Lou Hong, Boland Joseph F, Li Hongchuan, Burk Robert, Yeager Meredith, Anderson Stephen K, Wentzensen Nicolas, Schiffman Mark, Mirabello Lisa, Dean Michael
Cancer Genetics Research Laboratory, Division of Cancer Epidemiology and Genetics, Frederick National Laboratory for Cancer Research, Rockville, MD 20850, USA.
Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
Cancers (Basel). 2022 Oct 6;14(19):4895. doi: 10.3390/cancers14194895.
The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development.
人乳头瘤病毒16型（HPV-16）E7癌基因对致癌作用至关重要且高度保守。先前的研究发现，与宫颈癌患者相比，HPV-16阳性的无癌对照者中存在大量非同义E7变异体。为了研究E7变异体的功能，我们构建了全长HPV-16 E7基因，并对H9R、D21N、N29S、E33K、T56I、D62N、S63F、S63P、T64M、E80K、D81N、P92L和P92S位点（仅在对照者中发现）的变异体，以及D14E、N29H（宫颈上皮内瘤变2级，CIN2）和P6L、H51N、R77S（CIN3）进行了检测。我们测定了细胞质和细胞核中HPV-16 E7蛋白的稳态水平。对照者的所有变异体均显示E7蛋白水平降低，其中13个变异体中有7个蛋白水平较低。相比之下，CIN3癌前病变组的3个变异体中有2个的E7水平接近野生型。我们检测了稳定转染的NIH3T3细胞中代表性变异体的活性。在对照者中发现的H9R、E33K、P92L和P92S变异体的转化活性低于D14E和N29H变异体（CIN2），而R77S（CIN3）的活性仅比野生型E7略有降低。此外，在伤口愈合试验中，与H9R、E33K、P92L和P92S（对照者）以及D14E（CIN2）相比，R77S和野生型E7导致NIH3T3细胞迁移增加。这些数据表明，在HPV-16阳性的无癌女性中发现的E7变异体在转化和细胞迁移方面存在部分缺陷，进一步证明了完全活性的E7在癌症发展中的重要性。
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