Ubiquitin-specific peptidase 28 enhances STAT3 signaling and promotes cell growth in non-small-cell lung cancer

Abstract

Background and objectives: Ubiquitin-specific peptidase 28 (USP28) has been reported to play significant roles in several tumors, but its roles in non-small-cell lung cancer (NSCLC) is still unknown. In this study, we aimed to investigate the biological function and molecular mechanisms of USP28 in NSCLC.

Materials and methods: Immunoblotting analysis was used to detect relative proteins' expression. Luciferase assay was performed to explore the activation of signal transducer and activator of transcription 3 (STAT3). Immunoprecipitation was performed to assess whether USP28 interacted with STAT3 or deubiquitinated STAT3. Quantitative real-time PCR was performed to evaluate the relative mRNA levels of STAT3 and USP28. Cycloheximide chase assay was carried out to examine whether USP28 affected the half-life of STAT3 protein. Cell Counting Kit-8 assay and xenograft model were used to assess whether USP28 regulated NSCLC cell growth.

Results: In this study, the deubiquitinating enzyme USP28 was found to mediate STAT3 signaling in NSCLC cells. USP28 interacted with STAT3, and increased the stability of STAT3 by inducing its deubiquitination. Further studies showed that USP28 was upregulated in both the primary tissues and cell lines of NSCLC. The Kaplan-Meier plotter also indicated that USP28 predicted a poor prognosis of NSCLC patients. Moreover, knockdown of USP28 inhibited cell growth of NSCLC cells in vitro and delayed NSCLC tumor growth in vivo.

Conclusion: These results demonstrated that USP28 was functional in NSCLC cells, and promoted NSCLC cell growth by inducing STAT3 signaling. This suggests that USP28 could be a novel target for NSCLC therapy.

Keywords: STAT3; USP28; deubiquitinating enzyme; deubiquitination; non-small-cell lung cancer.

Figure 1

USP28 mediates STAT3 signaling in NSCLC cells. Notes: (A) The whole cell lysates of four NSCLC cell lines and one human bronchial epithelial cells were extracted, and USP28, p-STAT3, and STAT3 protein levels were measured by immunoblotting analysis. GAPDH was used as a loading control. (B) HBE, A549, H460, H1299 and H1975 cells were transfected with STAT3-Luc plasmids for 48 hours, followed by luciferase assay. (C) STAT3-Luc, Myc-USP28, Myc-USP25 or EV plasmids were transfected into H1299 cells along with the internal control vector Renilla for 48 hours. Then, the cells were prepared for luciferase assay by using a Dual-Luciferase^® reporter assay system. (D) H1299 cells were transfected with increased Myc-USP28 plasmids or EV for 48 hours, followed by immunoblotting against p-STAT3, STAT3, Myc, and GAPDH. (E) H1299 cells transfected with STAT3-Luc plasmids were infected with lentiviral shUSP28#1, shUSP28#2, or shNC for 48 hours, followed by luciferase assay. (F) H1299 cells were infected with lentiviral shUSP28#1, shUSP28#2, or shNC for 72 hours, followed by immunoblotting against p-STAT3, STAT3, USP28, and GAPDH. *P<0.05, **P<0.01. Abbreviations: EV, empty vector; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NSCLC, non-small-cell lung cancer; n.s., non-sense; NC, negative control; STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease.

Figure 2

USP28 interacts with STAT3 and decreases its polyubiquitination level. Notes: (A, B) Myc-USP28 or Flag-STAT3 plasmids were transfected into HEK293T for 36 hours, and then whole cell lysates were prepared for reciprocal Co-IP and immunoblotting against specific antibodies as indicated. (C) H1299 cells were lysed for Co-IP by using anti-STAT3 antibody, followed by immunoblotting against USP28 and STAT3. (D) HEK293T cells were transfected with Myc-USP28 or EV along with HA-Ub plasmids for 36 hours. Cell lysates were then prepared for IP using anti-IgG or anti-STAT3 antibody, followed by immunoblotting against STAT3 and Ub-K48 (using K48-linkage specific polyubiquitin antibody). In addition, whole cell lysates were also prepared for immunoblotting against STAT3, Myc and GAPDH. Abbreviations: EV, empty vector; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA-Ub, HA-ubiquitin; IB, immunoblotting; IP, immunoprecipitation; n.s., non-sense; STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease.

Figure 3

USP28 increases the stability of STAT3. Notes: (A, B) H1299 cells were transfected with increased Myc-USP28 plasmids for 48 hours, followed by immunoblotting against STAT3, Myc, and GAPDH (A), or qRT-PCR against STAT3 and GAPDH (B). (C) H1299 cells were transfected with increased Myc-USP28^C171A plasmids for 48 hours, followed by immunoblotting against STAT3, Myc, and GAPDH. (D, E) H1299 cells were infected with lentiviral shNC, shUSP28#1, or shUSP28#2 for 3 days, followed by immunoblotting against USP28, STAT3, and GAPDH (D), or qRT-PCR against STAT3 and GAPDH (E). (F) H1299 cells were transfected with Myc-USP28 or vector for 24 hours, followed by CHX chase assay. Immunoblotting analysis was performed against STAT3 and Myc. GAPDH was used as a loading control. (G) Statistical analysis of F. Abbreviations: CHX, cycloheximide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; qRT-PCR, quantitative real-time PCR; STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease; WT, wild-type.

Figure 4

USP28 is upregulated and predicts a poor prognosis in NSCLC. Notes: (A) USP28 expression levels in normal or lung squamous cell carcinoma tissues were retrieved from GEPIA database (http://gepia.cancer-pku.cn). (B) Fresh primary NSCLC tissues (T) and individual normal para-cancerous tissues (P) were analyzed for USP28 expression by qRT-PCR. (C) The whole cell lysates of four representative fresh primary NSCLC tissues (T) and individual normal para-cancerous tissues (P) were extracted, and USP28 and STAT3 protein levels were measured by immunoblotting analysis. GAPDH was used as a loading control. (D) The survival periods of NSCLC patients were estimated by Kaplan–Meier plotter (http://kmplot.com). *P<0.05, **P<0.01. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NSCLC, non-small-cell lung cancer; qRT-PCR, quantitative real-time PCR; STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease.

Figure 5

Knockdown of USP28 inhibits NSCLC cell growth. Notes: (A) A549 cells were stably infected with lentiviral shNC, shUSP28#1, or shUSP28#2 for indicated time, followed by CCK-8 assay at day 0, 1, 3, and 5. (B) H1299 cells were stably infected with lentiviral shNC, shUSP28#1, or shUSP28#2 for indicated time, followed by CCK-8 assay at day 0, 1, 3, and 5. (C) H1299 cells stably infected with lentiviral shNC or shUSP28#1 were subcutaneously injected into the right flank of each nude mouse. When tumors were palpable, tumor sizes were monitored every other day for 3 weeks. (D) Tumor weight was measured at the end of the experiment. (E) The excised tumors were prepared for immunoblotting against USP28 and GAPDH. *P<0.05, **P<0.01. Abbreviations: CCK-8, Cell Counting Kit-8; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; NSCLC, non-small-cell lung cancer; STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease.