Please wait a minute...
 Home  About the Journal Editorial Board Aims & Scope Peer Review Policy Subscription Contact us
Early Edition  //  Current Issue  //  Open Special Issues  //  Archives  //  Most Read  //  Most Downloaded  //  Most Cited
Aging and disease    2020, Vol. 11 Issue (1) : 44-59     DOI: 10.14336/AD.2019.0415
Orginal Article |
Predictive Value of Pin1 in Cervical Low-Grade Squamous Intraepithelial Lesions and Inhibition of Pin1 Exerts Potent Anticancer Activity against Human Cervical Cancer
Yan-Tong Guo, Yan Lu, Yi-Yang Jia, Hui-Nan Qu, Da Qi, Xin-Qi Wang, Pei-Ye Song, Xiang-Shu Jin, Wen-Hong Xu, Yuan Dong, Ying-Ying Liang, Cheng-Shi Quan*
Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, China.
Download: PDF(1926 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks    

Many oncogenes are involved in the progression from low-grade squamous intraepithelial lesions (LSILs) to high-grade squamous intraepithelial lesions (HSILs); which greatly increases the risk of cervical cancer (CC). Thus, a reliable biomarker for risk classification of LSILs is urgently needed. The prolyl isomerase Pin1 is overexpressed in many cancers and contributes significantly to tumour initiation and progression. Therefore, it is important to assess the effects of cancer therapies that target Pin1. In our study, we demonstrated that Pin1 may serve as a biomarker for LSIL disease progression and may constitute a novel therapeutic target for CC. We used a the novel Pin1 inhibitor KPT-6566, which is able to covalently bind to Pin1 and selectively target it for degradation. The results of our investigation revealed that the downregulation of Pin1 by shRNA or KPT-6566 inhibited the growth of human cervical cancer cells (CCCs). We also discovered that the use of KPT-6566 is a novel approach to enhance the therapeutic efficacy of cisplatin (DDP) against CCCs in vitro and in vivo. We showed that KPT-6566-mediated inhibition of Pin1 blocked multiple cancer-driving pathways simultaneously in CCCs. Furthermore, targeted Pin1 treatment suppressed the metastasis and invasion of human CCCs, and downregulation of Pin1 reversed the epithelial-mesenchymal transition (EMT) of CCCs via the c-Jun/slug pathway. Collectively, we showed that Pin1 may be a marker for the risk of progression to HSIL and that inhibition of Pin1 has anticancer effects against CC.

Keywords Pin1      SIL      cell death      EMT      C-Jun      KPT-6566      cervical cancer     
Corresponding Authors: Cheng-Shi Quan   
About author:

These authors contributed equally to this work.

Just Accepted Date: 07 May 2019   Issue Date: 15 January 2020
E-mail this article
E-mail Alert
Articles by authors
Yan-Tong Guo
Yan Lu
Yi-Yang Jia
Hui-Nan Qu
Da Qi
Xin-Qi Wang
Pei-Ye Song
Xiang-Shu Jin
Wen-Hong Xu
Yuan Dong
Ying-Ying Liang
Cheng-Shi Quan
Cite this article:   
Yan-Tong Guo,Yan Lu,Yi-Yang Jia, et al. Predictive Value of Pin1 in Cervical Low-Grade Squamous Intraepithelial Lesions and Inhibition of Pin1 Exerts Potent Anticancer Activity against Human Cervical Cancer[J]. Aging and disease, 2020, 11(1): 44-59.
URL:     OR
Figure 1.  The expression of Pin1 and c-Jun in SILs and cervical cancer patient tissues. Prognostic value of Pin1 in LSIL patients. (A-D) Low vs. high expression of Pin1/c-Jun in cervical cancer issue specimens; (E-H) Low vs. high expression of Pin1/c-Jun in squamous intraepithelial lesions specimens. (original magnification × 200). (I, J) The expression of Pin1 and c-Jun in normal cervical tissues and different stages of cervical cancer patients detected by western blot. (* compared with normal group, P<0.05).
