Identification and characterization of TROP-2 as a novel tumor suppressor gene in hepatocellular carcinoma

Abstract

As the second most lethal cancer worldwide, hepatocellular carcinoma (HCC) accounts for 85-90% of primary liver cancer. It has been shown that dysregulation of gene expression, including overexpression of oncogenes and down-regulation of tumor suppressor genes, plays a pivotal role in the initiation and progression of HCC. In this study, Tumor Associated Calcium Signal Transducer 2 (TACSTD2; also known as TROP-2) was shown to be frequently down-regulated in HCC by analysis of sequencing data obtained from The Cancer Genome Atlas (TCGA) database and qRT-PCR analysis of 205 pairs of non-tumor liver and HCC patient samples. Clinicopathological analysis found down-regulation of TROP-2 in HCC to be tightly associated with poor overall survival rate (P < 0.05), presence of adjacent organ invasion (P < 0.05) and poor differentiation (P < 0.05). Bisulfite genomic sequencing and methylation specific PCR indicated promoter hypermethylation of TROP-2, which provided possible explanation for the down-regulation of TROP-2 in HCC. Overexpression of TROP-2 was shown to inhibit cell proliferation, colony formation, migration, invasion in vitro and tumor growth and metastasis in vivo. TROP-2 overexpression also sensitized cancer cells to cisplatin induced cell apoptosis. Knockdown of this gene using shRNA appeared to have opposite effects. Given the membranous location of the TROP-2 protein and its possession of a large extracellular domain, it is possible that TROP-2 exerts its function through inhibiting the activity of other membrane receptors. To test this hypothesis and to dissect the downstream signaling of TROP-2 leading to its potent tumor suppressive functions, a human phospho-RTK array assay was performed. By comparing the signal intensities of 49 human RTKs between vector and shTROP-2 transfected cells, insulin-like growth factor 1 receptor (IGF-1R) and anaplastic lymphoma kinase (ALK) were shown to be stimulated by TROP-2 knockdown. To further investigate the functions of these two receptors in cancer, small molecule inhibitors AG1024 (IGF-1R inhibitor) and Crizotinib (ALK inhibitor) were used for treatment of cancer cells. In vitro functional assays showed that either AG1024 or Crizotinib could suppress cell viability, cell proliferation and migration, while a combination of the two inhibitors further strengthened such effects. In vivo tumor growth in tumor-bearing nude mice was also suppressed by injections of either AG1024 or Crizotinib, while the combined injection of the two drugs more effectively inhibited tumor growth. Immunoprecipitation assay demonstrated protein interactions between TROP-2 and the ligands of IGF-1R and ALK, namely IGF-1 and Midkine (MDK), respectively. Such protein bindings sequester IGF-1 and MDK from IGF-1R and ALK, inhibiting the activity of the two receptors, which in turn suppress tumorigenesis. The frequent down-regulation of TROP-2 in tumor tissues of HCC patients suggests that this gene may serve as a diagnostic marker for HCC. Upon TROP-2 down-regulation, IGF-1R and ALK will be hyperactivated, which will drive HCC progression. Therefore, dual inhibition of the two receptors with AG1024 and Crizotinib in TROP-2 down-regulated HCC patients may represent a novel molecular-targeted therapeutic option for future personalized medicine in HCC treatment.