In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9

Highlights

• Established CRISPR/Cas9 targeting promoter of HPV 16 and targeting E6, E7 transcript.

• CRISPR/Cas9 resulted in accumulation of p53 and p21, reduced the proliferation of cervical cancer cells.

• Finding inhibited tumorigenesis and growth of mice incubated by cells with CRISPR/Cas9.

• CRISPR/Cas9 will be a new treatment strategy, in cervical and other HPV-associated cancer therapy.

Abstract

Deregulated expression of high-risk human papillomavirus oncogenes (E6 and E7) is a pivotal event for pathogenesis and progression in cervical cancer. Both viral oncogenes are therefore regarded as ideal therapeutic targets. In the hope of developing a gene-specific therapy for HPV-related cancer, we established CRISPR/Cas9 targeting promoter of HPV 16 E6/E7 and targeting E6, E7 transcript, transduced the CRISPR/Cas9 into cervical HPV-16-positive cell line SiHa. The results showed that CRISPR/Cas9 targeting promoter, as well as targeting E6 and E7 resulted in accumulation of p53 and p21 protein, and consequently remarkably reduced the abilities of proliferation of cervical cancer cells in vitro. Then we inoculated subcutaneously cells into nude mice to establish the transplanted tumor animal models, and found dramatically inhibited tumorigenesis and growth of mice incubated by cells with CRISPR/Cas9 targeting (promoter+E6+E7)-transcript. Our results may provide evidence for application of CRISPR/Cas9 targeting HR-HPV key oncogenes, as a new treatment strategy, in cervical and other HPV-associated cancer therapy.

Introduction

Cervical cancer is the second most common type of malignancy among women worldwide and a leading cause of mortality among females in developing countries. It affects nearly half a million women each year, claiming a quarter of a million lives [1], [2]. According to several epidemiological and biological studies, human papilloma viruses (HPV) infection is the dominant etiological event in CC development [3], HPV DNA can be detected in more than 95% of cervical cancer and intraepithelial neoplasia biopsies [4], demonstrating the strong association of this cancer with HPV infection.

Human papillomaviruses (HPVs) are small DNA viruses with a genome of approximately 8 kb. A small subgroup, types 16, 18, 31, 33, and 45, has been designated high-risk HPVs and found to be associated with more that 90% of cervical cancers [5], of which HPV16 accounts for approximately 50% [6]. The oncogenic function of HPVs has been primarily attributed to E6 and E7, which are essential for malignant transformation and pivotal for maintenance of the malignant phenotype of cervical cancer [7], [8]. They are selectively expressed in HPV-related cancer cells to inactively tumor suppressor proteins such as P53 and pRb, leading to cell cycle disorder, telomerase activation, and cell immortalization. E6 is able to induce the degradation of p53, thereby inhibiting p53-dependent signaling, and contributing to tumorigenesis [9], [10]. In the case of E7, deregulation of the host cell cycle seems to be a major function of the oncoprotein. It is associated with the retinoblastoma family of proteins: pRb [11], [12], p107, and p130 [13], [14]), as well as cyclin-dependent kinase inhibitors of p21 [15], [16] and p27 [17], and prevents G1 arrest in response to a variety of antiproliferative signals, such as growth factor withdrawal, loss of cell adhesion, and DNA damage [18]. Because of the strong relationship between the expression of this group of HPV oncogenes and cervical cancer carcinogenesis, development of novel approaches directed against these oncogenes may provide a novel block therapy for cervical cancer.

Clustered regularly interspaced short palindromic repeats CRISPR-associated (Cas9) is a recently discovered, site-specific genome editing system that is part of the CRISPR–Cas bacterial acquired immune system, which cleaves foreign DNA [19], [20]. And the CRISPR/Cas9 system requires the design of only a single guide sequence that matches the DNA targeted for cleavage. Since the first report of the use of CRISPR/Cas9 for genome editing in human cells in 2013 [21], [22], [23], this technology has been used in vivo in human cells and other organisms. Molecular-targeting therapy with Cas9 is expected to prevent the development for disease caused by foreign genes, such as human immunodeficiency virus (HIV) infection, by inhibiting expression of genes [24]. And Fujii et al. [25] indicate the availability of CRISPR/Cas9 system for the large-scale deletion and imply the system is highly efficient for genome modification. Therefore, strategies for achieving specific and selective CRISPR/Cas9 to abrogate E6 and E7 function may be a rational therapeutic approach for treating HPV-positive cervical cancers.

In the hope of applying CRISPR/Cas9 as therapy for HPV-related cancer, in this study, we have designed a CRISPR/Cas9 system that targets E6 and E7. We have shown that a CRISPR/Cas9 system can effectively, specifically and stable suppress HPV-16 E6 and E7 expression in vitro and in vivo, and also demonstrated that the cervical tumor growth could be inhibited for mouse by CRISPR/Cas9 treatment.