TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy
Zheng Hu, … , Ding Ma, Hui Wang
Zheng Hu, … , Ding Ma, Hui Wang
Published December 15, 2014
Citation Information: J Clin Invest. 2015;125(1):425-436. https://doi.org/10.1172/JCI78206.
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Research Article Oncology

TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy

Abstract

Persistent HPV infection is recognized as the main etiologic factor for cervical cancer. HPV expresses the oncoproteins E6 and E7, both of which play key roles in maintaining viral infection and promoting carcinogenesis. While siRNA-mediated targeting of E6 and E7 transcripts temporarily induces apoptosis in HPV-positive cells, it does not eliminate viral DNA within the host genome, which can harbor escape mutants. Here, we demonstrated that specifically targeting E6 and E7 within host DNA with transcription activator–like effector nucleases (TALENs) induces apoptosis, inhibits growth, and reduces tumorigenicity in HPV-positive cell lines. TALEN treatment efficiently disrupted E6 and E7 oncogenes, leading to the restoration of host tumor suppressors p53 and retinoblastoma 1 (RB1), which are targeted by E6 and E7, respectively. In the K14-HPV16 transgenic mouse model of HPV-driven neoplasms, direct cervical application of HPV16-E7–targeted TALENs effectively mutated the E7 oncogene, reduced viral DNA load, and restored RB1 function and downstream targets transcription factor E2F1 and cycling-dependent kinase 2 (CDK2), thereby reversing the malignant phenotype. Together, the results from our study suggest that TALENs have potential as a therapeutic strategy for HPV infection and related cervical malignancy.

Authors

Zheng Hu, Wencheng Ding, Da Zhu, Lan Yu, Xiaohui Jiang, Xiaoli Wang, Changlin Zhang, Liming Wang, Teng Ji, Dan Liu, Dan He, Xi Xia, Tao Zhu, Juncheng Wei, Peng Wu, Changyu Wang, Ling Xi, Qinglei Gao, Gang Chen, Rong Liu, Kezhen Li, Shuang Li, Shixuan Wang, Jianfeng Zhou, Ding Ma, Hui Wang

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Figure 3

Efficacies of HPV subtype-specific TALENs in HPV-infected, precancerous, and cancer cells.

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Efficacies of HPV subtype-specific TALENs in HPV-infected, precancerous,...
(A and B) The representative images of (A) FISH and (B) γ-H2AX foci (red signals) in vector-, T27-, and T512-treated SiHa and S12 cells and in vector-, T34-, and T519-treated HeLa cells. Cell nuclei were indicated by DAPI staining (blue). n = 3 per group. Scale bars: 20 μm. (C–F) Detection of E6/E7 mutations using T7EI assays in SiHa, HeLa, and S12 cells. The sizes of the total PCR fragments (top one) and the cut fragments (bottom two) are marked with arrows. The numbers below the lanes represent the percentage of cleaved PCR products. Pos, T7EI-treated PCR fragments that contained a 2-bp deletion in the middle of the spacer region of the corresponding TALEN were mixed at 1:1 with PCR products that were amplified from TALEN-treated cells. Neg, PCR products that were amplified from TALEN-treated cells and not treated by T7EI. n = 3 per group. (G–J) Western blotting analysis of the E6 and E7 oncoproteins and their downstream proteins in SiHa or HeLa cells 48 and 72 hours after treatment with the corresponding TALENs. (G) HPV16 E6 and p53 in SiHa cells after treatment with HPV16-E6-T27. (H) HPV18 E6 and p53 in HeLa cells after treatment with HPV18-E6-T34. (I) HPV16 E7, RB1, and CDK2 in SiHa cells after treatment with HPV16-E7-T512. (J) HPV18 E7, RB1, and CDK2 in HeLa cells after treatment with HPV18-E7-T519. n = 3 per group.