The mechanistic study of acquired resistance to apatinib in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of common cause of cancer mortality worldwide. The treatment for HCC has entered a new era with the development of molecular-targeted therapies since the breakthrough discovery of sorafenib in 2007. Sorafenib, a multi­kinase inhibitor, blocks tumor cell proliferation by specifically targeting multiple growth factor pathways, and it exerts an anti-angiogenic effect. However, the survival benefit of sorafenib treatment is unsatisfactory due to development of acquired drug resistance. Recently, increasing body of evidence has shown that apatinib, a novel inhibitor of vascular endothelial growth factor receptor-2 (VEGFR2), has good safety and potent therapeutic effect on Chinese patients with advanced HCC. Due to these encouraging results, apatinib has received approval for treatment of advanced HCC in China. Similar to other tyrosine kinase inhibitors, HCC cells also acquire drug resistance to apatinib, which hinders its efficacy. To increase the treatment efficacy of apatinib for HCC patients, combination therapy targeting multiple signaling pathways may serve as a better treatment option by potentially circumventing drug resistance. In this study, we first established apatinib-resistant HCC cells in vitro through stepwise increase in apatinib dosage for 6 months. We afterward have characterized the resistant nature of these cells by morphological analysis and functional assays like Annexin V staining, migration and invasion assays and Western blotting. After verification, we have identified the molecules/pathways associated with the resistance by comparing the genetic profiles between apatinib-resistant HCC cells and their mock counterparts by RNA sequencing analysis. Upon analysis, we discovered that CPY1A1, a family member of cytochrome P450 superfamily of enzymes, was commonly upregulated in apatinib-resistant HCC cells with the highest fold-change in expression levels, which was further confirmed by qPCR and western blot analyses. Consistently, CPY1A1 enzymatic activity was also found to be upregulated in the resistant cells. We have successfully repressed the expression and activity of CYP1A1 in HCC cells through lentiviral-based gene knockdown. The CYP1A1-knockdown cells showed increased sensitivity towards apatinib treatment in Annexin V staining. To further understand the molecular mechanism of how CYP1A1 sensitizes the effect of apatinib, RNA sequencing analysis was performed to compare the genetic profiles between CYP1A1-knockdown cells and their non-target control (NTC) counterparts. Upon Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) analyses, we found that knockdown of CYP1A1 suppressed xenobiotic and bile acid metabolism, RhoGDI, SPINK1 and calcium signaling pathways. All in all, our study not only showed the potential tumor suppressive effect of apatinib on HCC cells, but also provides a mechanistic insight for the development of acquired apatinib resistance in HCC cells. In conclusion, targeting CYP1A1 and its downstream pathways in combination with apatinib may be the potential therapeutic regimen for treatment of advanced HCC patients.