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 4

Establishment and optimization of regional TALEN application to the uterine cervixes of K14-HPV16 transgenic mice.

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Establishment and optimization of regional TALEN application to the uter...
(A) FLAG-tagged HPV16-E7-T512 was fused with mRFP to monitor its expression and distribution. bGH pA, bovine growth hormone and polyadenylation signal. (B) Localized expression of HPV16-E7-T512 in the cervical epithelium. The right panel shows the boxed region in the left panel at higher magnification. Scale bars: 200 μm (left panel); 40 μm (right panel). n = 3 per group. (C) Optimization of the transfection efficiency through the use of a range of DNA-to-polymer ratios. Ten micrograms of the mRFP expression plasmids that were incubated with the corresponding volumes of polymer were transfected intravaginally. At days 2, 4, and 6, exfoliated cervical cells from the treated mice were gathered (with a process similar to a Pap smear test), and the mRFP-positive cells were counted. Data represent mean ± SD; n = 3 for each group of treated mice. (D) HPV16-E7-T512 was applied at 2-day, 3-day, or 6-day intervals for 24 days, and the E7 expression levels were assessed using IHC staining at treatment days 0, 12, 18, and 24. The integrated optical density (IOD) values were measured using Image-Pro PLUS (version 6.0). Data represent mean ± SD; *P < 0.05, **P < 0.01, 2-tailed Student’s t test; n = 3 per group. (E) Representative data from H&E staining and E7 IHC staining at treatment days 0, 12, 18, and 24. The mice were treated with HPV16-E7-T512 using the 3-day interval regimen. n = 3 per group. Scale bars: 80 μm.