CharacteristicsNo.OutcomeP value
LSILPin1 statusNegative373430.001
c-Jun statusNegative252050.599
Table 1  Pin1 and c-Jun statuses in patients on follow-up examination
CharacteristicsNo.Pin1 statusP valueC-jun statusesP value
Negative No. (%)Positive No. (%)Negative No. (%)Positive No. (%)
Age (years)<5010460.724190.662
Lymph node statusAbsent2210120.0392200.443
Tumor size≤4 cm259160.9272230.611
>4 cm31203
Table 2  Pin1 and c-Jun statuses according to Age, Lymph node status, Tumor size, P16 and Ki67.
Figure 2.  Genic or chemical downregulation of Pin1 suppressed cell proliferation in CCCs. (A) Chemical synthesis steps of KPT-6566. (B) Mass spectrum of KPT-6566, ESI-MS: m/z 466.0 [M+Na]+. (C) Hydrogen spectroscopy to confirm chemical structure of KPT-6566, 1H NMR (300 MHz,DMSO-d6) δ 8.14 - 8.04 (m, 2H), 8.03 -7.97 (m, 2H), 7.90 - 7.87 (s, 1H), 7.87 - 7.80 (m, 2H), 7.76 - 7.69 (m, 2H), 3.99 (s, 2H), 1.35 (s, 9H). (D) Cell viability assay of the Hela-shPin1/Hela-shNC and SiHa-shPin1/SiHa-shNC for 24 h. (E) Hela, SiHa or HUVEC cells were treated with KPT-6566 and the growth curves were plotted over concentration. (F) Representative micrographs of the colonies of Hela-shPin1/SiHa-shPin1 were counted and compared with that of NC. (G) Representative micrographs of the colonies of Hela/SiHa treated with KPT-6566 were counted and compared with that of treated with DMSO. Each assay was performed in triplicate. *P<0.05.
Figure 3.  KPT-6566 targeted approach brings better CCCs killing effects with less dose of cisplatin. (A) Electron microscope findings in Hela/SiHa treated with 5 μM KPT-6566, 4 μM DDP and combation of 5 μM KPT-6566 and 4 μM DDP. (B, C) Flow cytometry to measure apoptosis of Hela/SiHa cells after treated with gradient concentration of DDP, compared with the combinational treatment. Annexin V-APC and 7-ADD positive cells were representative apoptotic cells. Each assay was performed in triplicate. *P < 0.05. (D, E) Cleaved-caspase-3, cleaved-PARP expression in Hela/SiHa treated with the same dose of DDP and KPT-6566 by using western blot assay. Each assay was performed in triplicate. * P<0.05
CharacteristicsNo.c-JunP value
Negative No. (%)Positive No. (%)
LSILPin1 statusNegative3719180.002
HSILPin1 statusNegative4130.747
CCPin1 statusNegative10280.049
Table 3  Correlation between Pin1 and c-Jun in SILs and SCC.
Figure 4.  KPT-6566 blocked multiple cancer-driving pathways simultaneously in CCCs. (A-D) Hela/SiHa cells were treated with 5 μM KPT-6566. Expression of Pin1, c-Jun, β-catenin, cyclinD1, A KT-p473, ERK1/2, p65/NF-Κb, GSTP1 and H2AX were detected by western blot assay with specific antibodies. Each assay was performed in triplicate. *P<0.05.
Figure 5.  KPT-6566 inhibited CC tumor growth by targeting Pin1 in nude mice. (A) Xenograft tumors were harvested 8 weeks after implantation. (B) SiHa tumor volumes were measured weekly for 8 weeks and the curves of tumor volumes were plotted over time. *P < 0.05. (C) Data points are presented as the means ± SD for tumor weights of the tumors were analyzed. *P < 0.05. (D) Expression of Pin1 in xenograft tumors of nude mice were detected by IHC (original magnification × 200). (E) The xenograft tumor sections were subjected to H&E staining, and the percentage of the necrosis areas were counted. *P < 0.05. (F) The percentage of CC cell apoptosis internal xenograft tumor was counted through TUNEL. *P < 0.05. (G) The heart, liver, spleen, kidney and lung sections of combinational treatment nude mice were subjected to H&E staining.
