A constitutive active MAPK/ERK pathway due to BRAFV600E positively regulates AHR pathway in PTC

Abstract

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor mediating the toxicity and tumor-promoting properties of dioxin. AHR has been reported to be overexpressed and constitutively active in a variety of solid tumors, but few data are currently available concerning its role in thyroid cancer. In this study we quantitatively explored a series of 51 paired-normal and papillary thyroid carcinoma (PTC) tissues for AHR-related genes. We identified an increased AHR expression/activity in PTC, independently from its nuclear dimerization partner and repressor but strictly related to a constitutive active MAPK/ERK pathway. The AHR up-regulation followed by an increased expression of AHR target genes was confirmed by a meta-analysis of published microarray data, suggesting a ligand-independent active AHR pathway in PTC. In-vitro studies using a PTC-derived cell line (BCPAP) and HEK293 cells showed that BRAFV600E may directly modulate AHR localization, induce AHR expression and activity in an exogenous ligand-independent manner. The AHR pathway might represent a potential novel therapeutic target for PTC in the clinical practice.

Keywords: BRAF; aryl hydrocarbon receptor; gene expression; meta-analysis; papillary thyroid cancer.

Figure 1. AHR expression in PTC

Box plots of relative qPCR gene expression measurements of AHR in either all PTCs A. only the BRAF^V600E B. or BRAF^wt C. PTCs and the relative paired normal tissues. Each value was referred to a pool of normal thyroid tissues that was set to 1. In D. the AHR expression of BRAF^V600E vs BRAF^wt PTCs is shown. For each sample the reported value represents the fold increase in tumoral specimen compared to its normal counterpart, which was set to 1. Boxes indicate the range from lower to upper quartile values, with the line inside the box representing the median. The vertical lines mark the highest and lowest value observed within a distance of 1.5 times the inter-quartile range from the bottom and the top of the boxes, respectively. Each dot represents a single sample. E. Representative Western blot analysis of normal/tumoral match-pair samples for the expression of AHR, phospho-ERK and total ERK. Samples were corrected for protein loading by β-Actin and the BRAF mutational status was reported on the top. AHR immunostaining in BRAF^V600E F. and in BRAF^wt G. PTC samples. Normal thyroid areas surrounding cancer cells are also visible. Original magnification x40.

Figure 2. AHR expression in thyroid tumors derived cell lines

The PTC-derived BCPAP and K1, the medullary thyroid cancer cell line TT, and the 8505C established from an undifferentiated thyroid carcinoma were evaluated for AHR expression at both mRNA A. and protein level B. Samples were corrected for protein loading by β-Actin. Error bars represent standard deviations.

Figure 3. Effect of BRAF^V600E on AHR expression/activity in HEK293 cells

A. HEK293 cells were co-transfected with the XRE-luc reporter plasmid and either the human Flag-tagged BRAF^V600E plasmid or the empty vector pcDNA3.1 and treated with increasing concentration of the BRAF inhibitor SB590885. The relative activity was adjusted for transfection efficiency using pRL-TK. Error bars represent standard deviation. ** reflects p < 0.01 value for luciferase activity in mock versus BRAF^V600E transfected cells at basal level; # § reflect p < 0.01 values for treated versus untreated cells. B. Representative Western blot analysis of AHR in HEK293 cells transfected with either BRAF^V600E or pcDNA3.1. β-Actin was used to normalize for loading variations. C. Immunofluorescence staining of Hoechst (blue), Flag (red) and AHR (green) in BRAF^V600E transfected HEK293. Only one of three cells in the picture is positive for Flag-tagged BRAF^V600E, for which an increased in nuclear AHR could be observed. All experiments were performed at least in triplicate.

Figure 4. Effect of BRAF inhibitors on AHR expression and activity in thyroid cell lines

A. Representative Western blot analysis of BCPAP, 8505C and K1 cells treated with IC50 doses of RAF265 and SB590885 for the expression of AHR, phospho-ERK and total ERK. Samples were corrected for protein loading by β-Actin. B. Relative luciferase activity of BCPAP cells transfected with XRE-luc and treated with RAF265 and SB590885 with the same concentration as above. Error bars represent standard deviation *p < 0.05, compared with a group given no drug treatment (one-way ANOVA). All experiments were performed at least in triplicate.