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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.
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Abstract  

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   
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These authors contributed equally to this work.

Just Accepted Date: 07 May 2019   Issue Date: 15 January 2020
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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.
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http://www.aginganddisease.org/EN/10.14336/AD.2019.0415     OR     http://www.aginganddisease.org/EN/Y2020/V11/I1/44
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
LSILHSIL
LSILPin1 statusNegative373430.001
Positive352114
c-Jun statusNegative252050.599
Positive473512
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
≥5018612117
Lymph node statusAbsent2210120.0392200.443
Present60606
Tumor size≤4 cm259160.9272230.611
>4 cm31203
p16Negative000NA00NA
Positive281018226
Ki671%-25%0000.01400<0.001
25%-50%00000
50%-75%33021
>75%25718025
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
Positive35629
HSILPin1 statusNegative4130.747
Positive615
CCPin1 statusNegative10280.049
Positive18018
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.
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