Reduced CRYL1 expression in hepatocellular carcinoma confers cell growth advantages and correlates with adverse patient prognosis

Homozygous deletion screening has been widely utilized to define tumour suppressor genes (TSGs) in cancers. Although these biallelic deletions are infrequent, their identification has facilitated the discovery of many important TSGs. We have systematically examined the genome of hepatocellular carcinoma (HCC), a highly malignant tumour that is rapidly fatal, for the presence of homozygous deletions. Array-CGH analysis on early passage of HCC cultures and cell lines led us to identify six homozygous deleted (HD) regions. A high concordance between array-CGH and expression of HD genes was demonstrated, where crystallin Lambda1 (CRYL1; located on chromosome 13q12.11) displayed the most frequent down-regulation. We found that reduced mRNA expression of CRYL1 was common in HCC tumours when compared with their adjacent non-tumoural liver (p = 0.0097). Significant associations could also be drawn between repressed CRYL1 and advanced tumour staging, increased tumour size, and shorter disease-free survival of patients (P < 0.037). Moreover, homozygous deletions on CRYL1 could be detected in 36% of HCC cases, where recurrent HDs were identified on exons 1, 5, and 8. Examination of other causal events suggested histone deacetylation and promoter hypermethylation to be likely inactivating mechanisms as well. Re-expression of CRYL1 in the SK-Hep1 cell line, where biallelic loss of CRYL1 was found, induced profound inhibition of cellular proliferation and cell growth (p < 0.0015). By Annexin V staining, CRYL1 restoration readily increased pro-apoptotic cells with an induction of PARP cleavage. Flow cytometry further revealed that CRYL1 could prolong the G(2)-M phase, possibly through interruption of the Cdc2/cyclin B pathway. Given that regional chromosome 13q12-q14 loss is a causal genomic event in HCC tumourigenesis, our finding may have implications for identifying a novel TSG CRYL1 within this important locus. Copyright (C) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.