Ubiquitin-specific protease 44 inhibits cell growth by suppressing AKT signaling in non-small cell lung cancer

Abstract

Ubiquitin-specific protease 44 (USP44) has been reported as a tumor suppressor or promoter in some tumors, but its function in non-small cell lung cancer (NSCLC) is still unclear. In this study, USP44 was found significantly downregulated in both of NSCLC tissues and cell lines, and low expression of USP44 predicted a poor prognosis for NSCLC patients. Overexpression of USP44 markedly downregulated the expression levels of Cyclin D1 and CDK4, but upregulated p53 expression, as a result of which, suppressing the cell growth of NSCLC cells. Further studies indicated that overexpression of USP44 significantly inhibited the phosphorylation of AKT, and its down-stream signals, including mTOR and P70S6K. Moreover, overexpression of USP44 increased PTEN protein but not its mRNA levels, which suggested that USP44 inhibited AKT signaling by stabilizing PTEN in NSCLC cells. In conclusion, we demonstrated that USP44 showed prior evidence of a tumor suppressive function in NSCLC cells, and inhibited NSCLC cell growth by suppressing AKT signaling, suggesting that USP44 could be as a novel target for NSCLC therapy.

Keywords: AKT; Deubiquitinase; NSCLC; PTEN; USP44.

Figure 1

USP44 is lowly expressed in NSCLC and low USP44 predicts a poor prognosis for NSCLC patients. A, The expression levels of USP44 in normal or cancerous tissues from lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were analyzed by GEPIA based on TCGA database (http://gepia.cancer‐pku.cn). B,C, Sixteen pairs of fresh primary NSCLC tissues and individual normal para‐cancerous tissues were prepared for qRT‐PCR against USP44. GAPDH was used as an internal control. D, Four NSCLC cell lines and one human bronchial epithelial cells were prepared for qRT‐PCR against USP44. GAPDH was used as an internal control. E, The overall survival periods of lung cancer patients with low or high expression of USP44 were estimated by Kaplan‐Meier plotter (http://kmplot.com). ^* P < .05, ^** P < .01

Figure 2

Overexpression of USP44 inhibits cell growth in NSCLC cells. A, A549 and H1975 cells were stably infected with lentiviral PLVX‐NC or PLVX‐USP44, followed by immunoblotting against USP44. GAPDH was used as a loading control. B,C, A549 (B) and H1975 cells (C) stably infected with lentiviral PLVX‐NC or PLVX‐USP44 were cultured for indicated time, followed by CCK‐8 assay at day 0, 1, 3, or 5. D, A549 and H1975 cells were stably infected with lentiviral PLVX‐NC or PLVX‐USP44, followed by immunoblotting against Cyclin D1, CDK4, p53, and GAPDH. E, A549 and H1975 cells were infected with PLVX‐NC or PLVX‐USP44 for 4 days, followed by cell cycle analysis on a flow cytometer

Figure 3

USP44 inhibits AKT signaling in NSCLC cells. A, Four NSCLC cell lines and one human bronchial epithelial cells were prepared for immunoblotting against USP44, p‐AKT, AKT, and GAPDH. B, A549 and H1975 cells were transfected with empty vector (EV) or Myc‐USP44 plasmids for 48 hours, followed by immunoblotting against p‐AKT, AKT, and Myc. GAPDH was used as a loading control. C, A549 and H1975 cells were transfected with EV or Myc‐USP44 plasmids for 48 hours, followed by immunoblotting against p‐mTOR, mTOR, Myc, and GAPDH. D, A549 and H1975 cells were transfected with EV or Myc‐USP44 plasmids for 48 hours, followed by immunoblotting against p‐P70S6K, P70S6K, Myc, and GAPDH. E, Four representative pairs of fresh primary NSCLC tissues and individual normal para‐cancerous tissues were prepared for immunoblotting against USP44 and PTEN. GAPDH was used as an internal control

Figure 4

USP44 stabilizes PTEN protein in NSCLC cells. A,B, EV or Myc‐USP44 plasmids were transfected into A549 and H1975 cells for 48 hours, followed by immunoblotting against PTEN and Myc (A), or qRT‐PCR against PTEN (B). GAPDH was used as an internal control. C, A549 and H1975 cells were stably infected with lentiviral PLVX‐NC or PLVX‐USP44, followed by immunoblotting against USP44 and PTEN. GAPDH was used as a loading control. D, H1975 cells were transfected with EV or Myc‐USP44 plasmids. Forty‐eight hours later, cells were incubated with vehicle or 20 μM MG132 for 6 hours, followed by immunoblotting against PTEN, Myc, and GAPDH. E, Statistically analysis of D. F, A549 cells were transfected with Myc‐USP44 or empty vector (EV) for 24 hours, followed by CHX chase assay at indicated time. Then, immunoblotting was performed to assess the expression levels of PTEN and Myc‐USP44. GAPDH was used as a loading control. G, Statistically analysis of F