Gene expression profile of epithelial-mesenchymal transition mediators in papillary thyroid cancer.

PURPOSE: Platelet derived growth receptor alpha (PDGFRA) promotes the epithelial-mesenchymal transition (EMT) in thyroid follicular cells and is linked to lymphatic metastases in papillary thyroid cancer (PTC). We probed the regulatory network of genes linked to PDGFRA and EMT, comparing matched patient primary tumor and metastatic specimens, as well as engineered cell lines and ex vivo primary cultures with and without PDGFRA. METHODS: Freshly isolated thyroid tumors with or without metastases, with matching neighboring benign or normal tissue, was isolated for comparative transcriptional analysis using a TaqMan Low Density array (TLDA) assay with genes representing important markers of EMT, cellular adhesion, apoptosis, differentiation, senescence, and signal transduction pathways in thyroid cancer. Transfected primary cultures and immortalized cell lines were also analyzed with respect to PDGFRA expression and cell phenotype. RESULTS: We reveal the consistent upregulation of serine protease DPP4 and structural protein SPP1 with the progression of PTC to metastatic disease, as well as with PDGFRA expression. Conversely, epithelial integrity gene TFF3 and transcription factor SOX10 were strongly down-regulated. This gene network also includes important mediators of EMT including DSG1, MMP3, MMP9, and BECN. We observed similar genomic changes in ex vivo normal thyroid cells transfected with PDGFRA that also exhibited a partially dedifferentiated phenotype. In particular, we observed lamellopodia with induction of PDGFRA and illustrate that DPP4 and SPP1 were upregulated in this process, with decreased TFF3 and SOX10 as seen in tissue specimens. PDGFRA did decrease nuclear protein levels of differentiation factor TTF1, but not the transcription of TTF1 and PAX8. CONCLUSIONS: We demonstrate that PDGFRA activates EMT pathways and decreases expression of genes favoring epithelial integrity, pushing follicular cells toward a dedifferentiated phenotype. SPP1 and DPP4, previously linked with adverse outcomes in thyroid cancer, appear to be regulated by PDGFRA. PDGFRA expression promotes metastatic disease through multiple EMT levers that favor formation of an invasive phenotype and increased metalloproteinase expression.

Autocrine activation of platelet-derived growth factor receptor α in metastatic papillary thyroid cancer.

Metastatic dissemination of papillary thyroid cancer has been reported to be strongly associated with expression of platelet-derived growth factor (PDGFR) α and altered TTF1 function. However, the status of PDGF ligands in papillary thyroid cancer and the potential role of these ligands in metastatic disease are obscure. We assessed the prevalence of PDGF ligands in benign and malignant thyroid tumors to determine if ligand upregulation is associated with α-isoform (PDGF-AA or PDGF-BB) or the ß-isoform (PDGF-BB or PDGF-DD) of PDGFR in individual tumors. The immunohistochemical expression of PDGFRα, PDGF-AA, PDGF-BB, and PDGF-DD was surveyed in follicular adenomas (n=55), papillary thyroid carcinomas (103 with and 59 without nodal metastases), and lymph node metastasis (n=12). There is an augmented tendency for PDGF-AA expression in node-positive papillary thyroid cancer metastases (P<.0001). Although PDGF-BB and -DD were commonly identified, there was no relationship between the presence of these cytokines and malignant disease or metastases. Logistic regression demonstrated that PDGF-AA expression was significantly associated with the presence of PDGFRα (odds ratio=4.6, P=.004) and recurrent disease. When either PDGFRα or PDGF-AA was used to predict the presence of metastases, the sensitivity achieved was 86% and 88%, respectively, whereas specificities were lower at 71% and 61%, respectively. The augmented coexpression of PDGF-AA and PDGFRα in metastatic papillary thyroid cancers suggests that an autocrine signaling loop may contribute to nodal infiltration. Combined testing for the expression of PDGF-AA and PDGFRα may identify those patients with papillary thyroid cancer at risk of metastatic disease and resistance to therapy.

Hypoxia-increased expression of genes involved in inflammation, dedifferentiation, pro-fibrosis, and extracellular matrix remodeling of human bladder smooth muscle cells.

