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Interaction and global proteomics reveal the role of the pluripotency factor L1TD1 in embryonal tumor cell proliferation and mobility

ABSTRACT: Pluripotency factors are known to be abnormally expressed cancer cells, which often correlate with poor prognosis. The molecular mechanisms by which pluripotency factors contribute to tumor aggressiveness are yet to be elucidated. The pluripotency marker L1TD1 interacts with other pluripotency factors and is a marker for human embryonic stem cells. In cancer, L1TD1 expression has been detected in solid tumors, including Embryonal tumors of the Central Nervous System. Previously, we reported that, in medulloblastoma, L1TD1 expression correlates with metastasis and shorter overall patient survival. Here, we use Affinity purification-mass spectrometry and global proteomics to map the L1TD1 protein interaction network and assess proteins regulated by L1TD1 expression, respectively. Interactome and global proteome analysis led to the enrichment of overlapping Biological Processes attributed to L1TD1 including Cell proliferation, Cell death and cell migration. We also used L1TD1 overexpressing tumor cell populations for in vitro validation of the omic data. L1TD1 overexpressing cells presented higher cell mobility, enhanced filopodial formation and distinct cell morphology. These cells were overall more proliferative and resistant to cisplatin treatment in vitro.

INSTRUMENT(S): Orbitrap Fusion

DISEASE(S): Medulloblastoma

SUBMITTER: Brandon Coke

LAB HEAD: Rob Ewing

PROVIDER: PXD040803 | Pride | 2024-02-09

REPOSITORIES: Pride

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Project description:Medulloblastoma is a highly aggressive pediatric brain tumor, in which expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However, the contribution of OCT4 transcript variants to tumor aggressiveness is still poorly understood. In this study, we found that transcripts encoding OCT4A, but not OCT4B or OCT4B1, were significantly correlated with LIN28A expression, which encodes another well-known pluripotency factor. LIN28A was found to specifically bind OCT4A transcripts and interact with poly(A) binding protein and RNA helicase A in polysomal fractions of medulloblastoma cells, favoring increased OCT4A protein levels in these cells. Medulloblastoma cells stably overexpressing OCT4A displayed significantly enhanced clonogenic activity, tumorsphere generation and invasion capability, as well as increased tumorigenicity. In an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic effects of OCT4A were found to be expression-level dependent and accompanied by distinct subchromosomal aberrations and differential expression of newly discovered, still poorly characterized, non-coding RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer. Total RNA of 2-3 clones of each cell line (Control and OCT4A overexpression) was extracted with the RNeasy Mini kit (Qiagen), following the manufacturerâs protocol. Gene expression profiling was carried out independently for each sample using Affymetrix GeneChipÂ® Human Gene 2.0 ST whole-transcript arrays (Affymetrix, Santa Clara, CA, USA). The quality control and normalization of data were processed by AffymetrixÂ® Expression Console Software (Affymetrix). Differentially expressed genes were identified with the One-Way ANOVA, with a p-value cutoff of 0.05, using Transcriptome Analysis Console v3.0 (Affymetrix).

Project description:The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe an unbiased methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1,027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are prominently found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of under transcriptional arrest. In contrast, the corresponding di-methylation or protein expression level is not altered in expression, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA dataset in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase. Data analysis: All raw data analysis was performed with MaxQuant software suite version 1.2.6.20 supported by the Andromeda search engine. Data was searched against a concatenated target/decoy (forward and reversed) version of the UniProtKB Human database encompassing 71,434 protein entries. Mass tolerance for searches was set to maximum 7 ppm for peptide masses and 20 ppm for HCD fragment ion masses. Data was searched with carbamidomethylation as a fixed modification and protein N-terminal acetylation, methionine oxidation and mono-methylation on lysine and arginine as variable modifications. A maximum of three mis-cleavages was allowed while requiring strict trypsin specificity, and only peptides with a minimum sequence length of seven were considered for further data analysis. Peptide assignments were statistically evaluated in a Bayesian model on the basis of sequence length and Andromeda score. Only peptides and proteins with a false discovery rate (FDR) of less than 1% were accepted, estimated on the basis of the number of accepted reverse hits. Protein sequences of common contaminants such as human keratins and proteases used were added to the database.

			Action	DRS
	F001415.mgf	Mgf
	F001416.mgf	Mgf
	F001417.mgf	Mgf
	F001418.mgf	Mgf
	F001419.mgf	Mgf

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Interaction and global proteomics reveal the role of the pluripotency factor L1TD1 in embryonal tumor cell proliferation and mobility

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