HPV16 E6 Induces Chromosomal Instability due to Misaligned Chromosomes Caused by E6AP-Dependent Degradation of the Mitotic Kinesin CENP-E

Abstract

<h3>Purpose/Objective(s)</h3> Chromosome missegregation over multiple cell cycles, termed chromosomal instability (CIN), is common in tumors and often leads to aneuploidy, a hallmark of cancer. High risk human papillomavirus (HPV) causes anogenital and head and neck cancers (HNC). HPV has been shown to cause certain types of CIN, but the mechanisms are unclear and this has not been studied in HNC. Here we describe a specific, novel mechanism of CIN induced by the HPV16 oncoprotein E6, in a physiologically relevant HNC model and in patient tumors. <h3>Materials/Methods</h3> Normal oral keratinocytes (NOKs) were transduced with retrovirus expressing LXSN control vector, HPV16 E6, E7 or E6+E7 and were kept under selection to achieve stable cell lines. CIN was quantified by assessing mitotic defects using immunofluorescence microscopy or H&E-stained slides in the NOKs, HNC cell lines, patient-derived xenografts (PDX), and in patient HNC tumors. Quantitative immunofluorescence and western blotting were used to quantify CENP-E levels. Tetracycline (tet) inducible GFP-CENP-E was introduced into NOKs expressing the Tet repressor using the PiggyBac transposon system. p53 and E6AP null NOKs were created using CRISPR. Significant differences were determined using a two-tailed student's t-test. <h3>Results</h3> HPV+ HNC cell lines have a significant increase specifically in misaligned chromosomes during mitosis, as compared to HPV- cell lines (20% v. 5% of cells, n=4 cell lines each). Using NOKs expressing individual HPV oncogenes, we found that these defects are specifically induced by the HPV oncogene E6 rather than E7. Further studies revealed that HPV16 E6 causes the specific degradation of CENP-E, a mitotic kinesin that plays a critical role in chromosome congression during mitosis. Tetracycline induced over-expression of GFP-CENP-E in LXSN- and E6-expressing NOKs revealed appropriate localization to kinetochores in LXSN cells. Strikingly, E6 mediated degradation of CENP-E was so efficient that CENP-E levels (total and at kinetochores) did not increase even after tet-inducible expression. Degradation of CENP-E occurred post-translationally in a proteasome dependent manner. Proteasome inhibition in E6-expressing NOKs rescued CENP-E protein levels at the kinetochore and rescued the misaligned chromosome phenotype. Furthermore, E6-mediated CENP-E degradation required the ubiquitin ligase E6AP, as evidenced by CENP-E protein restoration and rescue of chromosome alignment in E6AP null NOKs expressing E6. Finally, a significantly increased incidence of misaligned chromosomes was confirmed in HPV+ tumors from HNC patients and HPV+ PDXs. <h3>Conclusion</h3> This study reveals a novel, specific mechanism by which HPV16 E6 induces CIN in HNC, which may play a role in HPV-induced tumor initiation, progression and radiation response. Additionally, there is exciting potential for synergy with a CENP-E inhibitor, currently in clinical trials, and radiation to increase CIN beyond a viable threshold to increase cell death.