SLC7A8 coding for LAT2 is associated with early disease progression in osteosarcoma and transports doxorubicin

Abstract

Background: Despite (neo) adjuvant chemotherapy with cisplatin, doxorubicin and methotrexate, some patients with primary osteosarcoma progress during first-line systemic treatment and have a poor prognosis. In this study, we investigated whether patients with early disease progression (EDP), are characterized by a distinctive pharmacogenetic profile. Methods and Findings: Germline DNA from 287 Dutch high-grade osteosarcoma patients was genotyped using the DMET Plus array (containing 1,936 genetic markers in 231 drug metabolism and transporter genes). Associations between genetic variants and EDP were assessed using logistic regression models and associated variants (p <0.05) were validated in independent cohorts of 146 (Spain and United Kingdom) and 28 patients (Australia). In the association analyses, EDP was significantly associated with an SLC7A8 locus and was independently validated (meta-analysis validation cohorts: OR 0.19 [0.06-0.55], p = 0.002). The functional relevance of the top hits was explored by immunohistochemistry staining and an in vitro transport models. SLC7A8 encodes for the L-type amino acid transporter 2 (LAT2). Transport assays in HEK293 cells overexpressing LAT2 showed that doxorubicin, but not cisplatin and methotrexate, is a substrate for LAT2 (p < 0.0001). Finally, SLC7A8 mRNA expression analysis and LAT2 immunohistochemistry of osteosarcoma tissue showed that the lack of LAT2 expression is a prognostic factor of poor prognosis and reduced overall survival in patients without metastases (p = 0.0099 and p = 0.14, resp.). Conclusion: This study identified a novel locus in SLC7A8 to be associated with EDP in osteosarcoma. Functional studies indicate LAT2-mediates uptake of doxorubicin, which could give new opportunities to personalize treatment of osteosarcoma patients.

Keywords: L-type amino acid transporter 2; doxorubicin; early disease progression; osteosarcoma; pharmacogenetics.

FIGURE 1

Flowchart of discovery and validation phase of this study. Details on quality control of genetic data are shown in Supplementary Figure S1. DMET, drug metabolizing enzymes and transporters; DFS, disease free survival; LD, linkage disequilibrium; IHC, immunohistochemistry.

FIGURE 2

Western blot (A) and functional validation (B,C) of the transport assay in HEK293 cells overexpressing the L-type amino acid transporter 2 (LAT2). Figure A shows a western blot with the anti-LAT2 antibody. Bands are only visible in LAT2-HEK293 cells and LAT2-4F2-HEK293 cells. Figure B confirms ³H-L-alanine as a substrate of LAT2 and LAT2-4F2, after exposure to 0.018 µM of ³H-l-Alanine for 2 min at 37˚C. Data is expressed as mean ± SD (N = 3). Figure C shows L-alanine (10 µM) uptake, in the presence of cisplatin (1 mM), doxorubicin (1 mM), methotrexate (1 mM) or BCH (1 mM) , after 1 min incubation at 37˚C. BCH is a known inhibitor of LAT2-4F2 and was therefore included as a positive control. Inhibition was expressed as a percentage ± SD of L-alanine uptake with solvent control only, which was fixed at 100% (N = 3).

FIGURE 3

Transport assay to assess if methotrexate (A), doxorubicin (B) or cisplatin (C) are substrates for LAT2-4F2. HEK293 cells were incubated with methotrexate (500 µM), doxorubicin (500 µM) and cisplatin (330 µM) for 30 min at 37˚C. No significant difference in uptake of methotrexate or cisplatin was found in LAT2-4F2-HEK293 cells (p = 0.1, p = 0.0028, resp.) compared control cells. Doxorubicin uptake was significant in LAT2-2F4-HEK293 cells (p <0.0001). All data is expressed as mean ± SD (N = 3).

FIGURE 4

Overall survival of osteosarcoma patients according to or LAT2 protein expression (A), stratified for the presence of metastasis, or SLC7A8 mRNA expression (B,C). Protein expression was determined by immunohistochemistry in the discovery cohort of this study and no patients with LAT2 expression and metastases at diagnosis were identified. mRNA expression was analyzed in an independent patient cohort (Kuijjer et al., 2012).