Proteomic profiling of cisplatin-resistant and cisplatin-sensitive germ cell tumour cell lines using quantitative mass spectrometry

A. Richter,D. Nettersheim,C. Oing,A. Fichtner,C. Lenz,S. Kueffer,O. Elakad,P. Ströbel,H. Bohnenberger,F. Bremmer

doi:10.1007/s00345-022-03936-1

Abstract

PurposeAdvanced testicular germ cell tumours (GCT) generally have a good prognosis owing to their unique sensitivity towards cisplatin-based chemotherapies. However, cisplatin-resistant GCT have a poor outcome. Further studies are mandatory to better understand resistance mechanisms and develop therapeutic strategies for refractory GCTs.MethodsProtein levels in cisplatin-resistant GCT cell lines of NTERA-2, NCCIT and 2102EP were analyzed by quantitative proteomic mass spectrometry (MS) in combination with stable isotope labelling by amino acids in cell culture (SILAC). Differentially abundant protein markers of acquired cisplatin resistance were validated by Western blotting. Comprehensive bioinformatical annotation using gene set enrichment analyses (GSEA) and STRING interaction analysis were performed to identify commonly affected pathways in cisplatin resistance and the data were compared to the GCT cohort of the ‘The Cancer Genome Atlas’.ResultsA total of 4375 proteins were quantified by MS, 144 of which were found to be differentially abundant between isogenic resistant and sensitive cell line pairs (24 proteins for NTERA-2, 60 proteins for NCCIT, 75 proteins for 2102EP). Western blotting confirmed regulation of key resistance-associated proteins (CBS, ANXA1, LDHA, CTH, FDXR). GSEA revealed a statistically significant enrichment of DNA repair-associated proteins in all three resistant cell lines and specific additional processes for individual cell lines.ConclusionHigh resolution MS combined with SILAC is a powerful tool and 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. Our study provides the largest proteomic in vitro library for cisplatin resistance in GCT, yet, enabling further studies to develop new treatment options for patients with refractory GCT.

Highlights

Testicular germ cell tumours (GCTs) are the most common malignant solid neoplasms in men between the age of 15 and 40 with an increasing incidence seen over the last 4 decades [1]
We provide a proteomic resource library and functional annotations determined by gene set enrichment analyses (GSEA), the STRING algorithm and DAVID Gene Ontology annotation of acquired cisplatin resistance in GCT cell lines and compared our findings to the ‘The Cancer Genome Atlas’ (TCGA) cohort of ‘testicular germ cell tumours’
Cisplatin resistance in GCTs rarely occurs in primary diseases, but in up to 15% of metastatic diseases with poor prognosis for the affected patient [5]

Summary

Introduction

Testicular germ cell tumours (GCTs) are the most common malignant solid neoplasms in men between the age of 15 and 40 with an increasing incidence seen over the last 4 decades [1]. Type II GCT are divided into seminomas (SEM) and nonseminomatous GCT (NSGCT) according to the WHO [2]. NSGCT comprise distinct subentities, e.g. embryonal carcinomas (EC), yolk sac tumours, A.

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Conclusion