Dataset Information

Neogenin, Defined as a GD3-associated Molecule by Enzyme-mediated Activation of Radical Sources, Confers Malignant Properties via Intracytoplasmic Domain in Melanoma Cells.

ABSTRACT: To investigate mechanisms for increased malignant properties in malignant melanomas by ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association between GD3 and neogenin was demonstrated by immunoblotting of the immunoprecipitates with anti-neogenin antibody from GD3+ cell lysates. The intracytoplasmic domain of neogenin (Ne-ICD) was detected in GD3+ cells at higher levels than in GD3- cells when cells were treated by a proteasome inhibitor but not when simultaneously treated with a ?-secretase inhibitor. Exogenous GD3 also induced increased Ne-ICD in GD3- cells. Overexpression of Ne-ICD in GD3- cells resulted in the increased cell growth and invasion activity, suggesting that Ne-ICD plays a role as a transcriptional factor to drive malignant properties of melanomas after cleavage with ?-secretase. ?-Secretase was found in lipid/rafts in GD3+ cells. Accordingly, immunocyto-staining revealed that GD3, neogenin, and ?-secretase were co-localized at the leading edge of GD3+ cells. All these results suggested that GD3 recruits ?-secretase to lipid/rafts, allowing efficient cleavage of neogenin. ChIP-sequencing was performed to identify candidates of target genes of Ne-ICD. Some of them actually showed increased expression after expression of Ne-ICD, probably exerting malignant phenotypes of melanomas under GD3 expression.

SUBMITTER: Kaneko K

PROVIDER: S-EPMC4974378 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Neogenin, Defined as a GD3-associated Molecule by Enzyme-mediated Activation of Radical Sources, Confers Malignant Properties via Intracytoplasmic Domain in Melanoma Cells.

Kaneko Kei K Ohkawa Yuki Y Hashimoto Noboru N Ohmi Yuhsuke Y Kotani Norihiro N Honke Koichi K Ogawa Mitsutaka M Okajima Tetsuya T Furukawa Keiko K Furukawa Koichi K

The Journal of biological chemistry 20160610 32

To investigate mechanisms for increased malignant properties in malignant melanomas by ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association ...[more]

PMID: 27288875

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Project description:Malignant melanoma (MM) is known to be intrinsically chemoresistant, even though only ~20% of MM carry mutations of the tumor suppressor p53. Despite improvement of systemic therapy the mortality rate of patients suffering from metastatic MM is still ~70%, highlighting the need for alternative treatment options or for the re-establishment of conventional therapeutic approaches, including chemotherapy. Screening the p53 mutation status in a cohort of 19 patient-derived melanoma samples, we identified one rarely described missense mutation of p53 leading to E285K amino acid exchange (mutp53(E285K)). Employing structural and computational analysis we revealed a major role of E285 residue in maintaining stable conformation of wild-type p53 (wtp53). E285K mutation was predicted to cause interruption of a salt-bridge network affecting the conformation of the C-terminal helix of the DNA-binding domain (DBD) thereby preventing DNA interaction. In this context, a cluster of frequently mutated amino acid residues in cancer was identified to putatively lead to similar structural effects as E285K substitution (E285 cluster). Functional analysis, including knockdown of endogenous p53 and reconstitution with diverse p53 missense mutants confirmed mutp53(E285K) to have lost transcriptional activity, to be localized in the cytosol of cancer cells, by both means conferring chemoresistance. Re-sensitization to cisplatin-induced cell death was achieved using clinically approved compounds aiming to restore p53 wild-type function (PRIMA1-Met), or inhibition of AKT-driven MAPK survival pathways (afuresertib), in both cases being partially due to ferroptosis induction. Consequently, active ferroptosis induction using the GPX4 inhibitor RSL3 proved superior in tumorselectively fighting MM cells. Due to high prevalence of the E285-cluster mutations in MM as well as in a variety of other tumor types, we conclude this cluster to serve an important function in tumor development and therapy and suggest new implications for ferroptosis induction in therapeutic applications fighting MM in particular and cancer in general.

Dataset Information

Neogenin, Defined as a GD3-associated Molecule by Enzyme-mediated Activation of Radical Sources, Confers Malignant Properties via Intracytoplasmic Domain in Melanoma Cells.

Publications

Neogenin, Defined as a GD3-associated Molecule by Enzyme-mediated Activation of Radical Sources, Confers Malignant Properties via Intracytoplasmic Domain in Melanoma Cells.

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