title: Expression of SLC22A1 variants may affect the response of hepatocellular carcinoma and cholangiocarcinoma to sorafenib.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), Biomedical Research Institute of Salamanca (IBSAL), University of Salamanca, Salamanca, Spain.
authors.forename: Elisa
authors.initials: E
authors.lastname: Herraez
authors.forename: Elisa
authors.initials: E
authors.lastname: Lozano
authors.forename: Rocio I R
authors.initials: RI
authors.lastname: Macias
authors.forename: Javier
authors.initials: J
authors.lastname: Vaquero
authors.forename: Luis
authors.initials: L
authors.lastname: Bujanda
authors.forename: Jesus M
authors.initials: JM
authors.lastname: Banales
authors.forename: Jose J G
authors.initials: JJ
authors.lastname: Marin
authors.forename: Oscar
authors.initials: O
authors.lastname: Briz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001650:Bile Duct Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D001653:Bile Ducts, Intrahepatic; D001706:Biopsy; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018281:Cholangiocarcinoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000627:therapeutic use; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D027702:Organic Cation Transporter 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010597:Pharmacogenetics; D010671:Phenylurea Compounds / Q000627:therapeutic use*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D000077157:Sorafenib; D016896:Treatment Outcome; D014982:Xenopus laevis
chemical_list: D000970:Antineoplastic Agents; D027702:Organic Cation Transporter 1; D010671:Phenylurea Compounds; D009536:Niacinamide; D000077157:Sorafenib