The role of Thyroid Transcription Factor-1 and Tumor differentiation in Resected Lung Adenocarcinoma

Abstract

To investigate the role of thyroid transcription factor-1 (TTF-1) and tumor differentiation in resected lung adenocarcinoma. A total of 520 patients with clinical early stage lung adenocarcinoma who underwent surgical resection were reviewed retrospectively. Clinical data and outcomes were evaluated with an average follow-up of 117 months. The results were validated via lung cancer cell line studies. The clinical parameters did not differ between relapse and nonrelapse patients. Exceptions were tumor differentiation, lymphovascular space invasion, F¹⁸-fluorodeoxyglucose maximum standard uptake value, tumor size, and pathological stage (p < 0.001). Poor tumor differentiation was the independent prognostic factor (odds ratio: 2.937, p = 0.026). The expression of TTF-1 was correlated with tumor differentiation in resected lung adenocarcinoma patients (p < 0.001). Five-year survival was 60.0% for score 1 TTF-1 expression patients, 80.1% for score 2 TTF-1 expression patients, and 86.1% for score 3 TTF-1 expression group patients. The lung cancer cell line study of knockdown and overexpression of TTF-1 revealed TTF-1 mediated High Mobility Group AT-Hook 2 (HMGA2) protein involved with epithelium-mesenchymal transformation. The chromatin immunoprecipitation revealed TTF-1 regulated HMGA2 via direct binding. TTF-1/HMGA2 axis was associated with tumor differentiation and mediated the aggressiveness of the tumor and prognosis.

Figure 1

(A) Patients relapsed with pulmonary adenocarcinoma also had poor postoperative outcome: five-year overall survival (OS) was 97.3% in the nonrelapse group and 38.4% in the relapse group (p < 0.001); (B)Tumor differentiation grade correlated with F¹⁸-fluorodeoxyglucose uptake, expressed as the maximum standard uptake value (SUVmax). The SUVmax of well-differentiated tumors was 2.66 ± 2.64, that of moderately differentiated tumors was 4.75 ± 3.64, and that of poorly differentiated tumors was 7.59 ± 5.25 (p < 0.001).

Figure 2

(A) Five-year overall survival of patients was 86.8% with well-differentiated tumors, 82.5% with moderately differentiated tumors, and 58.6% with poorly differentiated tumors (p < 0.001). (B) Five-year disease-free survival of patients was 95.1% with well-differentiated tumors, 66.5% with moderately differentiated tumors, and 50.4% with poorly differentiated tumors, p < 0.001; (C) The expression of TTF-1 IHC staining was scored after excluded the advanced stage patients (stage 3&4), Five-year OS was 60.0% in score 1 group, 80.1% in score 2 group, and 86.1% in score 3 TTF-1 group.

Figure 3

The tumor differentiation grade was also associated with thyroid transcription factor-1 (TTF-1) expression. (A) Well-differentiated tumors exhibited stronger TTF-1 expression in immunohistochemical staining (400X). (B) Moderately differentiated tumors showed focally positive TTF-1 expression(400X). (C) poorly differentiated tumors showed negative TTF-1 expression (400X). (D) The χ² test showed TTF-1 score was associated with tumor differentiation (p < 0.001).

Figure 4

(A) Reverse transcription-polymerase chain reaction revealed that TTF-1 expression was significantly higher in CL1-0 cells. (B) Western blot analysis validated the difference in tumor aggressiveness between CL1-0, CL1-5, and H1299 cells with different E-cadherin and Vimentin expression. (C) The less aggressive cell lines CL1-0 revealed higher TTF-1 expression and lower high-mobility group AT-hook 2 expression. There was reciprocal change in EGFR expression.

Figure 5

The expression of TTF-1, HMGA2, EMT markers and EGFR in CL1-0-SC, CL1-0-shTTF1, HCC827-SC, HCC827-shTTF1, CL1-5-Vector, CL1-5-TTF1, HCC827-GR-Vector and HCC827-GR-TTF1 cells were evaluated by western blotting. (A) The expression of HMGA2, mesenchymal markers and EGFR increased in CL1-0 and HCC827 cells but expression of epithelial markers decreased after knocked down TTF-1 by shRNA. (B) The expression of HMGA2, mesenchymal markers and EGFR decreased but expression of epithelial markers increased in CL1-5 and HCC827-GR cells following overexpression of TTF-1 by human TTF-1 cDNA ORF clone. The Western blotting was independently repeated three times, and the representative data are shown. GAPDH was served as a loading control.

Figure 6

The invasion abilities of CL1-0 and CL1-5 cells following knockdown/overexpression of TTF-1 were determined by transwell invasion assays. The invasion abilities were significantly inversely correlated with the expression of TTF-1. The transwell invasion assay was independently performed three times, and the representative data are shown. *Statistically significant differences between Scramble control/Vector and TTF-1 knockdown/overexpression group, p < 0.05 by Student’s t test. Error bars, S.E.M. of three independent experiments.

Figure 7

(A) Total lysates from CL1-0-SC, CL1-0-shTTF1, HCC827-SC and HCC827-shTTF1 cells were immunoprecipitated using 5 μg of anti-TTF-1 antibody or normal rabbit IgG and then incubated with magnetic beads. The precipitated chromatin was analyzed by PCR to investigate the interaction between TTF-1 and HMGA2 promoter. TTF-1 co-immunoprecipitated with HMGA2 promoter containing TTF1-recognition site and the effect was blocked after knockdown TTF-1 by TTF-1 shRNA. The results shown are representative of three independent experiments. (B) The miR33a expression was quantified by qPCR in CL1-0-SC, CL1-0-shTTF1, HCC827-SC, HCC827-shTTF1, CL1-5-Vector, CL1-5-TTF1, HCC827-GR-Vector and HCC827-GR-TTF1 cells. The expression of miR-33a was positively correlated with TTF-1 expression and this was validated by knockdown/overexpression of TTF-1. U6 was served as a loading control. Error bars, S.E.M. of three independent experiments.