Gremlin2 promotes the enrichment of CD44+ cancer stem cells after transarterial chemoembolization in the treatment of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. While HCC patients at early stages are eligible for liver resection and liver transplantation, patients diagnosed at late stages can only be treated with palliative therapies. Transarterial chemoembolization (TACE) is the standard treatment for un-resectable HCC, involving the induction of tumor ischemia and chemotherapy. It aims to reduce tumor growth and improve patient survival. However, the tumor response rate of TACE is often unsatisfactory.

Recently, the rise of the ‘cancer stem cell’ model has opened a new field for cancer research. This novel theory postulates that only a small fraction of cells can initiate tumor and they are named cancer stem cells (CSCs). They are characterized with the abilities in tumor initiation, chemoresistance and enhanced stem cell properties. It is suggested that they are attributed to the resistance of standard chemotherapies. Therefore, exploring effective treatment strategies to specifically target and eliminate CSCs will be the key to tackling HCC.

To investigate the underlying mechanisms by which HCC resists TACE, in vivo TACE was performed in the mouse model with orthotopic HCC. Herein, the enrichment of CD44+ hepatic CSCs was observed in TACE-treated HCC tumors in vivo. Moreover, CD44+ HCC cells possessed enhanced tumor-initiating capacity and increased ability in spheroid formation. These results suggested that CD44+ HCC cells are a potential population of CSCs.

We also examined the possible mechanism by which TACE leads to the enrichment of CD44+ CSCs. The gene expression profile of TACE treated tumors in comparison to that of untreated tumors was examined by RNA sequencing. Among all of the differentially expressed genes, Gremlin2, which is a secreted BMP antagonist, was found to be up-regulated in all treatment groups and the most significant increase iGremlin2 expression was observed in TACE-treated samples. Furthermore, the oncogenic roles of Gremlin2 were studied. Exogenous application of human recombinant Gremlin2 resulted in the enrichment of CD44+ CSCs and increased the expression of several stemness genes including SOX2, OLIGO2, NANOG and OCT4 in HCC cell lines. In addition, treatment with Gremlin2 reduced the activation of both endogenous and BMP ligands-induced Smad1/5 signalling in a dose-dependent and time-dependent manner. Besides, Gremlin2-overexpressing HCC cells showed CSC phenotypes along with enhanced tumorigenic potential characterized by increased abilities in cell growth, cell migration and invasion. Moreover, Gremlin2 but no other BMP antagonists, ligands or receptors was preferentially expressed in CD44+ CSCs with compromised BMP signalling. More importantly, Gremlin2 could antagonize the BMP ligands-induced Smad1/5 activation in CD44+ CSCs. Taken together, these findings suggested that Gremlin2 controls the maintenance of CSCs by suppressing BMP signalling.

In conclusion, this study demonstrated that the upregulation of Gremlin2 resulted from TACE may contribute to the enrichment of CSCs through attenuation of BMP signalling, which consequently promotes the growth of HCC tumors. These findings revealed a novel therapeutic target for eradicating CSCs in the treatment of HCC.