3). Expression of wildtype OCT1 induced quinine-sensitive TEA uptake by HCC and CGC cells (Fig. 4A-C). This ability was also

observed in S14F, L160F, G401S, and P197S variants, whereas it was partly or completely lost in the rest of detected variants. To validate this transport assay, wildtype OCT1 was also expressed in frog oocytes. This maneuver markedly enhanced their ability to take up, in a quinine-sensitive manner, both TEA (Fig. 4D) and sorafenib (Fig. 4E). Moreover, the expression of the novel variants in this model also confirmed the lack of ability of R61S fs*10 and C88A fs*16 to transport sorafenib, which this website was maintained in P197S (Fig. 4E). The effect of SNPs on the targeting to the plasma membrane was investigated by immunodetection of the V5-tag placed in the constructs. In this set of experiments, we also included C88R

and S189L, whose effects on protein targeting were not known, and G465R, whose functional consequences are controversial. Although G465R has been described as a loss-of-function variant,[24] our results indicate that when expressed in HCC and CGC cells this variant has a reduced, but not abolished, OCT1-mediated transport (Fig. 4A-C). When G465R was investigated in Alexander cells, similarly to wildtype OCT1, it was targeted to the plasma membrane (Fig. 5). In contrast, both C88R and S189L were mainly localized intracellularly BAY 73-4506 molecular weight (Fig. 5). This was consistent with the abolished ability of the latter two variants to mediate TEA uptake (Fig. 4). Regarding the novel OCT1 variants, both R61S fs*10 and C88A fs*16, which encode truncated proteins (Fig. 2B), resulted in impaired targeting to the plasma membrane (Fig. 5) and lack of the Farnesyltransferase ability to mediate sorafenib uptake by oocytes (Fig. 4E) and TEA uptake by transfected cells (Fig. 4A-C). In contrast, the functional variant P197S resulted in an entire OCT1 protein targeted to the plasma membrane (Fig. 5). Based on studies addressing the dose- (Fig. 3) and time- (Supporting Fig. 1) dependent sensitivity of Alexander cells to sorafenib, short-term (6 hours) exposure

of HCC and CGC cells to sorafenib was carried out (Fig. 6). Under these conditions only OCT1 variants with a relatively well-preserved ability to mediate TEA transport (Fig. 4) were able to induce sensitivity to sorafenib in all cells assayed (Fig. 6). Regarding the SNPs identified here, P197S, but not R61S fs*10 or C88A fs*16, enhanced the sensitivity to sorafenib in cells expressing these variants (Fig. 6). Interestingly, OCT1 inhibition with quinine reduced, in a dose-dependent manner, the sensitivity to sorafenib due to the expression of functional variants of this transporter. Selective identification of loss-of-function SNPs was performed by RT-QPCR in a larger series of HCC and CGC biopsies (Table 2). The abundance of each variant was normalized by the abundance of total OCT1 mRNA.