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USP22 promotes NSCLC tumorigenesis via MDMX up-regulation and subsequent p53 inhibition.

Ding F, Bao C, Tian Y, Xiao H, Wang M, Xie X, Hu F, Mei J - Int J Mol Sci (2014)

Bottom Line: Increasing evidence suggests that ubiquitin-specific protease 22 (USP22) has great clinicopathologic significance in oncology.Additionally, USP22 silencing downregulates MDMX protein expression and activates the p53 pathway.Taken together, our results suggest that USP22 promotes NSCLC tumorigenesis in vitro and in vivo through MDMX upregulation and subsequent p53 inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200092, China. drnail@sina.com.

ABSTRACT
Increasing evidence suggests that ubiquitin-specific protease 22 (USP22) has great clinicopathologic significance in oncology. In this study, we investigated the role of USP22 in human NSCLC tumorigenesis along with the underlying mechanisms of action. First, we determined the expression of USP22 in human NSCLC, as well as normal tissues and cell lines. We then studied the effects of USP22 silencing by shRNA on NSCLC cell growth in vitro and tumorigenesis in vivo, along with the effect on the p53 pathway. We found that USP22 is overexpressed in human NSCLC tissues and cell lines. USP22 silencing by shRNA inhibits proliferation, induces apoptosis and arrests cells at the G0/G1 phases in NSCLC cells and curbs human NSCLC tumor growth in a mouse xenograft model. Additionally, USP22 silencing downregulates MDMX protein expression and activates the p53 pathway. Our co-immunoprecipitation analysis shows that USP22 interacts with MDMX in NSCLC cells. Furthermore, MDMX silencing leads to growth arrest and apoptosis in NSCLC cells, and over-expression of MDMX reverses the USP22 silencing-induced effects. Taken together, our results suggest that USP22 promotes NSCLC tumorigenesis in vitro and in vivo through MDMX upregulation and subsequent p53 inhibition. USP22 may represent a novel target for NSCLC treatment.

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USP22 silencing activates the p53 pathway, down-regulates MDMX protein expression and interacts with MDMX in NSCLC cells. (A) The levels of p53 pathway proteins in A549 and NCl-H460 cells transfected with USP22 shRNA or SCR shRNA; (B,C) the mRNA and protein expressions of MDMX in A549 and NCl-H460 cells transfected with USP22 shRNA or SCR shRNA (n = 3). β-actin was used as an internal control. Data are expressed as the means ± SD. n.s. indicates no significant difference; (D) Immunofluorescence (IF) analysis of USP22, MDMX and p53 expression in A549 cells transfected with USP22 shRNA or SCR shRNA.
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ijms-16-00307-f003: USP22 silencing activates the p53 pathway, down-regulates MDMX protein expression and interacts with MDMX in NSCLC cells. (A) The levels of p53 pathway proteins in A549 and NCl-H460 cells transfected with USP22 shRNA or SCR shRNA; (B,C) the mRNA and protein expressions of MDMX in A549 and NCl-H460 cells transfected with USP22 shRNA or SCR shRNA (n = 3). β-actin was used as an internal control. Data are expressed as the means ± SD. n.s. indicates no significant difference; (D) Immunofluorescence (IF) analysis of USP22, MDMX and p53 expression in A549 cells transfected with USP22 shRNA or SCR shRNA.

Mentions: Having demonstrated that USP22 regulates cell proliferation in NSCLC cells, we investigated the possible underlying mechanisms. Abnormalities in p53 are frequently found in lung cancer, indicating its importance in this malignancy [21]. We noted that USP22 has been reported to antagonize the p53 pathway in previous studies [12,13]. To find out whether p53 is involved in the regulatory function of USP22 in NSCLC, we assessed p53 activation in USP22 shRNA-transfected cells by Western blot analysis. We found that USP22 silencing in A549 and NCI-H460 cells increased the protein expression of p53, p21 and Bax, the key p53 signal molecules (Figure 3A), suggesting that p53 activation plays a role in USP22 silencing-induced growth inhibition. MDM2 and MDMX promote ubiquitin-dependent p53 degradation and are the two major negative regulators of p53. It has been reported that USP22 antagonizes p53 in bladder cancer cells through up-regulating MDM2 [13]. Interestingly, we found that in A549 and NCI-H460 cells, USP22 silencing, while activating the p53 pathway, decreased MDMX protein expression (Figure 3C), which was confirmed with immunofluorescence (IF) analysis (Figure 3D). However, the mRNA expression of MDMX was not affected (Figure 3B). On the bases of these results, we speculated that USP22 silencing activates the p53 pathway in NSCLC cells by post-transcriptional down-regulation of MDMX.


