Quantitative LC-MS-based shot gun proteomics identifies deregulated proteins in gallbladder carcinoma

Gallbladder carcinoma (GBC) is a rare and understudied cancer entity. Radical surgery is the only potentially curative treatment option but due to late diagnosis few patients are eligible and 2-year survival rates of unresectable GBC is less than 10%. Therefore, the development of new treatment options, including targeted therapy for GBC is required to improve patient outcome.

We performed quantitative LC-MS-based proteomics of a total of 18 FFPE tumors and 5 healthy gallbladder specimens from a German GBC cohort. Quantitative proteomic analysis was performed and revealed differentially expressed proteins, which were further subjected to pathway analysis. Candidate genes representing potential tumor suppressors were selected from the significantly altered pathways. The role of these candidate genes in proliferation, migration and clonogenicity was investigated in GBC cell lines to evaluate their specific function and their potential as future treatment targets.

In the investigated human samples, we detected 611 proteins with a significant difference (adj. p-value < 0.05) between long and short surviving patients. Among the enriched pathways in patients with worse survival are the cellular defense response and regulated exocytosis and downregulated pathways are the extracellular matrix organization and cell/biological adhesion. We also found 1766 proteins to be differentially expressed between healthy gallbladder and GBC (adjusted p-value < 0.05). Interestingly, multiple pathways for RNA processing and inflammatory response were upregulated, whereas cytoskeleton organization and biological/cell adhesion were down regulated.

In order to identify potential tumor suppressor candidates, we selected genes of the significantly affected downregulated pathways with at least 4-fold decrease in tumors. This led to the identification of FHL1 which is strongly downregulated in GBC tumor samples, compared to healthy gallbladder (fold difference: 8.6, FDR = 0.008). Cell lines were infected with an inducible lentivirus to study the biological functions of FHL1. Expression of FHL1 in NOZ and G-415 cells significantly reduced cell viability supporting a tumor suppressive role of FHL1 in GBC.

Thus, proteomic profiling of a clinicopathologically well-characterized German GBC cohort identified multiple differentially expressed proteins and tumor-relevant pathways. Functional validation confirmed the tumor suppressive function of FHL1.