Clinical relevance, functional significance and therapeutic implication of annexin A3 in CD133⁺ liver cancer stem cells driven hepatocellular carcinoma

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, all of which have been reported to be associated with stem cell-like behaviors of cancer cells. We and others have previously reported that the tumor growth of HCC is fuelled in part by a subset of cells called cancer stem cells (CSCs) marked by the CD133 phenotype and bearing features that include the ability to self-renew, differentiate, initiate tumors in vivo and resist chemotherapy. Despite our growing knowledge of the importance of 〖CD133〗^+ liver CSCs in hepatocarcinogenesis, there is still a lack of diagnostic markers and therapeutic strategies targeted specifically at this subset of cells. Therefore, the identification of valid targets and the development of new therapies are urgently needed for the more effective clinical management of this deadly disease.

By RNA Sequencing profiling of sorted 〖CD133〗^+ liver CSCs and 〖CD133〗^- HCC cells isolated from Huh7 and PLC8024, we identified annexin A3 (ANXA3) to be the most commonly up-regulated gene which encodes for secretory proteins in the 〖CD133〗^+ subset. This observation was further validated in CD133 sorted cells isolated from additional HCC cell lines and clinical samples. Clinically, up-regulation of both endogenous and secretory ANXA3 was found to be tightly associated with advanced tumor stages. Importantly, the diagnostic value of secretory ANXA3 was found to be more superior to the commonly used biomarker alpha-fetoprotein (AFP). Subsequent functional studies involving lentiviral-based knockdown and overexpression found endogenous ANXA3 to regulate cancer and stem cell-like properties including tumor initiation, migration, invasion, metastasis, angiogenesis, self-renewal and resistance to chemotherapy and apoptosis. Likewise, co-culture of ANXA3-containing conditioned medium or recombinant ANXA3 potentiated cancer and stem cell-like properties in HCC cells. In view of the clinical significance and functional relevance of ANXA3 in HCC, we subsequently developed a novel monoclonal antibody specific against ANXA3 and tested its ability to inhibit oncogenic functions. Results from our in vitro functional studies showed that anti-ANXA3 monoclonal antibody could reduce the ability of HCC cells to confer cancer and stem cell-like features, while administration of the neutralizing antibody in combination with cisplatin in immunodeficient mice bearing HCC xenografts resulted in a significant reduction in tumor growth and self-renewal, concomitant with a decrease in CD133 expression. The mechanistic pathway by which ANXA3 drives HCC was subsequently elucidated. We found internalization of secretory ANXA3 in HCC cells to be dependent on caveolaemediated endocytosis. Gene expression profiling and molecular studies found internalized ANXA3 to drive HCC through a deregulated JNK pathway. This observation was further substantiated by rescue experiments involving a JNK inhibitor. Taken together, ANXA3 exerts oncogenic effects in 〖CD133〗^+ liver CSCs via the JNK pathway and anti-ANXA3 neutralizing antibody holds therapeutic potential for HCC patients. We believe that ANXA3 could serve as a novel diagnostic biomarker and therapeutic target in HCC.