Gene expression and functional evidence of epithelial-to-mesenchymal transition in papillary thyroid carcinoma invasion

Abstract

Papillary thyroid carcinomas (PTCs) that invade into local structures are associated with a poor prognosis, but the mechanisms for PTC invasion are incompletely defined, limiting the development of new therapies. To characterize biological processes involved in PTC invasion, we analyzed the gene expression profiles of microscopically dissected intratumoral samples from central and invasive regions of seven widely invasive PTCs and normal thyroid tissue by oligonucleotide microarray and performed confirmatory expression and functional studies. In comparison with the central regions of primary PTCs, the invasive fronts overexpressed TGF beta, NFkappaB and integrin pathway members, and regulators of small G proteins and CDC42. Moreover, reduced levels of mRNAs encoding proteins involved in cell-cell adhesion and communication were identified, consistent with epithelial-to-mesenchymal transition (EMT). To confirm that aggressive PTCs were characterized by EMT, 34 additional PTCs were examined for expression of vimentin, a hallmark of EMT. Overexpression of vimentin was associated with PTC invasion and nodal metastasis. Functional, in vitro studies demonstrated that vimentin was required both for the development and maintenance of a mesenchymal morphology and invasiveness in thyroid cancer cells. We conclude that EMT is common in PTC invasion and that vimentin regulates thyroid cancer EMT in vitro.

Fig. 1.

Analysis and confirmation of oligonucleotide expression microarrays. (A) Unsupervised cluster analysis of gene arrays comparing the ratios of center/normal and invasion/center was performed. Red indicates overexpression, and green indicates underexpression as determined by the ratio of center versus normal or invasion versus center for individual pairs. The total number of genes per cluster is denoted in parentheses. Columns are labeled as tumor number. (B) RUNX2, fibronectin 1 and vimentin immunohistochemistry of PTCs. Immunoactive RUNX2, fibronectin, and vimentin were low or absent in normal tissue and were up-regulated in tumors for each protein. Immunoactive RUNX2 and fibronectin 1 proteins were qualitatively increased the invasive regions of PTCs, whereas vimentin expression was similar in central and invasive areas.

Fig. 2.

Expression of mesenchymal proteins in PTC. (A) Immunostaining using antibodies against vimentin and osteopontin (OPN) was performed on thyroid samples from normal tissue, primary tumor, and nodal metastasis from individual patients. In comparison with normal tissue, vimentin and osteopontin were overexpressed in primary PTCs and in lymph node metastases in all examined cases. Representative data are shown. (B) Vimentin, OPN, and RUNX2 immunostaining was performed in PTCs that express RET/PTC or mutant BRAF as well as in patients whose tumors did not express either oncogene. Vimentin was overexpressed in all cases, whereas osteopontin and RUNX2 were overexpressed more dramatically in tumors with RET/PTC or a BRAF mutation.

Fig. 3.

EMT-related mRNA and protein expression in NPA, WRO, and ARO thyroid cancer cells. (A and B) Qualitative RT-PCR (A) and Western blots (B) demonstrate that only NPA cells express vimentin mRNA or protein. (C) Western blot reveals higher levels of osteopontin (OPN) and RUNX2 in the NPA cells. The arrow denotes the size of RUNX2.

Fig. 4.

Vimentin expression is required for thyroid cancer cell invasion. (A and B) Reduction of vimentin expression using siRNA versus scrambled sequence control is shown by immunofluorescent staining (A) and by Western blot analysis (B) using an anti-vimentin antibody. (C) NPA cell invasion is reduced by vimentin siRNA in comparison to control (scrambled sequence) transfectant. (D) Expression of vimentin in vimentin-null ARO cells induces a mesenchymal morphology (brown stained cells), similar to nontransfected NPA cells. (E) Vimentin expression in ARO cells increases their ability to invade in Boyden Chamber Assays.

Fig. 5.

Vimentin phosphorylation and perinuclear localization is associated with thyroid cancer cell invasion. Immunohistochemical staining was performed for ser-56 phospho–vimentin in NPA cells with endogenous vimentin expression (A) and in ARO cells transiently transfected with vimentin cDNA (B) before and after invasion. Immunofluorescense staining with nuclear envelope marker lamin A/C was performed for colocalization. In the overlap images, there is enhanced yellow staining, representing the colocalization of p-vimentin with lamin A/C in migrated cells.