Figure 6.  Downregulation of Pin1 potently inhibited migration and invasion of CCCs. (A-D) Representative images show the migration and invasion abilities of Hela/SiHa cells with Pin1 stably knocked down or negative control and Hela/SiHa cells treated with KPT-6566 or DMSO. The number of cells was quantified. *P < 0.05. (E, F) Hepatic metastasis model. The liver sections were subjected to H&E staining, and the metastatic nodules are indicated with arrows. The number of metastatic nodules and necrosis areas in the liver specimens were analysed (original magnification × 200). *P <0.05.
Figure 7.  Downregulation of Pin1 potently suppressed EMT of CCCs via c-Jun/slug pathway. (A, B) The expression of Pin1, c-Jun, slug and EMT associated proteins including E-cadherin, N-cadherin and vimentin in Hela/SiHa cells after downregulation of Pin1 by shRNA or KPT-6566 were detected by Western blot assay. *P < 0.05. (C) Western blotting was performed to detect the expression of Pin1, c-Jun, slug and EMT associated proteins in Hela-shPin1/SiHa-shPin1 after c-Jun overexpression or vector. *P<0.05.
[1] Chu TY, Shen CY, Lee HS, Liu HS (1999). Monoclonality and surface lesion-specific microsatellite alterations in premalignant and malignant neoplasia of uterine cervix: a local field effect of genomic instability and clonal evolution. Genes Chromosomes Cancer, 24:127-134.
[2] Costa RF, Longatto-Filho A, Pinheiro C, Zeferino LC, Fregnani JH (2015). Historical Analysis of the Brazilian Cervical Cancer Screening Program from 2006 to 2013: A Time for Reflection. PLoS One, 10:e0138945.
[3] Tempfer CB, Tischoff I, Dogan A, Hilal Z, Schultheis B, Kern P, et al. (2018). Neuroendocrine carcinoma of the cervix: a systematic review of the literature. BMC Cancer, 18:530.
[4] Sagheb K, Blatt S, Kraft IS, Zimmer S, Rahimi-Nedjat RK, Al-Nawas B, et al. (2017). Outcome and cervical metastatic spread of squamous cell cancer of the buccal mucosa, a retrospective analysis of the past 25 years. J Oral Pathol Med, 46:460-464.
[5] Guidi A, Codeca C, Ferrari D (2018). Chemotherapy and immunotherapy for recurrent and metastatic head and neck cancer: a systematic review. Med Oncol, 35:37.
[6] Takekuma M, Kasamatsu Y, Kado N, Kuji S, Tanaka A, Takahashi N, et al. (2017). The issues regarding postoperative adjuvant therapy and prognostic risk factors for patients with stage I-II cervical cancer: A review. J Obstet Gynaecol Res, 43:617-626.
[7] Shu XR, Wu J, Sun H, Chi LQ, Wang JH (2015). PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway. Diagn Pathol, 10:177.
[8] Rustighi A, Zannini A, Campaner E, Ciani Y, Piazza S, Del Sal G (2017). PIN1 in breast development and cancer: a clinical perspective. Cell Death Differ, 24:200-211.
[9] Zhou XZ, Lu KP (2016). The isomerase PIN1 controls numerous cancer-driving pathways and is a unique drug target. Nat Rev Cancer, 16:463-478.
[10] Lee YM, Shin SY, Jue SS, Kwon IK, Cho EH, Cho ES, et al. (2014). The role of PIN1 on odontogenic and adipogenic differentiation in human dental pulp stem cells. Stem Cells Dev, 23:618-630.
[11] Lu Z, Hunter T (2014). Prolyl isomerase Pin1 in cancer. Cell Res, 24:1033-1049.
[12] Wei S, Kozono S, Kats L, Nechama M, Li W, Guarnerio J, et al. (2015). Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer. Nat Med, 21:457-466.
[13] Tan X, Zhou F, Wan J, Hang J, Chen Z, Li B, et al. (2010). Pin1 expression contributes to lung cancer: Prognosis and carcinogenesis. Cancer Biol Ther, 9:111-119.
[14] Ryo A, Liou YC, Wulf G, Nakamura M, Lee SW, Lu KP (2002). PIN1 is an E2F target gene essential for Neu/Ras-induced transformation of mammary epithelial cells. Mol Cell Biol, 22:5281-5295.
