PERP-428 C / G單核苷酸多型性調節抗氧化活性影響肺癌風險之機制研究

Study on the Mechanism of PERP-428 C/G Single Nucleotide Polymorphism in Regulating Antioxidant Activity and Affecting Lung Cancer Risk

廖振毅(Chen-Yi Liao)

指導教授 ： 蔡菁華

中山醫學大學/醫學院/醫學研究所/博士(2021年)

https://doi.org/10.6834/csmu202100193

摘要

PERP (p53 Apoptosis Effect Related to PMP-22)為穿膜四次的細胞膜蛋白，先前的研究認為PERP是p53的下游基因，它的表現與p53執行細胞凋亡的路徑有關。雖然過去PERP被認為是一個腫瘤抑制基因，不過我們在肺癌中發現PERP的蛋白與mRNA都是呈現過度表現的情形。在CL1-5和A549細胞中使用G418篩選穩定抑制PERP的細胞，發現7天後會導致細胞死亡，但是若是短暫轉染抑制PERP時細胞則不受影響，表示PERP在癌細胞長時間的生存上是必要的因子。先前本實驗室透過cDNA cloning的方式，發現在數個肺癌細胞和患者的cDNA和genomic DNA中有PERP-428C轉變成PERP-428G的突變。並在NCBI的database中確認PERP-428為編號 rs648802 (C428G)之單核苷酸多型性 (Single nucleotide polymorphism)。接著我們先以304位肺癌患者及193位健康對照族群為樣本，研究PERP-428 SNP在肺癌與健康對照族群的差異，結果顯示與PERP-428 GG基因型相比，PERP-428 CC基因型罹患肺癌的風險較高 (OR = 5.38; 95% CI = 2.12-13.65, p < 0.001)，另外PERP-428 CG基因型罹患肺癌的風險也比PERP-428 GG基因型來的高 (OR = 2.34; 95% CI = 1.55-3.55, p < 0.001)。隨後透過實驗證實，過度表現PERP-428G基因型比PERP-428C基因型更能保護細胞不受到gallic acid和H2O2所誘導的壞死傷害。接著在本研究中，我們試圖去找尋表現PERP-428 SNP的細胞對ROS有不同的保護力之分子機制。我們發現不同PERP-428 SNP的p53穩定度並不相同，並且p53負調控抗氧化酵素Catalase (CAT)和Glutathione reductase (GR)，然後使其ROS含量改變。結果發現PERP-428C基因型比PERP-428G基因型有較高的p53穩定度，而PERP-428C基因型有較高的穩定度是因為受到MDM2表現的下降以及p53 Ser20磷酸化增加的影響。而且MDM2在PERP-428C基因型表現的減少是透過PTEN負調控MDM2 p1所造成的。另外我們也發現，p53無論是野生型或是R248W突變型，皆會去抑制抗氧化酵素CAT和GR的表現，進而改變ROS的含量。在ROS的誘導下，突變的p53R248W只有在ROS所誘導的細胞壞死上需要PARP-1的參與，然而野生型的p53在ROS誘導下，細胞凋亡以及細胞壞死皆會出現。本研究指出，表現不同PERP-428 SNP基因型的細胞透過調控PTEN-MDM2-p53路徑影響CAT和GR表現量，進而影響細胞內ROS的含量及抵抗ROS的能力。總結本研究來說，我們認為PERP-428C 基因型在保護細胞不受ROS所誘導之DNA損傷的能力較差，這是由於PERP-428C基因型具有高穩定度的p53，其抗氧化酵素含量比PERP-428G基因型來的少，因此比較不能夠消除過量的ROS，在罹患肺癌的風險上就有較高的可能性，這些發現提供了高風險族群一個潛在有價值的策略，透過平時避免對ROS導致的傷害，以預防罹患肺癌或是與ROS相關的慢性疾病。

