Twisted Gastrulation BMP Signaling Modulator 1 Regulates Papillary Thyroid Cancer Cell Motility and Proliferation

Abstract

Bone morphogenetic proteins (BMPs) are growth factors that have important functions in cell proliferation, migration and differentiation. To date, BMP pathway activation has been found in multiple human tumors and is associated with enhanced malignant tumor growth and metastasis. BMP activity is tightly regulated by a family of soluble extracellular secreted BMP modulators. Twisted gastrulation BMP signaling modulator 1 (TWSG1) is a direct BMP regulator that is required for the full signaling activity of BMPs. However, the functions and mechanisms of TWSG1 in papillary thyroid cancer (PTC) metastasis have not been reported. TWSG1 expression was detected in 44 PTC tissues with lymph node metastasis (LNM) and 56 PTC tissues without LNM using quantitative real-time polymerase chain reaction (qRT-PCR). Gain- and loss-of-function approaches were used to assess the biological function of TWSG1 in PTC cells. Matrigel assays demonstrated the effect of tumor cell-derived TWSG1 on endothelial cell function. Our results showed that TWSG1 expression was significantly enhanced in PTC with LNM compared to that in PTC without LNM. TWSG1 knockdown inhibited migration, invasion and proliferation of PTC cells. Additionally, TWSG1 suppression impaired the tumor cell-induced endothelial cell sprout formation. We found that TWSG1 signaling may be transduced by the BMP target transcription factor inhibitor of DNA binding 1 (Id1) and matrix metalloproteinases (MMPs) 2 and 9. In conclusion, TWSG1 was highly expressed in metastasized PTC; tumor growth, migration and invasion were dependent on TWSG1, and it may be a new diagnostic and therapeutic target for PTC.

Keywords: TWSG1; biomarker.; invasion; migration; papillary thyroid cancer.

Figure 1

TWSG1 is highly expressed in metastasized papillary thyroid cancer. qRT-PCR analysis of the expression levels of TWSG1 in PTC tissues (non-lymph node metastasis (non-LNM), n=56; lymph node metastasis (LNM), n=44) (A) and PTC cell lines (TPC1 and K1) (C). The values were normalized to GAPDH mRNA expression. Data were expressed as the mean ± S.D. of three independent experiments. ** indicates P<0.01. (B) Representative images of immunohistochemistry analysis of TWSG1 in non-metastasized and metastasized PTCs. The scale bar is 100 μm. (D) Western blot analyses of TWSG1 in TPC1 and K1 cells. GAPDH was used as a loading control. Representative images of three repeated experiments were shown. (E) Representative images of immunofluorescence analyses of TWSG1 in TPC1 and K1 cells. The scale bar is 20 μm.

Figure 2

Potential diagnostic value of TWSG1. The ROC curve of TWSG1 identifies the presence of LNM in PTC in terms of sensitivity and specificity. AUC: 0.762 (0.668-0.857), P<0.01.

Figure 3

TWSG1 knockdown suppresses the migration and invasion of K1 cells. (A) Wound healing assays were used to assess the migratory ability of K1 cells with knockdown of TWSG1. Representative images at 0 h and 24 h of three repeated experiments are shown. (B) Transwell assays were performed to determine the migratory ability of K1 cells with knockdown of TWSG1. Representative images of migrated cells in the lower chamber stained with crystal violet. (C) Transwell assays were performed to determine the invasive ability of K1 cells with knockdown of TWSG1. Representative images of invasive cells in the lower chamber stained with crystal violet. (D) The quantification of cell migration is presented as migrated cell numbers. All data are expressed as the mean ± S.D. of three independent experiments. ** indicates P<0.01. (E) The quantification of cell invasion is presented as invasive cell numbers. All data are expressed as the mean ± S.D. of three independent experiments. ** indicates P<0.01.

Figure 4

TWSG1 knockdown suppress the impact of K1 cells on endothelial cell function. (A) TWSG1 knockdown in K1 cells affects tube formation of the endothelial cells. HUVECs were incubated with the supernatant of K1 cells with or without TWSG1 knockdown. Thereafter, HUVECs were subjected to Matrigel assays. Representative micrographs of HUVECs stimulated with supernatants of siRNA-transfected K1 cells are shown. Scale bar= 100 μm. (B) Cumulative sprout length and (C) branch points of capillary-like structures were measured after 4 h and quantified. All data are expressed as the mean ± S.D. of three independent experiments. ** indicates P<0.01. (D) The expression levels of VEGF, endostatin, PAI-1 and thrombospondin-1 in K1 cells transfected with TWSG1-specific siRNA or ScrsiRNA supernatants measured by ELISA. All data are expressed as the mean ± S.D. of three independent experiments. ** indicates P<0.01.

Figure 5

The effect of rTWSG1 on endothelial cells. (A) rTWSG1 did not affect tube formation of the endothelial cells. HUVECs were incubated with 0, 1, 10, 100 ng/ml rTWSG1. Thereafter, HUVECs were subjected to Matrigel assays. Representative micrographs of HUVECs stimulated with rTWSG1 are shown. Scale bar= 100 μm. (B) Cumulative sprout length and (C) branch points of capillary-like structures were measured after 4h and quantified. All data are expressed as the mean ± S.D. of three independent experiments.

Figure 6

TWSG1 knockdown inhibits the expression of MMPs and the BMP target gene Id1. qRT-PCR analysis of the mRNA expression levels of MMP1 (A), MMP2 (B), MMP9 (C), MMP13 (D) and Id1 (E) in K1 cells with TWSG1 knockdown. The values were normalized to GAPDH mRNA expression. Data are expressed as the mean ± S.D. of three independent experiments. ** indicates P<0.01. (F) Western blot analysis of the expressions of MMP2, MMP9, Id1 and p-Smad1/5/8 in K1 cells with TWSG1 knockdown. Representative images of three repeated experiments are shown.

Figure 7

TWSG1 knockdown inhibits the proliferation of K1 cells. (A) CCK-8 assays were performed to measure K1 cell proliferation 12 h, 24 h, 36 h, 48 h and 72 h after transfection with TWSG1-specific siRNA. (B) Colony formation assays were performed to assess the proliferation of K1 cells transfected with TWSG1-specific siRNA. The colonies were identified and counted. (C) The colony formation assay results are presented as histograms. Data were from independent experiments performed in triplicate and are presented as the mean ± S.D. (D) Flow cytometry images of the cell cycle in K1 cells. (E) Cell cycle quantification is shown as a percentage of total cells. Data are expressed as the mean ± S.D. of three independent experiments. All data are expressed as the mean ± S.D. of three independent experiments. * indicates P<0.05, ** indicates P<0.01.