Cancer-Type Organic Anion Transporting Polypeptide 1B3 Is Localized in Lysosomes and Mediates Resistance against Kinase Inhibitors

Abstract

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3), a splice variant of the hepatic uptake transporter OATP1B3 (liver-type; Lt-OATP1B3), is expressed in several tumor entities including colorectal carcinoma (CRC) and breast cancer. In CRC, high OATP1B3 expression has been associated with reduced progression-free and overall survival. Several kinase inhibitors used for antitumor treatment are substrates and/or inhibitors of OATP1B3 (e.g. encorafenib, vemurafenib). The functional importance of Ct-OATP1B3 has not been elucidated so far. HEK293 cells stably overexpressing Ct-OATP1B3 protein were established and compared with control cells. Confocal laser scanning microscopy, immunoblot, and proteomics-based expression analysis demonstrated that Ct-OATP1B3 protein is intracellularly localized in lysosomes of stably-transfetced cells. Cytotoxicity experiments showed that cells recombinantly expressing the Ct-OATP1B3 protein were more resistant against the kinase inhibitor encorafenib compared to control cells [e.g. encorafenib (100 µM) survival rates: 89.5% vs. 52.8%]. In line with these findings, colorectal cancer DLD1 cells endogenously expressing Ct-OATP1B3 protein had poorer survival rates when the OATP1B3 substrate bromosulfophthalein (BSP) was coincubated with encorafenib or vemurafenib compared to the incubation with the kinase inhibitor alone. This indicates a competitive inhibition of Ct-OATP1B3mediated uptake into lysosomes by BSP. Accordingly, mass spectrometry-based drug analysis of lysosomes showed a reduced lysosomal accumulation of encorafenib in DLD1 cells additionally exposed to BSP. These results demonstrate that Ct-OATP1B3 protein is localized in the lysosomal membrane and can mediate transport of certain kinase inhibitors into lysosomes revealing a new mechanism of resistance.

Transcriptional Regulation of Liver-Type OATP1B3 (Lt-OATP1B3) and Cancer-Type OATP1B3 (Ct-OATP1B3) Studied in Hepatocyte-Derived and Colon Cancer-Derived Cell Lines

“…The Ct-OATP1B3 protein results from alternative splicing of the SLCO1B3 gene at which the Ct-SLCO1B3 gene possesses a first exon (designated as exon 1*), located in intron three of the Lt-SLCO1B3 gene [ 15 ]. Recently, we could demonstrate that in contrast to the plasma membranous localization of the Lt-OATP1B3 protein, the Ct-OATP1B3 variant is localized in the lysosomal membrane mediating the transport of substances from the intracellular compartment into these vesicles [ 17 ].…”

“…Total RNA isolation and the quantitative RT-PCR (qRT-PCR) was performed according to [ 17 ]. In brief, 1 × 10 7 cells were seeded on 10 cm cell culture dishes and the total RNA was isolated using the NucleoSpin RNA Plus Kit (MACHEREY-NAGEL GmbH & Co. KG; Düren, Germany).…”

“…The Ct-OATP1B3 protein encoded by this splice variant lacks the first 28 amino acids of the Lt-OATP1B3 protein [ 16 ]. Recently, we could demonstrate that in contrast to the localization of the Lt-OATP1B3 protein in the plasma membrane, the Ct-OATP1B3 protein is localized intracellularly in the membrane of lysosomes [ 17 ]. There, Ct-OATP1B3 is likely to mediate the transport of substances (e.g., the kinase inhibitor encorafenib) into lysosomes, reducing the cytoplasmic concentration of these drugs.…”

The Role of Solute Carrier Transporters in Efficient Anticancer Drug Delivery and Therapy

“…Furthermore, confirmation of the drug transporter localization on the plasma membrane is of great importance. For example, the cancer-type OATP1B3 transporter has a high expression in colorectal carcinomas, but its expression is mainly detected in lysosomes, and, thus, cannot facilitate the cell uptake of its substrates [ 155 , 156 ]. Similarly, knowledge of the PTMs of transporters in cancer cells is crucial due to their effect on the transporter function [ 157 ].…”

Understanding and targeting resistance mechanisms in cancer

“…Haberkorn et al. recently discovered that cancer‐type OATP1B3 protein, a splice variant of liver‐type OATP1B3, is localized in the lysosomal membrane of colorectal cancer (CRC) cells and contributes to the transport of encorafenib and vemurafenib into lysosomes, resulting in decreased drug concentrations in the cytoplasm and reduced drug efficacy 115 . Therefore, it is necessary to identify the genotype of intratumorally expressed SLCs in the process of confirming their roles in drug resistance.…”