關鍵字

肺癌 ； 單核苷酸多型性

並列摘要

PERP (p53 Apoptosis Effect Related to PMP-22)-a tetraspan protein is primarily localized in the plasma membrane. Previous studies have shown that PERP is a downstream target of p53 and its expression is related to p53-dependent apoptosis. Although historically considered a tumor suppressor, PERP is highly expressed at protein and mRNA levels in lung cancer. Stable knockdown of PERP in CL1-5 and A549 induced cell death on the 7th days of the G418 selection period, but transient knockdown has no effect on cell proliferation, indicating that PERP is essential for the long-term survival of lung cancer cells. During the cDNA cloning process, we found that PERP-428 C to G mutation were in the cDNA and genomic DNA of several lung cancer cells and patients. Based on NCBI database, PERP-428 was confirmed as a non-synonymous single nucleotide polymorphism. Therefore, we used 304 non-small cell lung cancer patients and 193 healthy controls to investigate the risk of lung cancer in different PERP. The result showed that relative to the PERP-428 GG genotype, PERP-428 CC was associated with the highest lung cancer risk (OR = 5.38; 95% CI = 2.12-13.65, p < 0.001), followed by the PERP-428 CG genotype (OR = 2.34; 95% CI = 1.55-3.55, p < 0.001). Our previous studies have shown that the ectopic expression of PERP-428G has better protection against gallic acid and H2O2 treatment compared with cells expressing PERP-428C. In this study, we aimed to investigate the molecular mechanism involved in the differential protection of gallic acid by PERP-428 SNP expressing cells. Mechanically, PERP-428 SNPs differentially regulate p53 protein stability. p53 negatively regulates the expression of antioxidant enzymes catalase (CAT) and glutathione reductase (GR), and then modulates the status of redox. The stability of p53 protein in cells expressing PERP-428C is higher than that in cells expressing PERP-428G, because the expression of MDM2 is decreased and p53 Ser20 phosphorylation is enhanced in cells expressing PERP-428C. The MDM2 mRNA level is decreased in PERP-428C cells via PTEN-mediated downregulation of the MDM2 constitutive p1 promoter. We also observed that both R248W mutant p53 and wild-type p53 are able to inhibit CAT and GR expression, but they induced different death phenotypes. Upon ROS treatment, R248W p53 mutant mainly induces necrosis via PARP-1, whereas the wild-type p53 induces both apoptosis and necrosis. This study indicated that in PERP-428C expressing cells, CAT/GR expression is decreased via the PTEN/MDM2/p53 pathway. In conclusion, we consider that PERP-SNP-mediated differential protection of cell from ROS damage would explain the possible link with lung cancer risk. Individuals with PERP-428 CC and PERP-428 CG genotypes have an increased risk of lung cancer. These findings provide a potentially valuable strategy for these high-risk individuals to prevent lung cancer or other ROS-related chronic diseases through preventive antioxidants and avoidance of ROS stressors.

並列關鍵字

PERP ； SNP ； ROS

參考文獻

Agarwal, A., Mahfouz, R.Z., Sharma, R.K., Sarkar, O., Mangrola, D., and Mathur, P.P. (2009). Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes. Reprod Biol Endocrinol 7, 143.

Aggarwal, V., Tuli, H.S., Varol, A., Thakral, F., Yerer, M.B., Sak, K., Varol, M., Jain, A., Khan, M.A., and Sethi, G. (2019). Role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements. Biomolecules 9, 735.

Aisner, D.L., Sholl, L.M., Berry, L.D., Rossi, M.R., Chen, H., Fujimoto, J., Moreira, A.L., Ramalingam, S.S., Villaruz, L.C., Otterson, G.A., Haura, E., Politi, K., Glisson, B., Cetnar, J., Garon, E.B., Schiller, J., Waqar, S.N., Sequist, L.V., Brahmer, J., Shyr, Y., Kugler, K., Wistuba, I.I., Johnson, B.E., Minna, J.D., Kris, M.G., Bunn, P.A., and Kwiatkowski, D.J.; LCMC2 investigators. (2018). The Impact of Smoking and TP53 Mutations in Lung Adenocarcinoma Patients with Targetable Mutations-The Lung Cancer Mutation Consortium (LCMC2). Clin Cancer Res 24, 1038-1047.

Alano, C.C., and Swanson, R.A. (2006). Players in the PARP-1 cell-death pathway: JNK1 joins the cast. Trends Biochem Sci 31, 309-311.

Alfieri, R., Giovannetti, E., Bonelli, M., and Cavazzoni, A. (2017). New Treatment Opportunities in Phosphatase and Tensin Homolog (PTEN) -Deficient Tumors: Focus on PTEN/Focal Adhesion Kinase Pathway. Front Oncol 7, 170.