Original article
Novel function of PERP-428 variants impacts lung cancer risk through the differential regulation of PTEN/MDM2/p53-mediated antioxidant activity

https://doi.org/10.1016/j.freeradbiomed.2021.02.017Get rights and content

Highlights

  • ․The PERP-428CC genotype is associated with a high risk of lung cancer.

  • ․Cells expressing PERP-428C stabilize the p53 protein by increasing phosphor Ser20 levels and decreasing MDM2 expression.

  • ․In PERP-428C cells, p53 inhibits catalase and GR mRNA levels, thereby increasing intracellular ROS and cancer risk.

Abstract

Lung cancer is the leading cause of cancer-related deaths worldwide. Identifying genetic risk factors and understanding their mechanisms will help reduce lung cancer incidence. The p53 apoptosis effect is related to PMP-22 (PERP), a tetraspan membrane protein, and an apoptotic effector protein downstream of p53. Although historically considered a tumor suppressor, PERP is highly expressed in lung cancers. Stable knockdown of PERP expression induces CL1-5 and A549 lung cancer cell death, but transient knockdown has no effect. Interestingly, relative to the PERP-428GG genotype, PERP-428CC was associated with the highest lung cancer risk (OR = 5.38; 95% CI = 2.12–13.65, p < 0.001), followed by the PERP-428CG genotype (OR = 2.34; 95% CI = 1.55–3.55, p < 0.001). Ectopic expression of PERP-428G, but not PERP-428C, protects lung cancer cells against ROS-induced DNA damage. Mechanistically, PERP-428 SNPs differentially regulate p53 protein stability. p53 negatively regulates the expression of the antioxidant enzymes catalase (CAT) and glutathione reductase (GR), thereby modulating redox status. p53 protein stability is higher in PERP-428C-expressing cells than in PERP-428G-expressing cells because MDM2 expression is decreased and p53 Ser20 phosphorylation is enhanced in PERP-428C-expressing cells. The MDM2 mRNA level is decreased in PERP-428C-expressing cells via PTEN-mediated downregulation of the MDM2 constitutive p1 promoter. This study reveals that in individuals with PERP-428CC, CAT/GR expression is decreased via the PTEN/MDM2/p53 pathway. These individuals have an increased lung cancer risk. Preventive antioxidants and avoidance of ROS stressors are recommended to prevent lung cancer or other ROS-related chronic diseases.

Introduction

The p53 apoptosis effect related to PMP-22 (PERP), located on 6q24, is a small (21.4 kDa) tetraspan membrane protein comprising 193 amino acids. PERP was first identified as an effector of p53 specifically involved in apoptosis but not in cell cycle arrest [1]. PERP appears to modulate apoptosis via the caspase-dependent pathway [2,3]. PERP not only functions as a downstream target of p53 but also stabilizes and activates p53 via enhanced phosphorylation of p53 on Ser20 and Ser46 in uveal melanoma [4].

PERP is a component of desmosomes in keratinocytes and is important for cell adhesion [5] and regulation of enamel formation [6]. C-terminal PERP truncation has been detected in patients with Olmsted syndrome and severe periorificial and palmoplantar keratoderma. Individuals with PERP mutations show epidermal hyperproliferation and immature desmosomes [7]. Knockdown of PERP expression causes desmosome deficiency, enhances cell survival, induces the expression of a set of inflammatory genes, and promotes skin cancer [8] and breast cancer [9]. Downregulation of PERP has been shown to be related to the aggressiveness of uveal melanoma [10]. However, compared with wild-type mice, mice with conditional knockout of PERP have been shown to exhibit resistance to papilloma development upon carcinogen induction [11]. Thus, PERP may play different roles in tumorigenesis in different contexts.

In this study we identified a new role of PERP in redox regulation. Specifically, the PERP-428C isoform was found to exhibit lower antioxidant capacity than the PERP-428G isoform, which explains why the PERP-428CC and PERP-428CG genotypes are associated with a higher risk of developing lung cancer than the PERP-428GG genotype. We demonstrated that the expression levels of catalase (CAT) and glutathione reductase (GR) are decreased in PERP-428C-expressing cells compared with those in PERP-428G-expressing cells via differential regulation of a novel JNK/PTEN/mouse double minute 2 homolog (MDM2)/p53 pathway. Thus, individuals with the PERP-428CC and PERP-428CG genotypes may be more susceptible to ROS-induced cell death. In such individuals, the use of preventive antioxidants may help to reduce cancer risk.

Section snippets

Study subjects, cell lines, and genotype analysis

Tissue microarrays established from 78 paraffin blocks of non-small cell lung cancer tissues were obtained from the Department of Pathology at Chung Shan Medical University Hospital. For PERP SNP analysis, genomic DNA was extracted from 304 non-small cell lung cancer specimens using a DNeasy Blood & Tissue Kit (Qiagen, CA, USA) and from 193 peripheral blood leukocytes of healthy controls using a QIAamp DNA Blood Mini Kit (Qiagen, CA, USA). PERP-428 polymorphisms were genotyped using a TaqMan

Expression of PERP in lung cancer specimens

Previous studies have demonstrated that PERP has a tumor suppressor function. However, we found that PERP mRNA was upregulated in lung cancer tissues compared with normal paired tissues in microarray (Fig. 1A) and real-time RT-PCR analyses from independent sets of lung cancer tissues (Fig. 1B). Moreover, in our immunohistochemical analysis, 98% of lung squamous cell carcinomas and 83% of lung adenocarcinomas overexpressed PERP protein (Fig. 1C; Table 1). Our results are supported by those of

Discussion

Intracellular ROS are signaling molecules that regulate various biological processes. The p53 protein functions as a master regulator of cellular redox homeostasis. p53 can play an antioxidant role under basal or low oxidative levels to support cell proliferation and survival, while it plays a pro-oxidant role under high levels of oxidative stress to promote cell senescence or death [19,20]. Under physiological conditions, p53 upregulates glutathione peroxidase (GPx) and SOD2 (also called MnSOD

Conclusion

The results of this study revealed a novel pathway in which PERP-428 SNPs differentially regulates basal MDM2 expression via PTEN and the p53 Ser 20 phosphorylation level, thereby modulating p53 stability, inhibiting CAT and GR expression and modulating intracellular ROS levels. Individuals with the PERP-428CC or PERP-428CG genotype are more susceptible to ROS challenge; this finding provides valuable insight for preventive medicine. Individuals with the PERP-428CC or PERP-428CG genotype should

Funding

This work was supported by Ministry of Science and Technology grants MOST 106-2320-B-040-019, MOST 107-2320-B040-023 and MOST 108-2320-B-040-004 (Taiwan, R.O.C) to Jinghua Tsai Chang.

Authors’ contributions

Study concept and design: JTC, CYL and SFY; Acquisition of data: CYL, TJW, CYH, HC, CHW and JCL; Statistical analysis: TJW, CYL and HC; Data analysis: JTC, CYL, TJW, CYH, CHW, JCL, SFY, HC, SLH, YFL and HL; Technical and material support: SFY, HC, CYH, YFL, SLH, HL and GTS; Writing: JTC; Critical revision of the manuscript: SFY, HC, YFL, HL and GTS; Study supervision: JTC. All authors read and approved the manuscript.

Declaration of competing interest

The authors claim that they have no conflicts of interest related to this study.

Acknowledgments

Results in Fig. 1D are based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.

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