Partial bladder outlet obstruction (pBOO) is characterized by exaggerated stretch, hydrodynamic pressure, and inflammation which cause significant damage and fibrosis to the bladder wall. Several studies have implicated hypoxia in its pathophysiology. However, the isolated progressive effects of hypoxia on bladder cells are not yet defined. Sub-confluent normal human bladder smooth muscle cells (hbSMC) were cultured in 3% O2 tension for 2, 24, 48, and 72 h. RNA, cellular proteins, and secreted proteins were used for gene expression analysis, immunoblotting, and ELISA, respectively. Transcription of hypoxia-inducible factor (HIF)1α and HIF2α were transiently induced after 2 h of hypoxia (p < 0.05), whereas HIF3 was upregulated after 72 h (p < 0.005). HIF1 and HIF3α proteins were significantly induced after 2 and 72 h, respectively. VEGF mRNA increased significantly after 24 and 72 h (p < 0.005). The inflammatory cytokines, TGFB (protein and mRNA), IL 1ß, 1L6, and TNFα (mRNA) demonstrated a time-dependent increased expression. Furthermore, the anti-inflammatory cytokine IL-10 was downregulated after 72 h (p < 0.05). Evidence of smooth muscle cell dedifferentiation included increased αSMA, vimentin, and desmin. Evidence of pro-fibrotic changes included increased CTGF, SMAD 2, and SMAD 3 as well as collagens 1, 2, 3, and 4, fibronectin, aggrecan, and TIMP 1 transcripts (p < 0.05). Total collagen proteins also increased time-dependently (p < 0.05). Together, these results show that exposure of hbSMC to low oxygen tension results in intense hypoxic cascade, including inflammation, de-differentiation, pro-fibrotic changes, and increased extracellular matrix expression. This elucidates mechanisms of hypoxia-driven bladder deterioration in bladder cells, which is important in tailoring in vivo experiments and may ultimately translate into improved clinical outcomes.

PDGFRα Regulates Follicular Cell Differentiation Driving Treatment Resistance and Disease Recurrence in Papillary Thyroid Cancer.

Dedifferentiation of follicular cells is a central event in resistance to radioactive iodine and patient mortality in papillary thyroid carcinoma (PTC). We reveal that platelet derived growth factor receptor alpha (PDGFRα) specifically drives dedifferentiation in PTC by disrupting the transcriptional activity of thyroid transcription factor-1 (TTF1). PDGFRα activation dephosphorylates TTF1 consequently shifting the localization of this transcription factor from the nucleus to the cytoplasm. TTF1 is required for follicular cell development and disrupting its function abrogates thyroglobulin production and sodium iodide transport. PDGFRα also promotes a more invasive and migratory cell phenotype with a dramatic increase in xenograft tumor formation. In patient tumors we confirm that nuclear TTF1 expression is inversely proportional to PDGFRα levels. Patients exhibiting PDGFRα at time of diagnosis are three times more likely to exhibit nodal metastases and are 18 times more likely to recur within 5years than those patients lacking PDGFRα expression. Moreover, high levels of PDGFRα and low levels of nuclear TTF1 predict resistance to radioactive iodine therapy. We demonstrate in SCID xenografts that focused PDGFRα blockade restores iodide transport and decreases tumor burden by >50%. Focused PDGFRα inhibitors, combined with radioactive iodine, represent an additional avenue for treating patients with aggressive variants of PTC.

Platelet-derived growth factor receptor-α promotes lymphatic metastases in papillary thyroid cancer.

Lymph node metastases are common in papillary thyroid cancer (PTC) and can be resistant to surgical extirpation or radioiodine ablation. We examined the role of platelet-derived growth factor receptor (PDGFR) in mediating lymph node metastases in PTC. Clinical specimens of PTC (n = 137) were surveyed in a tissue array and by western blots to examine the relationship between expression of the α and ß subunits of PDGFR and lymph node metastases. PDGFR-α was found at high levels in primary tumours with known lymphatic metastases but not in those tumours lacking nodal involvement (p < 0.0001). However, PDGFR-ß expression was not linked to metastatic disease (p = 0.78) as it was found in virtually all PTC specimens. A matching analysis in fresh PTC specimens (n = 13) confirmed that PDGFR-α expression was strongly linked to metastatic spread (p = 0.0047). PDGFR-α and -ß were not found in normal thyroid tissue (p < 0.0001). PTC cell lines selectively expressing PDGFR-α or -ß were assessed for invasive potential and activation of downstream signal transduction pathways. PTC cell lines expressing PDGFR-α responded to PDGF-BB stimulation with increased invasive potential and this process can be blocked by the tyrosine kinase receptor inhibitor sunitinib (p < 0.009). Cell lines with only PDGFR-ß, or no PDGFR, did not show significant changes in invasive potential. Activation of PDGFR-α led to downstream up-regulation of both the MAPK/ERK and PI3K/Akt pathways and disruption of either pathway is sufficient to block PDGFR-mediated increases in invasive potential. Thus, PDGFR-α is associated with lymph node metastases in papillary thyroid carcinoma and PDGFR-α promotes increased invasive potential in PTC cell lines. PDGFR-α is a strong candidate for a diagnostic biomarker to identify patients at risk of nodal metastases. Our results also strengthen the rationale for selection of tyrosine kinase receptor inhibitors that target PDGFR in the treatment of progressive, metastatic PTC.