Hepatoblastoma: glutamine depletion hinders cell viability in the embryonal subtype but high GLUL expression is associated with better overall survival

Andreas Schmidt,Martina Chiu,Steven W Warmann,Marcus O Scharpf,Philip Sander,Angela Armento,Jörg Fuchs,Verena Ellerkamp,Evi Schmid,Ovidio Bussolati

doi:10.1007/s00432-021-03713-4

Abstract

PurposeGlutamine plays an important role in cell viability and growth of various tumors. For the fetal subtype of hepatoblastoma, growth inhibition through glutamine depletion was shown. We studied glutamine depletion in embryonal cell lines of hepatoblastoma carrying different mutations. Since asparagine synthetase was identified as a prognostic factor and potential therapeutic target in adult hepatocellular carcinoma, we investigated the expression of its gene ASNS and of the gene GLUL, encoding for glutamine synthetase, in hepatoblastoma specimens and cell lines and investigated the correlation with overall survival.MethodsWe correlated GLUL and ASNS expression with overall survival using publicly available microarray and clinical data. We examined GLUL and ASNS expression by RT-qPCR and by Western blot analysis in the embryonal cell lines Huh-6 and HepT1, and in five hepatoblastoma specimens. In the same cell lines, we investigated the effects of glutamine depletion. Hepatoblastoma biopsies were examined for histology and CTNNB1 mutations.ResultsHigh GLUL expression was associated with a higher median survival time. Independent of mutations and histology, hepatoblastoma samples showed strong GLUL expression and glutamine synthesis. Glutamine depletion resulted in the inhibition of proliferation and of cell viability in both embryonal hepatoblastoma cell lines. ASNS expression did not correlate with overall survival.ConclusionGrowth inhibition resulting from glutamine depletion, as described for the hepatoblastoma fetal subtype, is also detected in established embryonal hepatoblastoma cell lines carrying different mutations. At variance with adult hepatocellular carcinoma, in hepatoblastoma asparagine synthetase has no prognostic significance.

Highlights

The amino acid glutamine (Gln) is involved in key cellular processes, such as energy metabolism, nucleotide biosynthesis, the balance of redox potential, and in various signaling pathways (DeBerardinis and Cheng 2010)
Since the tumor subtypes are different in gene expression and metabolism, we investigated the effect of glutamine depletion, GLUL expression, and GS abundance in the hepatoblastoma cell lines Huh-6 and HepT1, which differ in their mutations and, in contrast to the fetal-type HepG2, derive from tumors of the embryonal subtype (Crippa et al 2017; Koch et al 1999; Pietsch et al 1996)
Data of expression databases revealed that GLUL and asparagine synthetase (ASNS) are overexpressed in HB compared to non-tumor liver tissue

Summary

Introduction

The amino acid glutamine (Gln) is involved in key cellular processes, such as energy metabolism, nucleotide biosynthesis, the balance of redox potential, and in various signaling pathways (DeBerardinis and Cheng 2010). Therapeutic options that target Gln synthesis and metabolism are being investigated for selected tumors (Mates et al 2020; Schulte, 2018; Ye, 2018). The transcription of this gene is primarily regulated by the Wnt/β-catenin pathway. Activation of this signaling pathway through Wnt ligands prevents β-catenin degradation in the cytosol, allowing β-catenin to migrate into the nucleus where it increases the transcription of GLUL by interacting with the transcription factors TCF and LEF (Monga 2015; Willert and Nusse 1998). Mutations in the gene for β-catenin, CTNNB1, result in reduced degradation and, in increased GLUL expression and activity (Cadoret, 2002; Lopez-Terrada, 2009b)

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