Secretory ANXA3 endows liver cancer cells with tumorigenic and self-renewal abilities and promotes HCC development

Abstract

Hepatocellular carcinoma (HCC) is the third leading cause of mortality in Southeast Asia and in Hong Kong. Despite advances in treatment, the prognosis of advanced HCC patients remains dismal. Contemporary challenge in treating HCC has been the common chemotherapy resistance and recurrence after therapy.Therefore, the identification of valid targets and the development of new therapies are urgently needed for more effective clinical management of this deadly disease. We found secretory annexin A3 (ANXA3) to be up-regulated in HCC cell lines, compared with immortalized normal liver cell line MIHA. Clinically, secretory ANXA3 levels in the serum were found to be progressively elevated from non-HCC individuals to early and advanced HCC patients, with up-regulation of serum ANXA3 to be tightly associated with gender, number of tumor nodules, tumor sizes and tumor stages. More importantly, the diagnostic value of secretory ANXA3, as evident by ROC analysis, was found to be more superior to the commonly used biomarker alpha-fetoprotein (AFP). Subsequent functional studies found secretory ANXA3 to promote cancer and stem cell-like properties of HCC cells. Co-culture of conditioned medium from ANXA3-overexpressing cells with ANXA3-absent HCC cells potentiated the abilities of HCC cells to migrate, invade, self-renew, induce tube formation in endothelial cells, and resist apoptosis induced by staurosporine and chemotherapy. The in vivo tumor-initiating capacity was also significantly enhanced in HCC cells when co-cultured with ANXA3-containing conditioned medium compared with conditioned medium from cells transfected with empty vector control. Similar functional properties were also observed when MIHA cells were treated with recombinant ANXA3 proteins. In an effort to characterize the molecular mechanism by which ANXA3 drives cancer and stem cell-like properties in HCC, cDNA microarray profiling was performed to compare the gene expression between ANXA3-depleted and control cells. Pathway enrichment analysis found many of the deregulated genes to be closely associated with the JNK/AP-1 signaling cascade. Activation of JNK pathway was detected when cells were treated with recombinant ANXA3 proteins or ANXA3-containing conditioned medium. Collectively, our findings suggest that secretory ANXA3 plays pivotal roles in promoting tumor initiation, growth and chemoresistance through the activation of JNK signaling pathway in HCC.