Reduced drug uptake is an important mechanism of chemoresistance. Down-regulation of SLC22A1 encoding the organic cation transporter-1 (OCT1) may affect the response of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CGC) to sorafenib, a cationic drug. Here we investigated whether SLC22A1 variants may contribute to sorafenib chemoresistance. Complete sequencing and selective variant identification were carried out to detect single nucleotide polymorphisms (SNPs) in SLC22A1 complementary DNA (cDNA). In HCC and CGC biopsies, in addition to previously described variants, two novel alternative spliced variants and three SNPs were identified. To study their functional consequences, these variants were mimicked by directed mutagenesis and expressed in HCC (Alexander and SK-Hep-1) and CGC (TFK1) cells. The two novel described variants, R61S fs*10 and C88A fs*16, encoded truncated proteins unable to reach the plasma membrane. Both variants abolished OCT1-mediated uptake of tetraethylammonium, a typical OCT1 substrate, and were not able to induce sorafenib sensitivity. In cells expressing functional OCT1 variants, OCT1 inhibition with quinine prevented sorafenib-induced toxicity. Expression of OCT1 variants in Xenopus laevis oocytes and determination of quinine-sensitive sorafenib uptake by high-performance liquid chromatography-dual mass spectrometry confirmed that OCT1 is able to transport sorafenib and that R61S fs*10 and C88A fs*16 abolish this ability. Screening of these SNPs in 23 HCC and 15 CGC biopsies revealed that R61S fs*10 was present in both HCC (17%) and CGC (13%), whereas C88A fs*16 was only found in HCC (17%). Considering all SLC22A1 variants, at least one inactivating SNP was found in 48% HCC and 40% CGC. Conclusion: Development of HCC and CGC is accompanied by the appearance of aberrant OCT1 variants that, together with decreased OCT1 expression, may dramatically affect the ability of sorafenib to reach active intracellular concentrations in these tumors. (Hepatology 2013;53:1065–1073)

Bile acid accumulation in liver with cholangiolar neoplastic lesions may occur before cholestasis is clinically detected. Whether this favors intrahepatic cholangiocarcinoma development has been investigated in this study. The E. coli RecA gene promoter was cloned upstream from Luc2 to detect in vitro direct genotoxic ability by activation of SOS genes. This assay demonstrated that bile acids were not able to induce DNA damage. The genotoxic effect of the DNA-damaging agent cisplatin was neither enhanced nor hindered by the hepatotoxic and hepatoprotective glycochenodeoxycholic and glycoursodeoxycholic acids, respectively. In contrast, thioacetamide metabolites, but not thioacetamide itself, induced DNA damage. Thus, thioacetamide was used to induce liver cancer in rats, which resulted in visible tumors after 30 weeks. The effect of bile acid accumulation on initial carcinogenesis phase (8 weeks) was investigated in bile duct ligated (BDL) animals. Serum bile acid measurement and determination of liver-specific healthy and tumor markers revealed that early thioacetamide treatment induced hypercholanemia together with upregulation of the tumor marker Neu in bile ducts, which were enhanced by BDL. Bile acid accumulation was associated with increased expression of interleukin (IL)-6 and downregulation of farnesoid X receptor (FXR). Bile duct proliferation and apoptosis activation, with inverse pattern (BDL > thioacetamide + BDL >> thioacetamide vs. thioacetamide > thioacetamide + BDL > BDL), were observed. In conclusion, intrahepatic accumulation of bile acids does not induce carcinogenesis directly but facilitates a cocarcinogenic effect due to stimulation of bile duct proliferation, enhanced inflammation, and reduction in FXR-dependent chemoprotection. Implications: This study reveals that bile acids foster cocarcinogenic events that impact cholangiocarcinoma. Mol Cancer Res; 12(1); 91–100. ©2013 AACR.