[15] Liao XH, Zhang AL, Zheng M, Li MQ, Chen CP, Xu H, et al. (2017). Chemical or genetic Pin1 inhibition exerts potent anticancer activity against hepatocellular carcinoma by blocking multiple cancer-driving pathways. Sci Rep, 7:43639.
[16] Nogues L, Reglero C, Rivas V, Salcedo A, Lafarga V, Neves M, et al. (2016). G Protein-coupled Receptor Kinase 2 (GRK2) Promotes Breast Tumorigenesis Through a HDAC6-Pin1 Axis. EBioMedicine, 13:132-145.
[17] Nakatsu Y, Matsunaga Y, Ueda K, Yamamotoya T, Inoue Y, Inoue MK, et al. (2018). Development of Pin1 inhibitors and their potential as therapeutic agents. Curr Med Chem.
[18] Yang D, Luo W, Wang J, Zheng M, Liao XH, Zhang N, et al. (2018). A novel controlled release formulation of the Pin1 inhibitor ATRA to improve liver cancer therapy by simultaneously blocking multiple cancer pathways. J Control Release, 269:405-422.
[19] Campaner E, Rustighi A, Zannini A, Cristiani A, Piazza S, Ciani Y, et al. (2017). A covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action. Nat Commun, 8:15772.
[20] Min SH, Lau AW, Lee TH, Inuzuka H, Wei S, Huang P, et al. (2012). Negative regulation of the stability and tumor suppressor function of Fbw7 by the Pin1 prolyl isomerase. Mol Cell, 46:771-783.
[21] Jo A, Yun HJ, Kim JY, Lim SC, Choi HJ, Kang BS, et al. (2015). Prolyl isomerase PIN1 negatively regulates SGK1 stability to mediate tamoxifen resistance in breast cancer cells. Anticancer Res, 35:785-794.
[22] Wulf G, Ryo A, Liou YC, Lu KP (2003). The prolyl isomerase Pin1 in breast development and cancer. Breast Cancer Res, 5:76-82.
[23] Cao L, Sun PL, Yao M, Chen S, Gao H (2017). Clinical significance of CK7, HPV-L1, and koilocytosis for patients with cervical low-grade squamous intraepithelial lesions: a retrospective analysis. Hum Pathol, 65:194-200.
[24] Alli PM, Ali SZ (2003). Atypical squamous cells of undetermined significance--rule out high-grade squamous intraepithelial lesion: cytopathologic characteristics and clinical correlates. Diagn Cytopathol, 28:308-312.
[25] Guido R, Schiffman M, Solomon D, Burke L, Group ALTS (2003). Postcolposcopy management strategies for women referred with low-grade squamous intraepithelial lesions or human papillomavirus DNA-positive atypical squamous cells of undetermined significance: a two-year prospective study. Am J Obstet Gynecol, 188:1401-1405.
[26] Castle PE, Gravitt PE, Wentzensen N, Schiffman M (2012). A descriptive analysis of prevalent vs incident cervical intraepithelial neoplasia grade 3 following minor cytologic abnormalities. Am J Clin Pathol, 138:241-246.
[27] Cox JT, Schiffman M, Solomon D, Group A-LTS (2003). Prospective follow-up suggests similar risk of subsequent cervical intraepithelial neoplasia grade 2 or 3 among women with cervical intraepithelial neoplasia grade 1 or negative colposcopy and directed biopsy. Am J Obstet Gynecol, 188:1406-1412.
[28] Bansal N, Wright JD, Cohen CJ, Herzog TJ (2008). Natural history of established low grade cervical intraepithelial (CIN 1) lesions. Anticancer Res, 28:1763-1766.
[29] Chen EY, Tran A, Raho CJ, Birch CM, Crum CP, Hirsch MS (2010). Histological 'progression' from low (LSIL) to high (HSIL) squamous intraepithelial lesion is an uncommon event and an indication for quality assurance review. Mod Pathol, 23:1045-1051.
[30] Katki HA, Gage JC, Schiffman M, Castle PE, Fetterman B, Poitras NE, et al. (2013). Follow-up testing after colposcopy: five-year risk of CIN 2+ after a colposcopic diagnosis of CIN 1 or less. J Low Genit Tract Dis, 17:S69-77.
