TMEM17 depresses invasion and metastasis in lung cancer cells via ERK signaling pathway

Abstract

Transmembrane protein 17(TMEM17) is a newly identified protein, its expression pattern and clinicopathological relevance is still unclear. In this study, western blot assay was performed in 20 paired lung cancer samples and found that TMEM17 protein levels were lower in lung cancer tissues than that in the corresponding normal lung tissues (p=0.010). Immunohistochemistry staining in 143 cases lung cancer specimens also showed that TMEM17 expression in lung cancer tissues were significantly lower than adjacent normal lung tissues (35.7% vs 63.2%, p<0.001). And negative TMEM17 expression was significantly associated with poor histological differentiation (p=0.027), advanced TNM stages (p=0.006), positive lymph node metastasis (p=0.002) and poor prognosis (p=0.002). After overexpressing TMEM17, levels of p-ERK and its downstream molecules, p-P90RSK and Snail, were down-regulated, while levels of Occludin and Zo-1 were up-regulated, which result in the inhibition of invasion and migration ability of lung cancer cells. The effects were reversed by the incorporation of specific ERK inhibitor PD98059. In conclusion, loss of TMEM17 correlates with the development of non-small cell lung cancer (NSCLC) and predicts adverse clinical outcome of NSCLC patients. The effect of TMEM17 on inhibiting invasion and migration may attribute to restoring Occludin and Zo-1 expression through inactivating ERK-P90RSK-Snail pathway.

Keywords: ERK signaling; TMEM17; lung cancer; occludin and Zo-1; snail.

Figure 1. The expression of TMEM17 in NSCLC specimens

(A and B). TMEM17 was positively expressed in noncancerous tissues samples and only showed weak or negative expression in paired NSCLC samples. TMEM17 expression is significantly higher in normal lung tissue than that in NSCLC samples (P=0.010). (C, scale bar = 50 μm, insert scal bar=20μm) Representative images of IHC staining in cases 2, 10 and 11, TMEM17 showed cytosolic expression in all the cases. The expression of TMEM17 in HBE cells was higher than most of the NSCLC cell lines (except A549; D), and localized in the cytoplasm of HBE and A549 cells (E, left). Overexpressed TMEM17 is localized primarily in the cytoplasm in H1299 cells (detected by myc-tag, right). No positive signal is detected in non-transfected cells, 600× magnification

Figure 2. The expression and subcellular localization of TMEM17 in NSCLC tissues and cell lines

Positive expression of TMEM17 in normal bronchial (A) and alveolar epithelial (B), however, TMEM17 showed weak or negative expression in lung squamous cell carcinoma (C-D) and adenocarcinoma(E-F). In the same case, TMEM17 was positive expressed in adjacent normal bronchial cells, while only showed weak expression in lung carcinoma (red arrow; G,). In rare cases, TMEM17 can be found expressing in the nucleus (H, scale bar = 50 μm, insert scal bar=20μm). Kaplan–Meier survival analysis revealed that the overall survival for patients with negative TMEM17 expression was significantly shorter than for those with positive TMEM17 expression (I)

Figure 3. The effects on invasion and migration by overexpressing and depleting TMEM17

(A). The transfection efficiency after overexpressing TMEM17 in H1299 and SPC cells and depleting TMEM17 by siRNA in A549 and LK2 cells were tested by western blotting. After overexpressing TMEM17 in H1299 and SPC, the invasion (B) and migration (C) was significantly depressed, after knockdown TMEM17 in A549 and LK2 cells, the invasion (B, scale bar = 50 μm) and migration (C) was obviously enhanced.T17 was short for TMEM17 (*P<0.05;**P<0.01).

Figure 4. TMEM17 depressed NSCLC invasion and migration by ERK- p90RSK-Snail pathway

(A) When overexpressing TMEM17 in H1299 and SPC cells, Snail was downregulated, Occludin and Zo-1 were upregulated. When depleting TMEM17 in A549 and LK2 cells, Snail expression was upregulated and Occludin and Zo-1 were downregulated. (B) TMEM17 overexpression downregulated the levels of p-ERK and its downstream factor p-P90RSK, and their expression were increased after knockdown TMEM17. (C) Western blotting examination of p-ERK, p-p90RSK, Snail, Occludin and Zo-1protein levels after transfecting TMEM17-siRNA with or without incorporation of PD98059. Representative images of cells on the bottom of Transwell membranes show the changes in invasive cell numbers (×200, scale bar= 50 μm) (*P< 0.05). T17 was short for TMEM17