Phosphoproteomic Analysis of Metastatic and Non-Metastatic Uveal Melanoma

Abstract

Purpose : Phosphoproteomic analysis of primary uveal melanoma (pUM) was pursued for insights into molecular mechanisms of UM metastasis and potential therapeutic drug targets.

Methods : Quantitative phosphoproteomic analysis was pursued with 40 pUM (20 metastasizing and 20 non-metastasizing) and pooled normal choroid control specimens using methods and specimens defined in our recent UM proteomic study of 100 pUM (2021 Cancers 13, 3520). Tryptic peptides were quantified by AAA, labeled with unique iTRAQ tags and combined in 6 batches for LC MS/MS analysis. Each batch contained both metastasizing and non-metastasizing pUM specimens from Cleveland and Liverpool. Phosphopeptides were enriched using titanium dioxide and Fe-NTA IMAC. Following enrichment each batch was individually fractionated by RPHPLC and peptides analyzed by LC MS/MS using an Orbitrap Fusion Lumos Tribrid mass spectrometer. Protein identification utilized the UniProt human database, the Mascot search engine and ptmRS within Proteome Discoverer. Protein quantitation utilized the limma package in R. Bioinformatic analyses were performed with Ingenuity Pathway Analysis (IPA), the Reactome Pathway Browser and the UniProt Knowledge Base.

Results : 10,335 phosphopeptides derived from 2623 pUM phosphoproteins have been quantified, including 25 phosphoSites in 21 proteins differing significantly in abundance between metastasizing and non-metastasizing pUM after correction of multiplicity. PhosphoSites significantly more elevated in metastasizing pUM were detected in proteins encoded by TNS3, TYMP, LCP1, ACBD3, IFI16, EML4, and PEAK1. PhosphoSites significantly less abundant in metastasizing pUM were detected in proteins encoded by SRRM2, NACA, BZW2, UPF2, CD44, TJP2, PML, NELFB, MARCKS, GNG12, SLC16A1, MTSS2, ZRANB2, MITF. Combined with our recent UM proteomic study results, 5263 proteins have been quantified in pUM.

Conclusions : To date, pathway analyses implicate 9 of the 21 significant phosphoSite-containing proteins in 31 over-represented pathways. Several have been associated with other cancers, such as phosphorylation of Ser5 in L-plastin (LCP1) which has been reported to promote breast cancer invasiveness (2021 Cell Commun Signal 19, 22). We hypothesize that the identified phosphoSites that differ significantly between metastasizing and non-metastasizing pUM proteins may contribute to mechanisms of UM metastasis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.