TMEM196 inhibits lung cancer metastasis by regulating the Wnt/β-catenin signaling pathway

Abstract

Purpose: The TMEM196 protein, which comprises four membrane-spanning domains, belongs to the TMEM protein family. TMEM196 was identified as a candidate tumor suppressor gene in lung cancer. However, its role and mechanism in lung cancer metastasis remain unclear. Here, we study the role of TMEM196 in tumor metastasis to further verify the function in lung cancer.

Methods: In this study, we used qRT-PCR, western blot analysis and immunohistochemistry to examine the expression levels of TMEM196 and related proteins in lung cancer tissues and tumor cells. We utilized Transwell assays, xenograft nude mouse models, and TMEM196^-/- mouse models to evaluate the effects of TMEM196 on tumor invasion and metastasis. Finally, we used bioinformatics analysis and dual-luciferase reporter gene assays to explore the molecular mechanism of TMEM196 as a tumor suppressor.

Results: We found that TMEM196 mRNA and protein expression levels were significantly decreased in lung cancer tissues and cells. Low expression of TMEM196 in clinical patients was associated with poor prognosis. TMEM196 strongly inhibited tumor metastasis and progression in vitro and in vivo. The primary lung tumors induced by tail vein-inoculated B16 cells in TMEM196^-/- mice were significantly larger than those in TMEM196^+/+ mice. Mechanistically, TMEM196 inhibited the Wnt signaling pathway and repressed β-catenin promoter transcription. TMEM196 silencing in lung cancer cells and mice resulted in significant upregulation of the expression of β-catenin and Wnt signaling pathway downstream target genes (MMP2 and MMP7). Decreasing β-catenin expression in TMEM196-silenced cancer cells attenuated the antimetastatic effect of TMEM196.

Conclusions: Our results revealed that TMEM196 acts as a novel lung cancer metastasis suppressor via the Wnt/β-catenin signaling pathway.

Keywords: Lung cancer; Metastasis; TMEM196; Tumor suppressor; Wnt signaling.

Fig. 1

TMEM196 expression was significantly downregulated in lung cancer cells and tissues. A TMEM196 mRNA expression was downregulated in lung cancer cells compared with normal HBE cells, as determined by RT-PCR. B. IHC showed that TMEM196 protein expression was significantly decreased in lung cancer cells compared with normal HBE cells. C The immunohistochemistry results from the Human Protein Atlas database showed that the TMEM196 protein expression level was dramatically decreased in lung cancer tissues compared with adjacent normal tissues. D The protein expression of TMEM196 was assessed in adjacent and tumor lung samples by immunohistochemistry (IHC) on a tissue array. Original magnification × 200

Fig. 2

Low TMEM196 expression was associated with poor prognosis in lung cancer patients. A Kaplan–Meier survival curves showed that lung cancer patients with low TMEM196 expression had poorer survival than those with high TMEM196 expression based on the log-rank test (P < 0.001). B Kaplan–Meier curves of lung adenocarcinoma patients (P < 0.001). C Kaplan–Meier curves of lung squamous cell carcinoma patients (P< 0.01 ). D Kaplan–Meier curves of lung cancer patients in TNM stage I and II (P < 0.0001). E Kaplan–Meier curves of lung cancer patients in TNM stages III and IV (P = 0.0876)

Fig. 3

TMEM196 inhibits lung cancer cell migration and invasion. A Overexpression of TMEM196 in SPC-A-1 and LTEP-a-2 cells was confirmed by RT-PCR. B Knockdown of TMEM196 in A549 cells and SPC-A-1-TMEM196 stably expressing cells was confirmed by RT-PCR. The GAPDH gene served as a loading control. C Overexpression of TMEM196 inhibited lung cancer cell migration and invasion in vitro. D Low expression of TMEM196 promoted lung cancer cell migration and invasion in vitro. The error bars indicate the SEMs. **, P < 0.01; ***, P < 0.001; ****, P<0.0001

Fig. 4

Overexpression of TMEM196 repressed tumor metastasis in vivo. A Morphologic observations of nude mice injected with or without TMEM196-overexpressing lung cancer cells. B H&E staining of lung metastases from the TMEM196 and control groups. The arrows indicate metastasis loci. H&E, hematoxylin and eosin. C Statistical analysis of metastatic nodes in the lungs of nude mice. D Quantitative analyses of human GAPDH levels were used to quantify the number of metastatic human cancer cells in nude mouse lungs. **, P < 0. 01; ***, P < 0.001

Fig. 5

Knockout of TMEM196 enhanced tumor metastasis ability in vivo. A Knockout of TMEM196 was confirmed by qRT-PCR (WW: wild-type; KK: knockout). B Lung metastases were evaluated by tissue morphologic observation. C H&E staining of lung metastases of wild-type and TMEM196-null mice. D Statistical analysis of metastasis in nude mice. The arrows indicate metastasis loci. **, P < 0.01; ***, P < 0.001

Fig. 6

TMEM196 attenuated Wnt signaling by downregulating the expression of related genes. A TMEM196 is involved in cell–cell signaling through the Wnt signaling pathway. B TMEM196 inhibited TOP-flash (containing wild-type LEF/TCF-binding sites) reporter activation in LTEP-a-2 cells. C–F. Ectopic expression of TMEM196 significantly repressed the endogenous mRNA and protein expression of CTNNB1, MMP2, and MMP7, as determined by RT-qPCR (C) and WB analysis (E). TMEM196 knockdown significantly promoted the mRNA and protein expression of CTNNB1, MMP2, and MMP7, as determined by qRT-PCR (D) and WB analysis (F). GAPDH was used as an internal control. (G-H). WB and IHC analysis of protein expression in TMEM196 knockout and wild-type mice. Original magnification × 200. The error bars indicate the SEMs; *, P < 0.05; **, P < 0.01; ***, P <0.001

Fig. 7

TMEM196 inhibited tumor metastasis in a β-catenin-dependent manner. A Knockdown of TMEM196 and CTNNB1 in A549 cells was confirmed by RT-PCR. B TMEM196 inhibited tumor migration and invasion in a β-catenin-dependent manner. C TMEM196 targeted β-catenin in LTEP-a-2 cells and 293T cells, as determined by a luciferase reporter assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 8

TMEM196 acted as a suppressor gene dependent on the Wnt/β-catenin signaling pathway