Increased expression of AP2 and Sp1 transcription factors in human thyroid tumors: a role in NIS expression regulation?

Abstract

Background: Sodium/iodide symporter (NIS) is a key protein in iodide transport by thyroid cells and this activity is a prerequisite for effective radioiodide treatment of thyroid cancer. In the majority of thyroid cancers, however, iodide uptake is reduced, probably as a result of decreased NIS protein expression.

Methods: To identify the mechanisms that negatively affect NIS expression in thyroid tumors, we performed electrophoresis mobility shift assays and immunoblot analysis of nuclear protein extracts from normal and tumoral thyroid tissues from 14 unrelated patients.

Results: Two proteins closely related to the transcription factors AP2 and Sp1 were identified in the nuclear extracts. Expression of both AP2 and Sp1 in nuclear extracts from thyroid tumors was significantly higher than that observed in corresponding normal tissues.

Conclusion: These observations raise the possibility that NIS expression, and subsequently iodide transport, are reduced in thyroid tumors at least in part owing to alterations in the binding activity of AP2 and Sp1 transcription factors to NIS promoter.

Figure 1

Competition for binding between thyroid nuclear proteins and AP1 and Sp1 consensus oligonucleotides. 212 bp NIS probe was 5' end-labeled and used in gel retardation assays with 10 μg of thyroid tissue extracts. Lanes 1 and 8: probe alone; lanes 2 and 9: probe plus nuclear extract from tumoral thyroid tissue; lane 3: probe plus nuclear extract from tumoral thyroid tissue in the presence of a 200-fold molar excess of unlabeled AP2 consensus oligonucleotide; lane 4: probe plus nuclear extract from tumoral thyroid tissue in the presence of a 200-fold molar excess of unlabeled AP2 mutant oligonucleotide, lane 5: probe plus nuclear extract from tumoral thyroid tissue in the presence of a 200-fold molar excess of unlabeled Sp1 consensus oligonucleotide; lane 6: probe plus nuclear extract from tumoral thyroid tissue in the presence of a 200-fold molar excess of unlabeled Sp1 mutant oligonucleotide; lane 7: probe plus nuclear extract from tumoral thyroid tissue in the presence of a 200-fold molar excess of unlabeled AP2 and Sp1 consensus oligonucleotides. In lanes 10 and 11 the probe was incubated with nuclear extract from tumoral thyroid tissue were incubated in the presence of a specific anti-AP2 (lane 10) or anti-Sp1 (lane 11) antibody, respectively, thus inducing a supershift of the complex. Arrows show the position of free (DNA) and bound (DNA-P) probes.

Figure 2

Western blot analysis of nuclear extracts from normal and cancer tissues, using monoclonal anti-AP2 and anti-Sp1 antibodies. The cancerous tissues displayed higher levels of both transcription factors, compared with normal thyroid tissues. A representative of three separated assays is shown. N = normal thyroid tissue; T = tumoral thyroid tissue.

Figure 3

Binding between the NIS promoter region and nuclear extracts from thyroid tissues and correlation with NIS RNA levels. The probe was incubated with nuclear extracts from normal and tumoral thyroid tissues in the presence of 0.2 μg poly(dI-dC) with, and DNA protein complexes were resolved as shown in Fig. 1. Lanes: P, probe alone; N, probe plus nuclear extract from normal thyroid tissue; T, probe plus nuclear extract from cancer thyroid tissue. The levels of the NIS mRNA were assessed by a semi-quantitative RT-PCR based method, as previously described (12) and expressed as mean ± S.E.M. of three different experiments.