Trop2 enhances invasion of thyroid cancer by inducing MMP2 through ERK and JNK pathways

Abstract

Background: Mounting evidence has showed that Tumor-associated calcium signal transducer 2 (Trop2) is upregulated in various kinds of human cancers and plays important roles in tumorigenesis. However, the expression status and functional significance of Trop2 in thyroid cancer are largely unknown.

Methods: We first determined the expression of Trop2 by using RNAseqV2 data sets for thyroid cancer deposited on The Cancer Genome Atlas (TCGA) website. The expression of Trop2 was then confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry assays. Cell invasion and migration were assessed by conducting Transwell and wound healing assays. Furthermore, we explored the underlying mechanisms by using real-time RT-PCR, Western blot, zymography, and luciferase reporter assays.

Results: In this study, we demonstrated that the expression of Trop2 was significantly elevated in thyroid cancer and that its expression level was correlated with the tumor-node-metastasis (TNM) staging and N classification. Dysregulation of Trop2 altered the invasive capability of thyroid cancer cells. Further mechanistic study revealed that MMP2 expression was upregulated by Trop2. Moreover, we found that the effects of Trop2 were dependent on ERK and JNK pathways. The results from clinical specimens showed that Trop2 expression correlated with MMP2 expression in primary thyroid cancer.

Conclusion: The current study suggests that elevated expression of Trop2 may represent an important molecular hallmark that is biologically and clinically relevant to the progression of thyroid cancer.

Keywords: Invasion; MAPK; MMP2; Thyroid cancer; Trop2.

Fig. 1

Trop2 is overexpressed in thyroid cancer. a The expression of Trop2 in 59 pairs of primary tumors versus paired non-tumorous thyroid tissues using RNAseqV2 data sets deposited on TCGA website. b Expression of Trop2 in 18 paired tumors and adjacent non-tumorous thyroid tissues assessed by qRT-PCR. Experiments were repeated for three times in triplicate. c Representative images of IHC assays on Trop2 expression in thyroid lesions

Fig. 2

Knockdown of endogenous Trop2 suppresses the invasion and migration of thyroid cancer cells. a Trop2 knockdown was achieved by introducing specific shRNAs in thyroid cancer cells. The protein levels of Trop2 in indicated cells were assessed by WB. α-tubulin was used as a loading control. b Silencing of Trop2 led to significant decrease of invasive capability of thyroid cancer cells. The indicated cells traveled through the membrane by Transwell invasion assay (coated with Matrigel) were stained with crystal violet and imaged using a microscope. c The histogram showed the invading cells per field. The data are reported as mean ± SD of three independent experiments. *, P < 0.05. d Representative images of Transwell migration assay (coated without Matrigel) results of indicated cells. e Diagram of Transwell migration assay of indicated cells. The data are reported as mean ± SD of three independent experiments. *, P < 0.05. f Wound healing analyses of indicated cells. Streaks were created with a tip, and the representative phase-contrast images of the extent of cell migration into the wounded area at 24 h are shown. Experiments were repeated for three times

Fig. 3

Over-expression of Trop2 promotes the invasion and migration of thyroid cancer cells. a Overexpression of Trop2 in FTC-133 thyroid cancer cells was analyzed. The protein levels of Trop2 in indicated cells were assessed by WB and α-tubulin was used as a loading control. b Overexpression of Trop2 increased the invasive (with Matrigel) and migratory (without Matrigel) capability of FTC-133 cells. c The histogram showed the invading and migrating cells per field. The data are reported as mean ± SD of three independent experiments. *, P < 0.05. d Wound healing analyses of indicated cells. Streaks were created with a tip, and the representative phase-contrast images of the extent of cell migration into the wounded area at 24 h are shown. Experiments were repeated for three times

Fig. 4

Trop2 regulates the expression of MMP2. a Silencing of Trop2 in K1 and 8505C thyroid cancer cells resulted in down-regulation of MMP2 expression, but not MMP7, MMP9, MMP11, and MMP13 expression. The data are reported as mean ± SD of three independent experiments. *, P < 0.05. b Depletion of Trop2 in K1 and 8505C thyroid cancer cells resulted in inhibition of the MMP2 promoter reporter activity. The data are reported as mean ± SD of three independent experiments. *, P < 0.05. c Ectopic overexpression of Trop2 promotes the expression of MMP2 in FTC-133 thyroid cancer cells. The data are reported as mean ± SD of three independent experiments. *, P < 0.05. d Trop2 overexpression enhanced the MMP2 promoter reporter activity. The data are reported as mean ± SD of three independent experiments. *, P < 0.05. e Conditioned media were prepared by incubating vector-control or Trop2-knockdown cells in serum-free media for 24 h. MMP2 activities are analyzed by gelatin zymography. f Gelaltin zymography analysis of serum-free conditioned medium from FTC-133-vector, FTC-133-Trop2 cells

Fig. 5

ERK/JNK/AP1 signaling mediates the effect of Trop2 on MMP2 expression. a Comparison of AP1 reporter activity in indicated cells. The luciferase reporter showed decreased activation of AP1 reporter in Trop2 knocked-down K1 and 8505C cells. The data are reported as mean ± SD of three independent experiments. *, P < 0.05. b Overexpression of Trop2 promoted the AP1 reporter activity. The data are reported as mean ± SD of three independent experiments. *, P < 0.05. c and d AP1 ODNs significantly inhibited the effects of Trop2 on MMP2 expression (c) and MMP2 promoter reporter activity (d). The data are reported as mean ± SD of three independent experiments. *, P < 0.05. e WB analysis showed that deregulated expression of Trop2 significantly altered the phosphorylation of ERK and JNK. f and g Cells were treated with a concentration of 10 μM PD98059, 10 μM SP600125, or 10 μM SB203580 for 24 h. After incubation, MMP2 mRNA level was determined by qRT-PCR (f) and AP1 reporter activity was assessed by Dual-Luciferase reporter assays (g). The data are reported as mean ± SD of three independent experiments. *, P < 0.05

Fig. 6

Trop2 expression correlates with MMP2 expression in primary thyroid cancer. a Expression of Trop2 is associated with MMP2 expression levels in clinical thyroid cancer specimens. Two representative cases are shown. b Percentage of specimens showing low or high Trop2 expression in relation to the expression levels of MMP2. *, P < 0.05. c The association of Trop2 and MMP2 in 252 cases of thyroid cancer specimens were analyzed using RNAseqV2 data sets deposited on TCGA website. *, P < 0.05