USP22 promotes NSCLC tumorigenesis via MDMX up-regulation and subsequent p53 inhibition.

Ding F, Bao C, Tian Y, Xiao H, Wang M, Xie X, Hu F, Mei J - Int J Mol Sci (2014)

USP22 silencing activates the p53 pathway, down-regulates MDMX protein expression and interacts with MDMX in NSCLC cells. (A) The levels of p53 pathway proteins in A549 and NCl-H460 cells transfected with USP22 shRNA or SCR shRNA; (B,C) the mRNA and protein expressions of MDMX in A549 and NCl-H460 cells transfected with USP22 shRNA or SCR shRNA (n = 3). β-actin was used as an internal control. Data are expressed as the means ± SD. n.s. indicates no significant difference; (D) Immunofluorescence (IF) analysis of USP22, MDMX and p53 expression in A549 cells transfected with USP22 shRNA or SCR shRNA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307248&req=5

ijms-16-00307-f003: USP22 silencing activates the p53 pathway, down-regulates MDMX protein expression and interacts with MDMX in NSCLC cells. (A) The levels of p53 pathway proteins in A549 and NCl-H460 cells transfected with USP22 shRNA or SCR shRNA; (B,C) the mRNA and protein expressions of MDMX in A549 and NCl-H460 cells transfected with USP22 shRNA or SCR shRNA (n = 3). β-actin was used as an internal control. Data are expressed as the means ± SD. n.s. indicates no significant difference; (D) Immunofluorescence (IF) analysis of USP22, MDMX and p53 expression in A549 cells transfected with USP22 shRNA or SCR shRNA.
Mentions: Having demonstrated that USP22 regulates cell proliferation in NSCLC cells, we investigated the possible underlying mechanisms. Abnormalities in p53 are frequently found in lung cancer, indicating its importance in this malignancy [21]. We noted that USP22 has been reported to antagonize the p53 pathway in previous studies [12,13]. To find out whether p53 is involved in the regulatory function of USP22 in NSCLC, we assessed p53 activation in USP22 shRNA-transfected cells by Western blot analysis. We found that USP22 silencing in A549 and NCI-H460 cells increased the protein expression of p53, p21 and Bax, the key p53 signal molecules (Figure 3A), suggesting that p53 activation plays a role in USP22 silencing-induced growth inhibition. MDM2 and MDMX promote ubiquitin-dependent p53 degradation and are the two major negative regulators of p53. It has been reported that USP22 antagonizes p53 in bladder cancer cells through up-regulating MDM2 [13]. Interestingly, we found that in A549 and NCI-H460 cells, USP22 silencing, while activating the p53 pathway, decreased MDMX protein expression (Figure 3C), which was confirmed with immunofluorescence (IF) analysis (Figure 3D). However, the mRNA expression of MDMX was not affected (Figure 3B). On the bases of these results, we speculated that USP22 silencing activates the p53 pathway in NSCLC cells by post-transcriptional down-regulation of MDMX.

Bottom Line: Increasing evidence suggests that ubiquitin-specific protease 22 (USP22) has great clinicopathologic significance in oncology.Additionally, USP22 silencing downregulates MDMX protein expression and activates the p53 pathway.Taken together, our results suggest that USP22 promotes NSCLC tumorigenesis in vitro and in vivo through MDMX upregulation and subsequent p53 inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200092, China. drnail@sina.com.

ABSTRACT
Increasing evidence suggests that ubiquitin-specific protease 22 (USP22) has great clinicopathologic significance in oncology. In this study, we investigated the role of USP22 in human NSCLC tumorigenesis along with the underlying mechanisms of action. First, we determined the expression of USP22 in human NSCLC, as well as normal tissues and cell lines. We then studied the effects of USP22 silencing by shRNA on NSCLC cell growth in vitro and tumorigenesis in vivo, along with the effect on the p53 pathway. We found that USP22 is overexpressed in human NSCLC tissues and cell lines. USP22 silencing by shRNA inhibits proliferation, induces apoptosis and arrests cells at the G0/G1 phases in NSCLC cells and curbs human NSCLC tumor growth in a mouse xenograft model. Additionally, USP22 silencing downregulates MDMX protein expression and activates the p53 pathway. Our co-immunoprecipitation analysis shows that USP22 interacts with MDMX in NSCLC cells. Furthermore, MDMX silencing leads to growth arrest and apoptosis in NSCLC cells, and over-expression of MDMX reverses the USP22 silencing-induced effects. Taken together, our results suggest that USP22 promotes NSCLC tumorigenesis in vitro and in vivo through MDMX upregulation and subsequent p53 inhibition. USP22 may represent a novel target for NSCLC treatment.

Show MeSH
Related in: MedlinePlus