Expression ofSLC22A1variants 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. 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;58:1065-1073 H epatocellular carcinoma (HCC) and cholangiocarcinoma (CGC) are important causes of cancer-related death worldwide. Although surgery is potentially curative for patients with localized disease, these tumors are often in an advanced stage at the time of diagnosis, when surgery is no longer the

Differential Expression of Drug Uptake and Efflux Transporters in Japanese Patients with Hepatocellular Carcinoma

“…Because the organic cation uptake transporters OCT1 and OCT3 (Yonezawa et al, 2006;Herraez et al, 2013;Swift et al, 2013) and the ABC efflux transporters Fig. 4.…”

“…In the context of HCC therapy, the uptake transporters organic cation transporter (OCT) 1 (encoded by the SLC22A1 gene) and OCT3 (SLC22A3) as well as the ATP-binding cassette (ABC) efflux transporters MDR1/P-glycoprotein (ABCB1), multidrug resistance protein (MRP) 2 (ABCC2), and breast cancer resistance protein (BCRP; ABCG2) are of particular interest. They transport and confer resistance to anthracyclines, platinum drugs, and sorafenib (Cui et al, 1999;Burger et al, 2004;Yonezawa et al, 2006;Gillet and Gottesman, 2010;Herraez et al, 2013;Swift et al, 2013), which are commonly used in the treatment of HCC (El-Serag et al, 2008;Llovet et al, 2012). An increased expression of uptake transporters and a decreased expression of efflux transporters would favor the accumulation of cytostatic drugs within the tumor cells.…”

Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

“…Substrates of OATP1A2, which is highly expressed in cholangiocytes, include methotrexate, taxanes and imatinib, whose uptake by CCA cells in vitro can be impaired by OATP1A2 downregulation 272 or the expression of less active genetic variants 273 . Uptake of cationic drugs (for example, platinum derivatives and tyrosine kinase inhibitors) is mediated in part by organic cationic transporters (OCT), which are downregulated (OCT3) 274 or very poorly expressed (OCT1) 275 in CCA. Gemcitabine and 5-fluorouracil are taken up through nucleoside transporters, equilibrative nucleoside transporters (ENT) and concentrative nucleoside transporters (CNT).…”

The lack of the organic cation transporter OCT1 at the plasma membrane of tumor cells precludes a positive response to sorafenib in patients with hepatocellular carcinoma

“…Due to frequent nonsense mutations and aberrant splicing, an important proportion of OCT1 mRNA found in HCC is expected to generate non-functional truncated peptides [13]. Thus, to analyze the presence of OCT1 in patient samples we have used two antibodies raised against the N-terminal region, i.e., the head of Patients (n=39) were diagnosed of suffering from hepatocellular carcinoma and were included in the study based on eligibility criteria described in detail in Method section.…”

“…The mechanism of action of sorafenib depends on its access to the intracellular site of action on transmembrane tyrosine kinase receptors, which may be affected by changes in the expression and activity of transporters accounting for its uptake. The organic cation transporter-1 (OCT1, gene symbol SLC22A1) has been suggested to play a major role in this process [13,14]. OCT1 functions as an electrogenic, sodium-and protonindependent bidirectional polyspecific transporter [15].…”