TRIM27 acts as an oncogene and regulates cell proliferation and metastasis in non-small cell lung cancer through SIX3-β-catenin signaling

Abstract

The Wnt/β-catenin pathway plays vital roles in diverse biological processes, including cell differentiation, proliferation, migration, and insulin sensitivity. A recent study reported that the DNA-binding transcriptional factor SIX3 is essential during embryonic development in vertebrates and capable of downregulating target genes of the Wnt/β-catenin pathway in lung cancer, indicating negative regulation of Wnt/β-catenin activation. However, regulation of the SIX3-Wnt/β-catenin pathway axis remains unknown. We measured the expression of TRIM27 and SIX3 as well as investigated whether there was a correlation between them in lung cancer tissue samples. Herein, we found that the E3 ubiquitin ligase, TRIM27, ubiquitinates, and degrades SIX3. TRIM27 induces non-small cell lung cancer (NSCLC) cell proliferation and metastasis, and the expression of β-catenin, S100P, TGFB3, and MMP-9 were significantly inhibited by SIX3. Furthermore, XAV939 is a selective β-catenin-mediated transcription inhibitor that inhibited TRIM27- and SIX3-mediated NSCLC cell proliferation, migration, and invasion. Clinically, lung tissue samples of cancer patients showed increased TRIM27 expression and decreased SIX3 expression. Taken together, these data demonstrate that TRIM27 acts as an oncogene regulating cell proliferation and metastasis in NSCLC through SIX3-β-catenin signaling.

Keywords: SIX3; TRIM27; lung cancer; ubiquitination; β-catenin.

Figure 1

TRIM27 and SIX3 expression levels and correlation analysis in lung cancer cells and tissues. (A) A549 cell lysates underwent co-immunoprecipitation with anti-SIX3 or control IgG antibody. The immunoprecipitates were then immunoblotted with the indicated antibodies. TRIM27, SMURF2, and SIX3 expression in lung cancer tissues (n = 138) and adjacent normal lung tissues (n = 30) in our independent hospital cohort was detected by quantitative real-time PCR (B) and immunohistochemistry (D). (C, E) Correlation analysis of SIX3, SMURF2, and TRIM27 in lung cancer tissues (n = 138). Statistical analyses were performed using the Chi-square test. (F) TRIM27 and SIX3 expression in cells from the NSCLC cell lines H1975, A549, and H358, and the human bronchial epithelial cell line 16HBE were measured by western blot analysis. Survival probability of patients with lung cancer from a Kaplan-Meier Plotter database (G) and our independent hospital cohort (H, I). Scale bar: 50 μm. All experiments were repeated at least three times, and data are represented as mean ± SD. (B) ***P < 0.001 (Mann-Whitney U test). (F) *P < 0.05, ***P < 0.001 (one-way ANOVA followed by Dunnett’s test).

Figure 2

Identification of the binding motifs involved in TRIM27-SIX3 interaction. Schematic representation of FLAG-tagged full-length (A) TRIM27 or (C) SIX3 (FL), along with its various deletion mutants (D1 and D2). CC, coiled-coil domain. A549 cells were cotransfected with the (B) indicated HA-tagged SIX3 constructs along with those encoding FLAG-tagged TRIM27 or the (D) indicated FLAG-tagged TRIM27 constructs along with those encoding HA-tagged SIX3. Interaction between TRIM27 and SIX3 was determined by immunoprecipitation and immunoblotting. (E–G) A549 cells infected with pLVX-Puro-TRIM27 or blank pLVX-Puro vector were treated with DMSO or 10 μM MG132 for 4 h, and SIX3 expression was determined by quantitative real-time PCR and western blot analysis. (H, I) A549 cells infected with pLVX-Puro-TRIM27 or blank pLVX-Puro vector were treated with CHX (100 μg/mL), and SIX3 expression was determined by western blot analysis. (J) SIX3 was immunoprecipitated and immunoblotted in A549 cells transfected with siTRIM27-1 or siNC. (K) A549 cells were cotransfected with the SIX3 (WT) or mutant SIX3 constructs along with myc-TRIM27 and His-Ub constructs, and a pull-down assay was performed. All experiments were repeated at least three times, and data are represented as mean ± SD. (F) ***P < 0.001 (two-way ANOVA followed by Dunnett’s test).

Figure 3

SIX3 regulates NSCLC cell proliferation, invasion, and migration. H1975 cells were transfected with siSIX3-1, siSIX3-2, or siNC, and cell proliferation (A), migration (B), and invasion (C) were determined by CCK-8 and transwell assay. A549 cells were infected with pLVX-Puro-SIX3 or blank pLVX-Puro vector, and cell proliferation (D), migration (E), and invasion (F) were determined by CCK-8 and transwell assay. All experiments were repeated at least three times, and data are represented as mean ± SD. ***P < 0.001 (two-way ANOVA followed by Dunnett’s test).

Figure 4

SIX3 overexpression inhibits NSCLC cell proliferation, migration, and invasion induced by TRIM27 overexpression. H1975 cells were infected with blank pLVX-Puro vector, pLVX-Puro-TRIM27, or pLVX-Puro-SIX3, and cell proliferation (A), migration (B), invasion (C), and related protein expression (D, E) were determined by CCK-8, transwell assay, and western blot analysis. All experiments were repeated at least three times, and data are represented as mean ± SD. ***P < 0.001 (two-way ANOVA followed by Dunnett’s test).

Figure 5

The roles of TRIM27 and SIX3 on lung metastasis in vivo. (A) Histology of single lung lobe from mice intravenously injected with A549 cells stably infected with blank pLVX-Puro vector, pLVX-Puro-TRIM27, or pLVX-Puro-SIX3. Scale bars: 500 μm. (B) Quantification of microscopic nodules in the lungs of each group. (C) The incidence of metastasis in mice after intravenous tail injection of each cell type is shown in the table. All experiments were repeated at least three times, and data are represented as mean ± SD. *P < 0.05, ***P < 0.001 (two-way ANOVA followed by Dunnett’s test).

Figure 6

TRIM27 and SIX3 regulate NSCLC cell migration and invasion partly through the Wnt/β-catenin pathway. H1975 cells transfected or infected with siSIX3-1, pLVX-Puro-TRIM27, or both siNC and blank pLVX-Puro vector were treated with DMSO or 10 μM XAV939, and cell migration, invasion, and related protein expression were determined by transwell assay (A–C) and western blotting (D). All experiments were repeated at least three times, and data are represented as mean ± SD. ***P < 0.001 (two-way ANOVA followed by Dunnett’s test).