Aptamer-coupled Doxorubicin Overcomes Sorafenib Resistance in Hepatocellular Carcinoma

Abstract

Sorafenib is a potent multi-kinase inhibitor and is the only FDA-approved first-line agent for the treatment of advanced hepatocellular carcinoma (HCC). Liver cancer stem cells (LCSCs) represent a small subset of cells within the HCC tumour mass with an important role in the tumour initiation, progression, recurrence and metastasis. Importantly, LCSCs are also the key cell types that are responsible for the acquisition of sorafenib resistance in HCC. Hence, LCSCs are an ideal target for overcoming sorafenib resistance and HCC therapy. Aptamers (also known as ‘chemical antibodies’) are a group of single-stranded nucleic acid (DNA or RNA) oligonucleotides. As aptamers have distinctive features, particularly favourable tumourpenetrating capacities, low toxicity and immunogenicity, they have been frequently used to deliver therapeutic payloads to cancer cells, and have achieved encouraging anti-tumour effects in various cancers. In this study, we observed that two sorafenib-resistant HCC cell lines derived from Huh7 and Hep3B cells were highly enriched with LCSCs. We developed aptamers specific to LCSC surface marker epithelial cell adhesion molecule (EpCAM) and CD133. These aptamers were then conjugated with Doxorubicin (Dox) to generate the therapeutic complexes EpCAM-apt- Dox (EP-apt-Dox) and CD133-apt-Dox (CD-apt-Dox). We have demonstrated that these therapeutic complexes can be specifically delivered to the EpCAM+ or CD133+ cells in vitro and the xenograft tumours derived from the sorafenib-resistant HCC cells in nude mice. In vitro studies have shown that combination of either EP-apt-Dox or CD-apt-Dox with sorafenib could not only significantly inhibit the proliferation and colony-forming ability but also induce high rates of apoptosis in sorafenib-resistant HCC cells. In animal studies, EP-apt-Dox or CDapt- Dox could sensitize the resistant xenograft tumours to sorafenib treatment. Importantly, the combination of EP-apt-Dox or CD-apt-Dox with sorafenib could exert synergistic anti-tumour effect as demonstrated by significant reduction in the rate of tumour growth and metastasis, and a significant improvement in animal survival rate. These therapeutic effects are likely due to the successful interruption of the self-renewal capacity and ultimately the depletion of the LCSCs. Moreover, treatment of EP-apt-Dox or CD-apt-Dox exhibited much lower toxicity in normal organs (such as heart and liver) than free Dox or sorafenib alone. This study may provide novel evidence that LCSCs are a major subset of cells that are responsible for sorafenib resistance, and that aptamer-based targeting of LCSCs possesses a great potential for overcoming sorafenib resistance and improving treatment outcomes in patients with advanced HCC.