Testicular cancer (TC) accounts for approximately 1% of adult cancers and 5% of all urological cancers diagnosed, with germ cell tumors (GCTs) being the most common histopathological subtype (90-95% of cases). The rapid metastasis of TC to retroperitoneal lymph nodes, mediastinum, lung, brain, liver, and bones necessitates a deeper understanding of the mechanisms driving this aggressive behavior. Elucidating these mechanisms could highlight significant changes in cell signaling or modulation of the immune system.

To explore the molecular alterations underlying TC, we conducted comprehensive proteomics and phosphoproteomics analyses on tissue samples. Proteins were trypsin digested, separated using a nano-LC system coupled with Orbitrap Fusion MS, and processed with PEAKS 11 software. Phosphoproteins were enriched using Ti-IMAC beads and similarly analyzed. Our study quantified over 5000 proteins and more than 1000 phosphoproteins across 10 samples.

Our findings reveal a significant upregulation in the tumor of biochemical pathways associated with the cell cycle, cell signaling, and cell-cell interactions mediated by tyrosine kinase receptors. Proteins responsible for neutrophil activation as well as the phagocytosis process also show increased expression. However, in cancer we observe reduced coagulation and prothrombin activation as well as reduced activity of processes related to cholesterol management.

Phosphoproteomics identified 400 proteins differentiating cancerous tissue from controls. The identified proteins are responsible for the regulation of cell structures, cell metabolic processes, mRNA splicing, regulation of mRNA processing. Phosphorylated proteins indicate the involvement of small GTPases, as well as MAPK family proteins in the carcinogenesis process. The obtained phosphoproteomics results confirm the global observation of changes occurring in cancer cells affecting cell signaling, disturbances in glycolysis or the cell cycle, as well as cell-cell interaction resulting from disturbances in Gap junctions.

These insights enhance our understanding of Testicular Cancer at molecular level and may inform the development of targeted therapies and diagnostic/prognostic markers.