Identification and characterization of two oncogenes SPOCK1 and AZIN1 in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC), which constitutes 75%-80% of primary liver cancer, is one of the most common malignancies worldwide. Hepatocarcinogenesis is a complicated and slow process accumulating multiple genetic and epigenetic alterations. In spite of its prolonged pre-malignant stage, HCC is usually diagnosed late and of high aggressiveness. A better understanding of the genetic and epigenetic changes during HCC development and progression is of great importance to early diagnosis and treatment of HCC.

Gain of chromosome 1q21 is one of most frequent genetic alteration in HCC and chromodomain helicase DNA binding protein 1-like (CHD1L) was recently identified to be responsible for this amplification. As a family member of SNF-2 like transcription factors, CHD1L plays an important role in HCC development via regulation of various downstream targets. In this study, a novel oncogene, sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1 (SPOCK1), was identified as a CHD1L target. CHD1L protein directly bound to the promoter region (nt -1662 to +34) of SPOCK1 and activated its transcription. Clinically, overexpression of SPOCK1 was detected in 60% of human HCC samples and was significantly associated with advanced clinical stage (P=0.020), shorter overall survival (P=0.011) and poorer disease-free survival of patients (P=0.039). Functionally, the ectopic expression of SPOCK1 in HCC cells conferred strong tumorigenic ability, while shRNA-mediated SPOCK1 silencing abolished this effect. Further study showed that the SPOCK1-enhanced cell survival could be attributed to its anti-apoptotic effects. SPOCK1 could suppress HCC cell apoptosis through the activation of AKT and subsequent inhibition of the (cytochrome c)-(caspase-9)-(caspase-3) pathway. In addition to its tumorigenic roles, the overexpression of SPOCK1 in HCC cells conferred strong metastatic ability via MMP9-mediated extracellular matrix remodeling.

In addition to genetic alterations, epigenetic changes also get increasing attentions due to their profound effects on gene activity and expression. A-to-I RNA editing is a post-transcriptional epigenetic modification which converts a site-selective adenosine nucleotide into inosine. The importance of RNA editing has long been underestimated because most of RNA editing modifications occur in a subtle way. The next-generation sequencing provides enough depth to unravel this mystery. The transcriptome sequencing data obtained from this study identified an A-to-I RNA editing at codon 367 (Ser→Gly) of antizyme inhibitor 1 (AZIN1). A high modification rate of AZIN1 was found to be prevalent in HCC specimens and closely associated with HCC pathogenesis. Adenosine deaminase acting on RNA-1 (ADAR1), but not ADAR2 or ADAR3, was responsible for AZIN1 RNA editing. This recoding editing event conferred “gain-of-function” phenotypes as manifested by augmented tumorigenic capabilities and higher aggressive potentials. Compared with wild-type AZIN1 protein, the edited form possessed stronger affinity to antizyme. As a result, edited AZIN1 demonstrated higher protein stability and ensuing neutralization of the antizyme-mediated degradation of ODC and CCND1 oncoproteins. The rescued ODC and CCND1 robustly accelerated cell proliferation thereby promoting HCC development.

In conclusion, two novel molecular mechanisms, (CHD1L)-(SPOCK1)-(AKT) and (ADAR1)-(edited AZIN1)-(ODC/CCND1), were delineated during HCC initiation and progression. Also, a causal link between RNA hyper-editing activity and cancer development was established for the first time in this study.