TIPE3 differentially modulates proliferation and migration of human non-small-cell lung cancer cells via distinct subcellular location

Abstract

Background: TIPE3 (TNFAIP8L3), a transfer protein for lipid second messengers, is upregulated in human lung cancer tissues. The most popular lung cancer is non-small cell lung cancer (NSCLC) with high incidences and low survival rates, while the roles of TIPE3 in NSCLC remain largely unknown.

Methods: TIPE3 expression was examined in tissue chips from patients with NSCLC using immunohistochemistry; the correlation of plasma membrane expression of TIPE3 with T stage of NSCLC was analyzed. After endogenous TIPE3 was silenced via siRNA, or TIPE3 with N or C-terminal flag was overexpressed via transient or stable transfection, human NSCLC cells were assayed for the proliferation and migration, respectively. NSCLC cells, in which TIPE3 with C-terminal flag was stably transfected, were inoculated into mice to establish xenograft tumors, the tumor growth and the expression of TIPE3 in tumor tissues were examined.

Results: TIPE3 was broadly expressed in lung tissues of patients with NSCLC. The plasma membrane expression of TIPE3 was positively correlated with the T stage of NSCLC. Knockdown of endogenous TIPE3, which was predominantly expressed in the plasma membrane, inhibited the proliferation and migration of NSCLC cells. While transient overexpression of TIPE3 with N-terminal flag, which was mostly trapped in the cytoplasm, inhibited the growth and migration of NSCLC cells accompanied by inactivation of AKT and ERK. In contrast, stable overexpression of TIPE3 with C-terminal flag, which could be localized in the plasma membrane, markedly promoted the growth and migration of NSCLC cells through activation of AKT and ERK. Notably, in xenograft tumor models established with NSCLC cells, stable overexpression of TIPE3 with C-terminal flag in NSCLC cells significantly promoted the tumor growth and enhanced the expression and plasma membrane localization of TIPE3 in tumor tissues.

Conclusion: This study demonstrates that human TIPE3 promotes the proliferation and migration of NSCLC cells depending on its localization on plasma membrane, whereas cytoplasmic TIPE3 may exert a negative function. Thus, manipulating the subcellular location of TIPE3 can be a promising strategy for NSCLC therapy.

Keywords: Migration; Non-small-cell lung cancer; Proliferation; TIPE3.

Fig. 1

Expression of TIPE3 on tissues and cell lines from NSCLC. a TIPE3 expression was detected on lung tissue chips from patients with primary NSCLC by immunohistochemistry. b The mRNA of TIPE3 in NSCLC cell lines H1975 and A549 were detected by PCR. c The expression of endogenous TIPE3 and Na⁺/K⁺-ATPase was detected using confocal laser microscopy

Fig. 2

Silence of endogenous TIPE3 attenuates the proliferation and migration of lung cancer cells. a Endogenous TIPE3 was knocked down in H1975 cells using siTIPE3. b The growth curve of H1975 cells after silence of TIPE3 was determined by CCK8 assay (n = 4 per timepoint). c, d The migration of H1975 cells after silence of TIPE3 was determined. Representative (c) and statistic (d) data are shown (n = 5 per group). Bars represent mean ± s.e.m. **P < 0.01, ***P < 0.001

Fig. 3

Overexpression of TIPE3 with N-terminal flag inhibits the proliferation and migration of lung cancer cells. a Long or short TIPE3 with N-terminal flag was transiently overexpressed in A549 and H1975 cells by plasmid transfection, and detected by western-blot. b, c The growth curves of A549 (b) and H1975 (c) cells overexpressing TIPE3 were determined by CCK8 assay (n = 4 per timepoint). d-f The migration of A549 and H1975 cells overexpressing TIPE3 was determined. Representative (d) and statistic (e, f) data are shown (n = 5 per group). Bars represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Exogenous TIPE3 with N-terminal flag is mainly located in the cytoplasm of lung cancer cells. Exogenous short TIPE3 with N-terminal flag was transiently overexpressed in A549 cells by plasmid transfection. a The subcellular location of TIPE3 was detected using confocal laser microscopy. b The levels of p-AKT and p-MEK were determined by western-blot

Fig. 5

Overexpression of exogenous TIPE3 promotes the proliferation and migration of lung cancer cells. a, b TIPE3 with C-terminal flag was stably overexpressed in A549 cells by lentivirus transfection. GFP was detected by immunofluorescence (a), and flag was detected by western-blot (b). c The growth curves of A549 cells overexpressing TIPE3 were determined by CCK8 assay (n = 6 per timepoint). d, e The migration of A549 cells overexpressing TIPE3 was determined. Representative (d) and statistic (e) data (n = 5 per group) are shown. f, g The migration of A549 cells overexpressing TIPE3 was determined in the absence or presence of siTIPE3. Representative (f) and statistic (g) data are shown (n = 5 per group). Bars represent mean ± s.e.m. *P < 0.05, ***P < 0.001

Fig. 6

Exogenous TIPE3 with C-terminal flag is mainly located in the plasma membrane of lung cancer cells. Exogenous short TIPE3 with C-terminal flag was stably overexpressed in A549 cells by lentivirus transfection. a The subcellular location of TIPE3 was detected using confocal laser microscopy. b The levels of p-AKT and p-MEK were determined by western-blot

Fig. 7

Exogenous TIPE3 with C-terminal flag promotes tumor growth in vivo. A549 cells (3 × 10⁶ cells in 100 μl PBS per mice) transfected with mock or recombinant (carrying C-terminal flag-tagged human TIPE3) lentivirus were subcutaneously injected into nu/nu mice (n = 5 per group) to establish xenograft tumors. a-d The growth curve of tumors (a), tumor size (b, c) and weight (d) were determined. e TIPE3 expression was evaluated in sections of tumor tissues by immunohistochemistry. *P < 0.05, ***P < 0.001