[31] Herfs M, Parra-Herran C, Howitt BE, Laury AR, Nucci MR, Feldman S, et al. (2013). Cervical squamocolumnar junction-specific markers define distinct, clinically relevant subsets of low-grade squamous intraepithelial lesions. Am J Surg Pathol, 37:1311-1318.
[32] Paquette C, Mills AM, Stoler MH (2016). Predictive Value of Cytokeratin 7 Immunohistochemistry in Cervical Low-grade Squamous Intraepithelial Lesion as a Marker for Risk of Progression to a High-grade Lesion. Am J Surg Pathol, 40:236-243.
[33] Mills AM, Paquette C, Terzic T, Castle PE, Stoler MH (2017). CK7 Immunohistochemistry as a Predictor of CIN1 Progression: A Retrospective Study of Patients From the Quadrivalent HPV Vaccine Trials. Am J Surg Pathol, 41:143-152.
[34] Huang EC, Tomic MM, Hanamornroongruang S, Meserve EE, Herfs M, Crum CP (2016). p16ink4 and cytokeratin 7 immunostaining in predicting HSIL outcome for low-grade squamous intraepithelial lesions: a case series, literature review and commentary. Mod Pathol, 29:1501-1510.
[35] Cheng CW, Leong KW, Tse E (2016). Understanding the role of PIN1 in hepatocellular carcinoma. World J Gastroenterol, 22:9921-9932.
[36] Singh S (2015). Cytoprotective and regulatory functions of glutathione S-transferases in cancer cell proliferation and cell death. Cancer Chemother Pharmacol, 75:1-15.
[37] Chatterjee A, Gupta S (2018). The multifaceted role of glutathione S-transferases in cancer. Cancer Lett, 433:33-42.
[38] Gnoni A, Russo A, Silvestris N, Maiello E, Vacca A, Marech I, et al. (2011). Pharmacokinetic and metabolism determinants of fluoropyrimidines and oxaliplatin activity in treatment of colorectal patients. Curr Drug Metab, 12:918-931.
[39] Felix LM, Vidal AM, Serafim C, Valentim AM, Antunes LM, Monteiro SM, et al. (2018). Ketamine induction of p53-dependent apoptosis and oxidative stress in zebrafish (Danio rerio) embryos. Chemosphere, 201:730-739.
[40] F DOM, Gomes BC, Rodrigues AS, Rueff J (2017). Genetic Susceptibility in Acute Pancreatitis: Genotyping of GSTM1, GSTT1, GSTP1, CASP7, CASP8, CASP9, CASP10, LTA, TNFRSF1B, and TP53 Gene Variants. Pancreas, 46:71-76.
[41] Cabral BLS, da Silva ACG, de Avila RI, Cortez AP, Luzin RM, Liao LM, et al. (2017). A novel chalcone derivative, LQFM064, induces breast cancer cells death via p53, p21, KIT and PDGFRA. Eur J Pharm Sci, 107:1-15.
[42] Brzozowa-Zasada M, Piecuch A, Michalski M, Segiet O, Kurek J, Harabin-Slowinska M, et al. (2017). Notch and its oncogenic activity in human malignancies. Eur Surg, 49:199-209.
[43] Han X, Yoon SH, Ding Y, Choi TG, Choi WJ, Kim YH, et al. (2010). Cyclosporin A and sanglifehrin A enhance chemotherapeutic effect of cisplatin in C6 glioma cells. Oncol Rep, 23:1053-1062.
[44] Wang T, Liu Z, Shi F, Wang J (2016). Pin1 modulates chemo-resistance by up-regulating FoxM1 and the involvements of Wnt/beta-catenin signaling pathway in cervical cancer. Mol Cell Biochem, 413:179-187.
[45] Fukushima K, Okada A, Oe H, Hirasaki M, Hamori M, Nishimura A, et al. (2018). Pharmacokinetic-Pharmacodynamic Analysis of Cisplatin with Hydration and Mannitol Diuresis: The Contribution of Urine Cisplatin Concentration to Nephrotoxicity. Eur J Drug Metab Pharmacokinet, 43:193-203.
