Yonsei Med J.  2015 Nov;56(6):1503-1514. 10.3349/ymj.2015.56.6.1503.

Hypoxia Induces Epithelial-Mesenchymal Transition in Follicular Thyroid Cancer: Involvement of Regulation of Twist by Hypoxia Inducible Factor-1alpha

• Yang YJ
• Na HJ
• Suh MJ
• Ban MJ
• Byeon HK
• Kim WS
• Kim JW
• Choi EC
• Kwon HJ
• Chang JW
• Koh YW

• KMID: 2345876
• DOI: http://doi.org/10.3349/ymj.2015.56.6.1503

Abstract

PURPOSE
Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poor prognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroid cancer. We showed that hypoxia inducible factor-1alpha (HIF-1alpha) induced aggressiveness in FTC cells and identified the underlying mechanism of the HIF-1alpha-induced invasive characteristics.
MATERIALS AND METHODS
Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on HIF-1alpha, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-time PCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functional character of EMT. The involvement of HIF-1alpha and Twist in EMT were studied using gene overexpression or silencing. After orthotopic nude mouse model was established using the cells transfected with lentiviral shHIF-1alpha, tissue analysis was done.
RESULTS
Hypoxia induces HIF-1alpha expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentin expression. We showed that overexpression of HIF-1alpha via transfection resulted in the aforementioned changes without hypoxia, and repression of HIF-1alpha with RNA interference suppressed hypoxia-induced HIF-1alpha and EMT. Furthermore, we also observed that Twist expression was regulated by HIF-1alpha. These were confirmed in the orthotopic FTC model.
CONCLUSION
Hypoxia induced HIF-1alpha, which in turn induced EMT, resulting in the increased capacity for invasion and migration of cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategy to prevent invasive and metastatic FTC.

Keyword

Hypoxia; hypoxia inducible factor-1alpha; epithelial-mesenchymal transition; Twist; orthotopic thyroid cancer model; follicular thyroid cancer

MeSH Terms

Adenocarcinoma, Follicular/*genetics/metabolism
Animals
Anoxia/*genetics
Cadherins/genetics
Epithelial-Mesenchymal Transition/*genetics
Gene Expression Regulation, Neoplastic
Hypoxia-Inducible Factor 1, alpha Subunit/*genetics/metabolism
Lymphokines
Mice
Neoplasm Invasiveness
Phenotype
Real-Time Polymerase Chain Reaction
Signal Transduction/drug effects
Thyroid Neoplasms/*genetics/metabolism
Transcriptional Activation
Twist Transcription Factor/*genetics/metabolism
Vimentin/metabolism
Cadherins
Hypoxia-Inducible Factor 1, alpha Subunit
Lymphokines
Twist Transcription Factor
Vimentin

Figure

• 

  Fig. 1 Hypoxia-induced HIF-1α expression in cell lines derived from different types of thyroid cancers. All cells were harvested after 16 h under normoxic or hypoxic conditions (1% O2), and HIF-1α expression was analyzed using the following methods. (A) Western blot analysis of HIF-1α in thyroid cancer cell lines derived from follicular (FTC133 and ML1), papillary (BCPAP and TPC1), and anaplastic (8505C and FRO) carcinomas. (B) Total RNA isolated from each selected thyroid cancer cell line. Cell lines with high protein expression of HIF-1α under hypoxic conditions (FTC133, TPC1, 8505C) were analyzed for the presence of HIF-1α transcripts using real-time PCR with primers specific for HIF-1α. (C) Immunofluorescence staining of HIF-1α expression in thyroid cancer cells. Cells were incubated under normoxic or hypoxic conditions for 16 hours, followed by immunofluorescence analysis. HIF-1α, green; DAPI, blue. Hypoxia-induced HIF-1α expression was highest in FTC133 cells. Scale bar=50 µm. Each figure is representative of triplicate experiments. FTC, follicular thyroid cancer; PTC, papillary thyroid cancer; ATC, anaplastic thyroid cancer; HIF-1α, hypoxia inducible factor-1α.

• 

  Fig. 2 Hypoxia-induced EMT in thyroid cancer cell lines (FTC133, TPC1, and 8505C). All cells were incubated under normoxia or hypoxia (1% O2) for 16 h. (A) External cell morphology was analyzed under a phase-contrast microscope. After exposure to hypoxic conditions, cells appeared to be flatter, elongated, and ramified with long extending processes. Loss of cell-to-cell contact was also noted after hypoxia (×200). Scale bar=50 µm. (B) Western blot analysis of signals related to EMT using the indicated antibodies revealed that the expression of a general epithelial marker (E-cadherin) decreased and that of mesenchymal markers (N-cadherin and vimentin) increased under hypoxic conditions. In addition, a downstream transcription factor, Twist, was also increased after hypoxia exposure. (C) Immunofluorescence assays using Alexa 568 (red)- or Alexa 488 (green)-labeled antibodies (1:250) to visualize E-cadherin (red) or vimentin (green), respectively, in each cell line; DAPI was used to label cell nuclei (blue). Changes consistent with the Western blotting results were noted using confocal microscopy and were most intense in FTC133 cells. Scale bar=50 µm. Each figure is representative of triplicate experiments. HIF-1α, hypoxia inducible factor-1α; FTC, follicular thyroid cancer; EMT, epithelial-to-mesenchymal transition. (D) Transwell invasion assays were performed to evaluate the invasive ability of cells. FTC133, TPC1, and 8505C cells were seeded on a filter (pore size, 8 coated µm) coated with Matrigel in the upper compartment and exposed to a normoxic or hypoxic environment. After 12 h, the cells in the pores or cells attached to the undersurface of the membrane were counted, while those attached to the lower section were stained with crystal violet (panel) and counted using a light microscope (graph) (×200). Scale bar=250 µm. The data represent mean±SD of three independent experiments. **p<0.01. EMT, epithelial-to-mesenchymal transition; FTC, follicular thyroid cancer.

