expression of Notch pathway components were measured using qPCR and Western blot in uveal melanoma cell lines. Expression of Jag2 ligand was upregulated using Jag2-GFP-MSCV constructs or downregulated by sh-Jag2 in the uveal melanoma cell lines Mel285, Mel290, 92.1, and OMM1, and the effects on growth and invasion were assessed.. Jag2 was introduced into Mel285 and Mel290 cells, which have low baseline levels of both this ligand and Notch activity. Overall growth of the Jag2-expressing cultures increased somewhat, and a significant 3-fold increase in clonogenic growth in soft agar was also noted. Introduction of Jag2 increased motility in both wound-healing and transwell invasion assays. We also observed a significant increase in Jag2 and Hes1 mRNA in invasive OMM1 cells that had passed through a Matrigel-coated filter in the transwell assay when compared with noninvading cells. Loss-of-function studies performed in 92.1 and OMM1 lines using Jag2 shRNAs showed that downregulation of the ligand significantly suppressed cellular growth, invasion, and migration.. Our data suggest that Jag2 may play an important role in promoting Notch activity, growth, and metastasis in uveal melanoma.

Topics: Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Cell Line, Tumor; Cell Movement; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Jagged-2 Protein; Ligands; MAP Kinase Signaling System; Melanoma; Membrane Proteins; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptors, Notch; RNA, Messenger; Signal Transduction; Transcription Factor HES-1; Up-Regulation; Uveal Neoplasms

MAP2 is a neuron-specific microtubule-associated protein that binds and stabilizes dendritic microtubules. Previously, we showed that MAP2 expression is (a) activated in cutaneous primary melanoma and (b) inversely associated with melanoma tumor progression. We also showed that ectopic expression of MAP2 in metastatic melanoma cells inhibits cell growth by inducing mitotic spindle defects and apoptosis. However, molecular mechanisms of regulation of MAP2 gene expression in melanoma are not understood. Here, we show that in melanoma cells MAP2 expression is induced by the demethylating agent 5-aza-2'-cytidine, and MAP2 promoter is progressively methylated during melanoma progression, indicating that epigenetic mechanisms are involved in silencing of MAP2 in melanoma. In support of this, methylation of MAP2 promoter DNA in vitro inhibits its activity. Because MAP2 promoter activity levels in melanoma cell lines also correlated with activating mutation in BRAF, a gene that is highly expressed in neurons, we hypothesized that BRAF signaling is involved in MAP2 expression. We show that hyperactivation of BRAF-MEK signaling activates MAP2 expression in melanoma cells by two independent mechanisms, promoter demethylation or down-regulation of neuronal transcription repressor HES1. Our data suggest that BRAF oncogene levels can regulate melanoma neuronal differentiation and tumor progression.

Topics: Amino Acid Substitution; Animals; Azacitidine; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Cell Differentiation; Cell Line, Tumor; CpG Islands; DNA Methylation; Down-Regulation; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; MAP Kinase Signaling System; Melanoma; Mice; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase Kinases; Mutant Proteins; Neurons; Promoter Regions, Genetic; Proto-Oncogene Proteins B-raf; Response Elements; Transcription Factor HES-1; Transcription, Genetic