[46] Oh GS, Kim HJ, Shen A, Lee SB, Khadka D, Pandit A, et al.(2014). Cisplatin-induced Kidney Dysfunction and Perspectives on Improving Treatment Strategies. Electrolyte Blood Press, 12:55-65.
[47] Rocha CRR, Silva MM, Quinet A, Cabral-Neto JB, Menck CFM (2018). DNA repair pathways and cisplatin resistance: an intimate relationship. Clinics (Sao Paulo), 73:e478s.
[48] Kaestner B, Spicher K, Jaehde U, Enzmann H (2017). Effects of sorafenib and cisplatin on preneoplastic foci of altered hepatocytes in fetal turkey liver. Toxicol Res (Camb), 6:54-62.
[49] Galluzzi L, Vitale I, Michels J, Brenner C, Szabadkai G, Harel-Bellan A, et al. (2014). Systems biology of cisplatin resistance: past, present and future. Cell Death Dis, 5:e1257.
[50] Li X, Wei LC, Zhang Y, Zhao LN, Li WW, Ping LJ, et al. (2016). The Prognosis and Risk Stratification Based on Pelvic Lymph Node Characteristics in Patients With Locally Advanced Cervical Squamous Cell Carcinoma Treated With Concurrent Chemoradiotherapy. Int J Gynecol Cancer, 26:1472-1479.
[51] Kim MR, Choi HK, Cho KB, Kim HS, Kang KW (2009). Involvement of Pin1 induction in epithelial-mesenchymal transition of tamoxifen-resistant breast cancer cells. Cancer Sci, 100:1834-1841.
[52] Bong AHL, Monteith GR (2017). Breast cancer cells: Focus on the consequences of epithelial-to-mesenchymal transition. Int J Biochem Cell Biol, 87:23-26.
[53] Hagemann T, Bozanovic T, Hooper S, Ljubic A, Slettenaar VI, Wilson JL, et al. (2007). Molecular profiling of cervical cancer progression. Br J Cancer, 96:321-328.
[54] Kozono S, Lin YM, Seo HS, Pinch B, Lian X, Qiu C, et al. (2018). Arsenic targets Pin1 and cooperates with retinoic acid to inhibit cancer-driving pathways and tumor-initiating cells. Nat Commun, 9:3069.
[55] Hennig L, Christner C, Kipping M, Schelbert B, Rucknagel KP, Grabley S, et al. (1998). Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone. Biochemistry, 37:5953-5960.
[56] Uchida T, Takamiya M, Takahashi M, Miyashita H, Ikeda H, Terada T, et al. (2003). Pin1 and Par14 peptidyl prolyl isomerase inhibitors block cell proliferation. Chem Biol, 10:15-24.
[1] Supplementary data Download
[1] Jiangbo Song, Min Chen, Zhiquan Li, Jianfei Zhang, Hai Hu, Xiaoling Tong, Fangyin Dai. Astragalus Polysaccharide Extends Lifespan via Mitigating Endoplasmic Reticulum Stress in the Silkworm, Bombyx mori[J]. Aging and disease, 2019, 10(6): 1187-1198.
[2] Blokh David, Stambler Ilia. Information Theoretical Analysis of Aging as a Risk Factor for Heart Disease[J]. Aging and disease, 2015, 6(3): 196-207.
[3] Zhang Fan, Ho YuanWan, Fung* Helene H.. Learning from Normal Aging:Preserved Emotional Functioning Facilitates Adaptation among Early Alzheimer’s Disease Patients[J]. Aging and disease, 2015, 6(3): 208-215.
[4] Piano Amanda, Titorenko Vladimir I.. The Intricate Interplay between Mechanisms Underlying Aging and Cancer[J]. Aging and disease, 2015, 6(1): 56-75.
[5] Pedro Garrido. Aging and Stress: Past Hypotheses, Present Approaches and Perspectives[J]. Aging and Disease, 2011, 2(1): 80-99.
Full text



Copyright © 2014 Aging and Disease, All Rights Reserved.
Address: Aging and Disease Editorial Office 3400 Camp Bowie Boulevard Fort Worth, TX76106 USA
Fax: (817) 735-0408 E-mail:
Powered by Beijing Magtech Co. Ltd