Amplification of GEP at chromosome 17q21 and its overexpression in human liver cancer

Abstract

Introduction: Hepatocellular carcinoma (HCC) is the 3rd most lethal cancer worldwide. Curative treatments, such as surgical resection, are limited to only a small group of patients. Granulin-epithelin precursor (GEP), which is a secretory growth factor, has been shown to enhance the growth, invasion, metastasis and chemo-resistance of HCC. GEP was found to overexpress in tumors compared to the non-tumor counterpart, and the overexpression correlates to poor prognosis in HCC patients. Antibody targeting GEP inhibited tumor growth in vivo. However, the overexpression mechanisms in HCC are not clear. Hypothesis: Gene amplification may be one of the mechanisms for GEP overexpression as its locus, 17q21, is frequently amplified. Method: Quantitative Microsatellite Analysis (QuMA) was used to determine the copy number alteration of the GEP gene locus. In this method, quantitative PCR was used to quantify the GEP and microsatellite loci. The genomic stable microsatellite loci were used as reference. Copy number variations were determined by normalization of HCC data to the copy numbers of blood samples from healthy donors, which were defined as diploid 2N. GEP mRNA level was quantified by real-time RT-PCR. Correlations between GEP copy number and its mRNA expression level were analyzed. Results: GEP DNA copy numbers were quantified in a panel of clinical HCC (n=60). Amplification of GEP locus was observed in 30% (20/60) HCC, and the amplification frequency was comparable to published reports on chromosome 17q. Overall, GEP copy number correlated to the mRNA overexpression levels (n=60, r=0.267, P=0.039). For HCC with GEP gene locus amplification (n=20), tight correlation with GEP expression levels were observed (r=0.646, P=0.002). Summary: We showed that GEP gene was frequently amplified in HCC, and this amplification correlates to the expression level of GEP. Fluorescent in situ hybridization (FISH) will be performed to further investigate the amplification status.