WT1-interacting protein inhibits cell proliferation and tumorigenicity in non-small-cell lung cancer via the AKT/FOXO1 axis

Abstract

Lung cancer is the most common cancer and the leading cause of cancer-related death worldwide; hence, it is imperative that the mechanisms underlying the malignant properties of lung cancer be uncovered in order to efficiently treat this disease. Increasing evidence has shown that WT1-interacting protein (WTIP) plays important roles both physiologically and pathologically in humans; however, the role of WTIP in cancer is unknown. Here, we investigated the role and mechanism of WTIP in cell proliferation and tumorigenesis of non-small-cell lung cancer (NSCLC). We report that WTIP is a tumor suppressor in human NSCLC. We found that WTIP expression was significantly reduced in both NSCLC cell lines and clinical specimens compared to that in normal controls; this reduction was largely attributed to promoter hypermethylation. Downregulation of WTIP significantly correlates with poor prognosis and predicts a shorter overall survival and progression-free survival among NSCLC patients. Moreover, ectopic overexpression of WTIP dramatically inhibits cell proliferation and tumorigenesis in vitro and in vivo; conversely, depletion of WTIP expression shows the opposite effects. Mechanistically, WTIP impairs AKT phosphorylation and activation, leading to enhanced expression and transcriptional activity of FOXO1, which further increases p21Cip1 and p27Kip1, and decreases cyclin D1, which consequently results in cell cycle arrest. Collectively, the results of the current study indicate that WTIP is an important proliferation-related gene and that WTIP expression may represent a novel prognostic biomarker for NSCLC.

Keywords: AKT; FOXO1; NSCLC; WTIP; cell proliferation; non-small cell lung cancer.

Figure 1

Promoter methylation leads to WTIP downregulation in NSCLC. Western blotting (A, C) and real‐time PCR (B, D) analysis of WTIP expression in NSCLC cell lines and paired NSCLC tumor and nontumor tissues. (E) Real‐time PCR analysis of WTIP mRNA levels in the indicated cells treated with vehicle or 5‐Aza‐dC for 72 h. Error bars represent the mean ± SD obtained from three independent experiments. *P < 0.05, unpaired t‐test. (F) Bisulfite genomic sequencing of three individual clones reveals the methylation status of CpG islands in the WTIP promoter in the indicated specimens. The methylation status of the CpG sites from three individual clones was distinguished by different shades of blue as indicated. N, nontumor. T, tumor.

Figure 2

Downregulation of WTIP correlates with poor prognosis of NSCLC patients. (A) Representative cases of patient specimens with different levels of WTIP staining. Scale bars, 50 μm. (B, C) Kaplan–Meier analyses of OS (B) and PFS (C) of NSCLC patients stratified by WTIP expression levels. (D) Online Kaplan–Meier analyses of OS, PFS, and PPS of lung cancer patients with high or low levels of WTIP mRNA. Plots were generated using the publicly accessible tool KM Plotter. P‐value was calculated by log‐rank test.

Figure 3

Overexpression of WTIP inhibits cell proliferation and induces cell cycle arrest in NSCLC cells. (A) Western blotting analysis of WTIP in the indicated cells with stable overexpression of WTIP. (B) MTT assay analysis of cell growth. Error bars represent the mean ± SD obtained from three independent experiments. *P < 0.05, unpaired t‐test. (C) Representative pictures of cell colonies originating from the indicated cells and stained with crystal violet. (D) Representative pictures of BrdU staining of the indicated cells. Pictures were taken at 400× magnification. Scale bars, 20 μm. (E) Cell cycle distribution as analyzed by flow cytometry. (F) Western blotting analyses of the expression of the cell cycle regulators p21, p27, cyclin D1, phosphorylated Rb (p‐Rb), and total Rb in vector control‐expressing and WTIP‐overexpressing cells. α‐Tubulin served as a loading control.

Figure 4

Depletion of WTIP promotes cell proliferation and cell cycle progression. (A) Western blotting analysis of WTIP in cell lines with stable expression of specific shRNAs. (B) MTT assay analysis of cell growth. Error bars represent the mean ± SD obtained from three independent experiments. *P < 0.05, unpaired t‐test. (C) Representative pictures of cell colonies originating from the indicated cells and stained with crystal violet. (D) Representative pictures of BrdU staining. Pictures were taken at 400× magnification. Scale bars, 20 μm. (E) Cell cycle distribution as analyzed by flow cytometry. (F) Western blotting analyses of the expression of p21, p27, cyclin D1, phosphorylated Rb (p‐Rb), and total Rb in the indicated cells. α‐Tubulin served as a loading control.

Figure 5

WTIP inhibits tumorigenicity of NSCLC in vitro and in vivo. Representative pictures (A) and quantification (B) of colony formation as analyzed by anchorage‐independent growth. Pictures were taken at 40× magnification. Scale bars, 0.2 mm. Error bars represent the mean ± SD obtained from three independent experiments. *P < 0.05, unpaired t‐test. (C–F) Xenograft model in nude mice. Different groups of A549 cells were subcutaneously injected into nude mice. (C) Growth curve of the tumor volumes measured on the indicated days. Error bars represent the mean ± SD. *P < 0.05, unpaired t‐test. (D and E) Representative pictures of tumor growth (D) and tumor weights (E) 42 days after inoculation. Error bars represent the mean ± SD. *P < 0.05, unpaired t‐test. (F) Representative pictures of H&E staining and IHC staining of WTIP and Ki‐67 in the indicated xenografted tumors. Scale bars, 50 μm.

Figure 6

WTIP expression correlates with FOXO1 activation in NSCLC. (A) GSEA plot showing that WTIP expression is positively correlated with the AKT‐downregulated gene signature (AKT_UP. V1_DN) and FOXO1‐targeted gene signature (V$FOXO1_01) in the gene expression profiles of LUAD and LUSC patients from the TCGA database. (B) Western blotting analyses of the expression of phosphorylated AKT (p‐AKT), total AKT, phosphorylated FOXO1 (p‐FOXO1), total FOXO1, phosphorylated GSK‐3β (p‐ GSK‐3β), and total GSK‐3β. α‐Tubulin served as a loading control. (C) Relative FOXO1 luciferase reporter activity in 293FT and A549 cells transfected with the indicated plasmids. *P < 0.05, unpaired t‐test. (D) Representative pictures of FOXO1 immunostaining. White arrows, cell with nucleus retained FOXO1. Pictures were taken at 630× magnification. Scale bars, 20 μm. (E) Western blotting analyses of the expression of FOXO1, p21, p27, and cyclin D1. α‐Tubulin served as a loading control. (F) MTT assay analysis of cell growth of the indicated cells. Error bars represent the mean ± SD obtained from three independent experiments. *P < 0.05, unpaired t‐test. (G) IHC staining analysis of WTIP, FOXO1, and Ki‐67 expression in NSCLC patient specimens.