DPPA2 is a potential marker for cancer stem cells in hepatocellular carcinoma

Abstract

Liver cancer is the second leading cause of tumor-related deaths worldwide and one of the few solid tumors with increasing rates in both incidence and mortality. Accounting for 90% of primary liver cancer, hepatocellular carcinoma (HCC) is difficult to diagnose at an early stage and frequently relapses after treatment due to the existence of a small subgroup of cells within the tumor that are resistant to chemotherapy and radiotherapy. Therefore, it is crucial to identify and eliminate these cells for tumor eradication. Previous studies suggested that therapy resistance was indeed an intrinsic characteristic of cancer stem cells (CSCs), along with the ability of self-renewal, differentiation, and tumor initiation. According to the CSC theory, tumor growth and progression are fueled by these small population of dedicated stem cells, which have been successfully identified in multiple types of cancer. Besides, CSCs reside at the apex of the developmental hierarchy, accounting for the heterogeneity of malignant tumors. This CSC theory inspires a new treatment concept that specifically targets CSCs rather than differentiated tumor cells. However, the developmental hierarchy of CSCs and the underlying mechanism are still poorly understood in most solid tumors. In light of the strong similarity between CSCs and embryonic stem cells (ESCs), we hypothesized that pluripotent genes, which control the differentiation of ESCs, might also play vital roles in tumor initiation, metastasis, and drug resistance in HCC. Using an in vitro model of hepatocyte differentiation and analyzing samples at multiple differentiation stages, we compared gene expression patterns with classical pluripotent genes, including SOX2 and OCT4. Developmental pluripotency factor-2 (DPPA2) was identified as a candidate pluripotent gene since it showed the highest expression at the undifferentiated stage and decreased dramatically upon differentiation. Clinically, DPPA2 was sporadically overexpressed in 28% (34/121) of HCC clinical samples and HCC cell lines at both RNA and protein levels. More importantly, the upregulation of DPPA2 was significantly correlated with shorter overall survival time by Kaplan Meier analysis. Based on further analysis of clinicopathologic features, high expression of DPPA2 was associated with more advanced tumor stages, more tumor nodules, poorer differentiation stages along with the existence of tumor thrombus. Regulatory factors for DPPA2 expression were also identified, including CpG methylation and sex hormone. Next, multiple functional studies related to stem cell-like properties were adopted. Results showed that exogenous introduction of DPPA2 expression in the nontumorigenic hepatocytes cell line MiHA and HCC cell line Hep3B could significantly improve cell ability of self-renewal, tumorigenic, metastasis, and resistance to chemotherapy. Furthermore, the overexpression of DPPA2 also enhanced the expression of stemness and EMT markers. Mechanistically, RNA-sequencing results and further validation by qRT-PCR suggested that DPPA2 could induce the expression of WNT9A potentially through binding to its promoter. In summary, DPPA2 is a potential functional marker for CSCs in HCC. This novel finding sheds light on the potential and importance of pluripotent proteins in tumor initiation, metastasis, and drug resistance. Further investigation might identify DPPA2 as a potential prognostic biomarker or therapeutic target for the treatment of HCC or other tumors.