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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Respiratory Research 1/2017

ABCB1 and ABCG2 drug transporters are differentially expressed in non-small cell lung cancers (NSCLC) and expression is modified by cisplatin treatment via altered Wnt signaling

Zeitschrift:
Respiratory Research > Ausgabe 1/2017
Autoren:
M. Vesel, J. Rapp, D. Feller, E. Kiss, L. Jaromi, M. Meggyes, G. Miskei, B. Duga, G. Smuk, T. Laszlo, I. Karner, J.E. Pongracz
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12931-017-0537-6) contains supplementary material, which is available to authorized users.

Abstract

Background

Lung cancer (LC) is still the most common cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all LC cases but is not a single entity. It is now accepted that, apart from the characteristic driver mutations, the unique molecular signatures of adeno- (AC) and squamous cell carcinomas (SCC), the two most common NSCLC subtypes should be taken into consideration for their management. Therapeutic interventions, however, frequently lead to chemotherapy resistance highlighting the need for in-depth analysis of regulatory mechanisms of multidrug resistance to increase therapeutic efficiency.

Methods

Non-canonical Wnt5a and canonical Wnt7b and ABC transporter expressions were tested in primary human LC (n = 90) resections of AC and SCC. To investigate drug transporter activity, a three dimensional (3D) human lung aggregate tissue model was set up using differentiated primary human lung cell types. Following modification of the canonical, beta-catenin dependent Wnt pathway or treatment with cisplatin, drug transporter analysis was performed at mRNA, protein and functional level using qRT-PCR, immunohistochemistry, immune-fluorescent staining and transport function analysis.

Results

Non-canonical Wnt5a is significantly up-regulated in SCC samples making the microenvironment different from AC, where the beta-catenin dependent Wnt7b is more prominent. In primary cancer tissues ABCB1 and ABCG2 expression levels were different in the two NSCLC subtypes. Non-canonical rhWnt5a induced down-regulation of both ABCB1 and ABCG2 transporters in the primary human lung aggregate tissue model recreating the SCC-like transporter pattern. Inhibition of the beta-catenin or canonical Wnt pathway resulted in similar down-regulation of both ABC transporter expression and function. In contrast, cisplatin, the frequently used adjuvant chemotherapeutic agent, activated beta-catenin dependent signaling that lead to up-regulation of both ABCB1 and ABCG2 transporter expression and activity.

Conclusions

The difference in the Wnt microenvironment in AC and SCC leads to variations in ABC transporter expression. Cisplatin via induction of canonical Wnt signaling up-regulates ABCB1 and ABCG2 drug transporters that are not transporters for cisplatin itself but are transporters for drugs that are frequently used in combination therapy with cisplatin modulating drug response.
Zusatzmaterial
Additional file 1: Figure S1. ABCG2 and Wnt7b immunohistochemistry images of primary adeno (AC) and squamous cell carcinoma (SCC) tissues. Representative images of ABCG2 (B) and Wnt7b (B) immunochemistry of primary AC and SCC tissues (n = 5, each). Scale bar is 100 μm at 20× magnification and 50 μm at 40× magnification images. (DOCX 26321 kb)
12931_2017_537_MOESM1_ESM.docx
Additional file 2: Figure S2. Establishing a 3D lung tissue aggregate co-culture to model drug transporter expression and activity. Relative mRNA expression of ABCB1 and ABCG2 drug transporters in A) 2D monocultures, 3D co-culture aggregates (NS: NHLF-SAEC, HNS: HMVEC-L-NHLF-SAEC), B) in 3D co-culture aggregate (HNS: HMVEC-L-NHLF-SAEC) and normal, healthy lung tissue. mRNA expression is relative to beta-actin. In 2D cultures drug transporter expressions are much lower than in the controls. These expression levels increase in 3D culture conditions and all three cell types are needed to become similar to normal lung tissue expression levels. Data are presented as mean ± SEM, n = 3 C) representative image of ABCG2 protein expression in 3D co-culture aggregate (HNS: HMVEC-L-NHLF-SAEC), scale bar 50 μm, magnification; D) functional activity of ABCB1 and ABCG2 drug transporters in 3D HMVEC-L-NHLF-SAEC co-culture aggregates. Data are presented as mean ± SEM of multidrug resistance activity factor values (MAF), n = 3. MAF values ≥ 20 are considered as active transporter function. (DOCX 655 kb)
12931_2017_537_MOESM2_ESM.docx
Additional file 3: Figure S3. Effects of cisplatin in 3D lung tissue tumor cell line aggregates. Relative mRNA expression of ABCC1 and ABCC2 drug transporters of cisplatin treatment of 3D co-culture aggregates of adenocarcinoma cell line A549-NHLF (A) and (B); 3D co-culture aggregates of squamous cell line H520-NHLF (C) and (D). Data are presented as mean±SEM, n=3. (DOCX 55 kb)
12931_2017_537_MOESM3_ESM.docx
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