Ubiquitin specific peptidase 49 inhibits non-small cell lung cancer cell growth by suppressing PI3K/AKT signaling

Abstract

Ubiquitin specific peptidase 49 (USP49) has been reported as a tumor suppressor in several tumors, but its function and molecular mechanism in non-small cell lung cancer (NSCLC) are still unknown. In this study, USP49 was found downregulated in NSCLC primary tissues and cell lines, and high USP49 predicted a positive index for the overall survival of NSCLC patients. Overexpression of USP49 downregulated the expression levels of Cyclin D1, and upregulated p53 expression. Further flow cytometry analysis showed that overexpressed USP49 induced cell cycle arrest at G0/G1 phase. As a result, overexpression of USP49 significantly inhibited cell growth of NSCLC cells. In mechanism, overexpression of USP49 inhibited PI3K/AKT signaling, but knockdown of USP49 enhanced this signaling. Further studies indicated that USP49 deubiquitinated PTEN and stabilized PTEN protein, which suggested that USP49 inhibited PI3K/AKT signaling by stabilizing PTEN in NSCLC cells. In conclusion, we demonstrated that USP49 was functional in NSCLC cells, and inhibited NSCLC cell growth by suppressing PI3K/AKT signaling, suggesting that USP49 could be as a novel target for NSCLC therapy.

Keywords: AKT; Deubiquitination; PTEN; USP49; non-small cell lung cancer.

Figure 1

USP49 is lowly expressed in NSCLC, and predicts positive index for NSCLC patients. A, The expression levels of USP49 in normal or cancerous tissues from lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were analyzed by GEPIA online, which was based on the TCGA database (http://gepia.cancer‐pku.cn). B, Twenty pairs of fresh primary NSCLC tissues and individual normal para‐cancerous tissues were prepared for qRT‐PCR against USP49. GAPDH was used as an internal control. ^* P < .05, ^** P < .01. C, Three representative fresh primary NSCLC tissues and individual normal tissues were prepared for immunoblotting against USP49 and β‐actin. D, Four NSCLC cell lines and one human bronchial epithelial cells were prepared for immunoblotting against USP49 and β‐actin. E, The overall survival periods of NSCLC patients were estimated by Kaplan‐Meier plotter (http://kmplot.com). NSCLC, non‐small cell lung cancer [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Overexpression of USP49 inhibits cell growth by suppressing cell cycle progression in NSCLC cells. A. H322 cells were stably infected with lentiviral PLVX‐NC or PLVX‐USP49 for indicated time, followed by CCK‐8 assay at day 0, 2, 4, and 6. The expression level of USP49 was also detected by immunoblotting. B, H1792 cells were stably infected with lentiviral PLVX‐NC or PLVX‐USP49 for indicated time, followed by CCK‐8 assay at day 0, 2, 4, and 6. The expression level of USP49 was also detected by immunoblotting. C, H322 and H1792 cells were infected with PLVX‐NC or PLVX‐USP49 for 4 days, and then they were lysed for immunoblotting against Cyclin D1, p53, and USP49. β‐actin was used as a loading control. D, H322 cells were infected with PLVX‐NC or PLVX‐USP49 for 4 days, followed by cell cycle analysis on a flow cytometer. NSCLC, non‐small cell lung cancer

Figure 3

USP49 regulates PI3K/AKT signaling in NSCLC cells. A, H322 cells were transfected with empty vector (EV) or Myc‐USP49 plasmids for 72 hours, followed by immunoblotting against p‐p85, PI3K p85, PTEN, and Myc. β‐actin was used as a loading control. B, H322 cells transfected with EV or Myc‐USP49 plasmids were also prepared for immunoblotting against p‐AKT, AKT, p‐mTOR, mTOR, Myc and β‐actin. C, H322 cells were infected with PLVX‐NC or PLVX‐USP49 for 4 days, followed by immunoblotting against p‐p85, PI3K p85, PTEN, USP49, and β‐actin. D, Above cells were also prepared for immunoblotting against p‐AKT, AKT, p‐mTOR, mTOR, USP49, and β‐actin. NSCLC, non‐small cell lung cancer

Figure 4

USP49 deubiquitinates and stabilizes PTEN in NSCLC cells. A, The correlation analysis between PTEN and USP49 was retrieved from GEPIA database (http://gepia.cancer‐pku.cn). B, HEK293T cells were transfected with Myc‐USP49 or Flag‐PTEN along with HA‐Ub plasmids. Forty‐eight hours later, cells were lysed for immunoprecipitation against Flag, followed by immunoblotting against HA and Flag. C, Above whole cell lysates were also prepared for immunoblotting against Flag, Myc, and β‐actin. D, H322 cells were transfected with Myc‐USP49 or EV for 24 hours, followed by CHX chase assay at indicated time. Then, immunoblotting was carried out to assess the expression levels of PTEN and Myc‐USP49. β‐actin was used as a loading control. E, Statistically analysis of figure D. NSCLC, non‐small cell lung cancer