Kennedy Edward M, Kornepati Anand V R, Goldstein Michael, Bogerd Hal P, Poling Brigid C, Whisnant Adam W, Kastan Michael B, Cullen Bryan R
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
J Virol. 2014 Oct;88(20):11965-72. doi: 10.1128/JVI.01879-14. Epub 2014 Aug 6.
High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas and are linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence. Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in the induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16- and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide a proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers. Importance: Human papillomaviruses (HPVs) are the causative agents of almost all cervical carcinomas and many other tumors, including many head and neck cancers. In these cancer cells, the HPV DNA genome is integrated into the cellular genome, where it expresses high levels of two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability. Here, we demonstrate that the recently described bacterial CRISPR/Cas RNA-guided endonuclease can be reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed by HPV, resulting in cell cycle arrest, leading to cancer cell death. We propose that viral vectors designed to deliver E6- and/or E7-specific CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and eliminate HPV-induced cancers.
高危型人乳头瘤病毒（HPV），包括HPV - 16和HPV - 18，是宫颈癌的致病因子，且与肛门生殖器和口咽区域的其他几种肿瘤有关。大多数HPV诱导的肿瘤含有通常为游离型的HPV基因组的整合拷贝，这些拷贝总是保留两个HPV癌基因E6和E7的完整形式。E6诱导细胞肿瘤抑制因子p53的降解，而E7使视网膜母细胞瘤（Rb）蛋白不稳定。先前的研究表明，宫颈癌细胞中E6功能的丧失会诱导p53表达以及诱导细胞凋亡和细胞周期停滞的下游效应分子。同样，E7的缺失会使Rb表达增加，导致细胞周期停滞和衰老。在此，我们证明，细菌Cas9 RNA引导的核酸内切酶与针对E6或E7的单向导RNA（sgRNA）一起表达，能够诱导HPV基因组的切割，从而导致E6或E7基因中引入失活的缺失和插入突变。这导致p53或Rb的诱导，进而导致细胞周期停滞并最终导致细胞死亡。发现HPV - 16和HPV - 18转化的细胞均对靶向HPV基因组特异性DNA切割有反应。这些数据为载体递送的Cas9 / sgRNA组合可能代表HPV诱导的癌症的有效治疗方式这一观点提供了原理证明。重要性：人乳头瘤病毒（HPV）是几乎所有宫颈癌以及许多其他肿瘤（包括许多头颈癌）的致病因子。在这些癌细胞中，HPV DNA基因组整合到细胞基因组中，在那里它表达高水平的两种病毒癌基因，即E6和E7，这是癌细胞生长和存活所必需的。在此，我们证明，最近描述的细菌CRISPR / Cas RNA引导的核酸内切酶可以重新编程，以靶向和破坏HPV转化的宫颈癌细胞中的E6或E7基因，导致细胞周期停滞，从而导致癌细胞死亡。我们提出，设计用于将E6和/或E7特异性CRISPR / Cas递送至肿瘤细胞的病毒载体可能代表一种治疗和消除HPV诱导的癌症的新型高效工具。
