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, enzymemediated 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.It has been long known that malignant transformation of cells brings about changes in the carbohydrate structures in the glycoproteins and glycolipids expressed on the cell surface (1).In particular, there have been a number of reports on the expression of unique glycosphingolipids in neuroectoderm-derived cancer cells (2), osteosarcomas (3, 4), small lung cancer cells (5, 6), and T-cell leukemia cells (7-9). Some of them have been utilized in the clinical field as tumor markers (10) or target molecules of antibody therapy (11).Because ganglioside GD3 was reported to be neo-antigen in melanomas (12-14), it has attracted the interests of researchers in cancer immunology field, and its expression in normal and cancer tissues has been analyzed (15). The effects of anti-GD3 antibody therapy in melanomas (16) and various aspects of its roles in melanomas have been also reported (17-19). These results suggest that GD3 plays important roles in the enhancement of malignant properties of melanomas (20, 21) by regulating glycolipid-enriched microdomain (GEM) 2 /rafts (22). However, precise mechanisms for GD3 function in cancers have not yet been clarified.To investigate how gangliosides are involved in the regulation of cell signaling, we have applied enzyme-mediated activation of radical sources (EMARS) method (23) to identify membrane molecules interacting wi...

New era of research on cancer‐associated glycosphingolipids

“…An EMARS/MS analysis (Figure ) with melanoma cells using anti‐GD3 mAb revealed various membrane molecules as candidate molecules that associate with GD3 . Among them, Neogenin‐1 was defined as a GD3‐associating molecule and also as a GEM/raft‐residing molecule exclusively in GD3 + cells . Neogenin‐1 was shown to be involved in enhancement of the malignant properties of melanomas such as increased cell growth, invasion, and migration, indicating that Neogenin‐1 might be an effector molecule exerting the effects of melanoma‐specific ganglioside, GD3 (Figure A).…”

“…Consequently, GD3 expression resulted in the shift of Neogenin‐1 (Figure B) but also of γ‐secretase to lipid rafts from the nonraft compartment, leading to increased cleavage of the intracytoplasmic domain of Neogenin‐1. Neogenin intracytoplasmic domain promoted expression of multiple genes such as GPR126 , STXBP5 , MMP16 , SPATA31A1 , and S6K , as identified by ChIP‐sequencing, possibly playing central roles in enhancing the malignant properties of melanomas (Figure A).…”

ASC amino acid transporter 2, defined by enzyme‐mediated activation of radical sources, enhances malignancy of GD2‐positive small‐cell lung cancer

“…C‐2, C‐6, and C‐8 are GD2 − SK‐LC‐17 transfectants (Figures A, ). Tumor‐specific gangliosides coordinately work with plasma membrane proteins in cancer cells, which leads to enhancement of malignant phenotypes . However, it is not well known what kind of plasma membrane proteins work together with GD2 in SCLC cells.…”

“…Moreover, neogenine, a membrane protein that promotes the development of the nervous system, was identified as a GD3‐associating molecule in melanoma cells. Neogenin enhanced malignant phenotypes in melanoma cells through its intracellular domain as a transcription factor . In this study, we undertook EMARS and MS to clarify how GD2 works and what kind of molecules GD2 works with in SCLC cells.…”

“…Although roles of GD2 in malignant phenotypes of SCLC have been analyzed, there is no information about molecules interacting with GD2 in GEM/rafts. To date, it has been reported that GD3 interacts with membrane proteins in melanomas and glioma cells, which promotes malignant phenotypes of individual cancer cells . In this study, we aimed to reveal the molecular mechanisms by which GD2 expressed on the membrane of SCLC cells induces malignant phenotypes, then used EMARS combined with MS to address this aim.…”