• 

  Fig. 3 HIF-1α overexpression promotes EMT in FTC133 cells even under normoxia. FTC133 cells were transfected transiently with either empty vector (Control) or pcDNA3-HIF-1α (pcHIF-1α) and maintained under normoxia. (A) After a 48-hr transfection period, morphological changes in cells were analyzed under a phase-contrast microscope (×200). HIF-1α overexpression induced typical EMT morphologic changes, including increased cytoplasmic projection (black arrowheads). Scale bar=50 µm. E-cadherin, vimentin, and Twist expression was evaluated by (B) Western blot analysis, (C) real-time PCR using GAPDH as an internal control, and (D) immunofluorescence analysis in HIF-1α-transfected FTC133 cells. DAPI was used to label cell nuclei (blue). Scale bar=50 µm. (E) Transwell invasion assay results revealed that HIF-1α overexpression significantly increased the invasive ability of FTC133 cells. The panel is representative of triplicate experiments. Scale bar=250 µm. Graphs show quantification of the transwell assay. The data represent mean±SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. HIF-1α, hypoxia inducible factor-1α; EMT, epithelial-to-mesenchymal transition; FTC, follicular thyroid cancer.

• 

  Fig. 4 Knockdown of HIF-1α in FTC133 cells reverses hypoxia-induced EMT. Stable silencing of HIF-1α was performed using shRNA lentiviral particles (shHIF-1α). The lentiviral vector was used as the empty control vector (LVcontrol). All cells were incubated under normoxia or hypoxia (1% O2) for 16 h. (A) shHIF-1α transduction repressed hypoxia-induced HIF-1α activation in FTC133 cells. HIF-1α and EMT marker (E-cadherin, vimentin, and Twist) expression was evaluated by (B) Western blot analysis, (C) real-time PCR using GAPDH as an internal control, and (D) immunofluorescence analysis in HIF-1α knockdown FTC133 cells. DAPI was used to label cell nuclei (blue). Scale bar=20 µm. (E) Transwell invasion assay and (F) scratch-wound healing assay revealed that HIF-1α suppression significantly decreased the invasion and migration of FTC133 cells. Each panel was representative of triplicate experiments. Scale bars=250 µm and 200 µm, respectively. Graphs show quantification of each assay. The data represent mean±SD of three independent experiments. *p<0.05, **p<0.01. HIF-1α, hypoxia inducible factor-1α; EMT, epithelial-to-mesenchymal transition; FTC, follicular thyroid cancer; LVcontrol, lentiviral control; NS, not significant.

• 

  Fig. 5 Knockdown of HIF-1α by specific shRNA reduces EMT in an orthotopic FTC murine model. To determine whether the aforementioned results were evident in vivo, stable shHIF-1α FTC133 cells (1×105/mouse) were injected into the right side of the thyroid gland in 6-week-old male nude mice (n=5). Twenty-eight days after inoculation with FTC133 cells, tumors were harvested for Western blotting and histologic evaluations including H&E staining and immunohistochemical analysis. (A) Representative image of tumors. Tumors derived from shHIF-1α cells showed significantly decreased tumor volume and weight compared with that from LVcontrol cells. Scale bar=10 mm. (B) H&E stain of tumor derived LVcontrol cells showed more invasive features than that from shHIF-1α cells. Upper panel (×100): tumors derived from shHIF-1α cells had a clear boundary between the tumor and adjacent nontumor tissue (arrowheads), whereas tumors induced by LVcontrol cells demonstrated irregular invasive fronts. Lower panel (×200): tumor cell invasions into blood vessel forming tumor embolis (arrows) are noted in the tissue from LVcontrol group, whereas no definite tumor emboli in that from shHIF-1α group. Scale bar=50 µm. (C) Representative immunohistochemical results showed more intense staining of E-cadherin, but weaker staining of vimentin and Twist, supporting the in vitro results. Scale bar=100 µm. (D) Western blot analysis of HIF-1α, E-cadherin, vimentin, and Twist showed similar changes to those seen in vitro. **p<0.01, ***p<0.001. M, muscle; Tu, tumor; V, vessel; HIF-1α, hypoxia inducible factor-1α; EMT, epithelial-to-mesenchymal transition; FTC, follicular thyroid cancer; LVcontrol, lentiviral control; H&E, hematoxylin and eosin.

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