HPV 5 and 8 E6 Abrogate ATR Activity Resulting in Increased Persistence of UVB Induced DNA Damage

The role of the E6 oncoprotein from high-risk members of the α human papillomavirus genus in anogenital cancer has been well established. However, far less is known about the E6 protein from the β human papillomavirus genus (β-HPVs). Some β-HPVs potentially play a role in non-melanoma skin cancer development, although they are not required for tumor maintenance. Instead, they may act as a co-factor that enhances the carcinogenic potential of UV damage. Indeed, the E6 protein from certain β-HPVs (HPV 5 and 8) promotes the degradation of p300, a histone acetyl transferase involved in UV damage repair. Here, we show that the expression of HPV 5 and 8 E6 increases thymine dimer persistence as well as the likelihood of a UVB induced double strand break (DSB). Importantly, we provide a mechanism for the increased DNA damage by showing that both extended thymine dimer persistence as well as elevated DSB levels are dependent on the ability of HPV 8 E6 to promote p300 degradation. We further demonstrate that HPV 5 and 8 E6 expression reduces the mRNA and protein levels of ATR, a PI3 kinase family member that plays a key role in UV damage signaling, but that these levels remain unperturbed in cells expressing a mutated HPV 8 E6 incapable of promoting p300 degradation. We confirm that the degradation of p300 leads to a reduction in ATR protein levels, by showing that ATR levels rebound when a p300 mutant resistant to HPV 8 mediated degradation and HPV 8 E6 are co-transfected. Conversely, we show that ATR protein levels are reduced when p300 is targeted for degradation by siRNA. Moreover, we show the reduced ATR levels in HPV 5 and 8 E6 expressing cells results in delayed ATR activation and an attenuated ability of cells to phosphorylate, and as a result accumulate, p53 in response to UVB exposure, leading to significantly reduced cell cycle arrest. In conclusion, these data demonstrate that β-HPV E6 expression can enhance the carcinogenic potential of UVB exposure by promoting p300 degradation, resulting in a reduction in ATR levels, which leads to increased thymine dimer persistence and increased UVB induced DSBs.高危α人乳头瘤病毒属（α human papillomavirus genus）成员的E6癌蛋白（E6 oncoprotein）在肛门生殖器癌（anogenital cancer）发生中的作用已得到充分阐明。然而，人们对β人乳头瘤病毒属（β human papillomavirus genus, β-HPVs）的E6蛋白的了解却极为有限。部分β-HPV可能参与非黑色素瘤皮肤癌（non-melanoma skin cancer）的发生发展，尽管其并非肿瘤维持所必需。相反，它们可能作为辅助因子（co-factor）增强紫外线损伤（UV damage）的致癌潜能。诚然，特定β-HPV（如HPV 5和8型）的E6蛋白可促进p300的降解——p300是一种参与紫外线损伤修复（UV damage repair）的组蛋白乙酰转移酶（histone acetyl transferase）。本研究证实，HPV 5和8型E6的表达会延长胸腺嘧啶二聚体（thymine dimer）的存留时间，并增加紫外线B（UVB）诱导的双链断裂（double strand break, DSB）发生概率。重要的是，我们阐明了DNA损伤加剧的分子机制：延长的胸腺嘧啶二聚体存留时间与升高的DSB水平，均依赖于HPV 8型E6促进p300降解的能力。我们进一步证实，HPV 5和8型E6的表达会降低ATR的信使RNA（mRNA）与蛋白水平——ATR是磷脂酰肌醇3-激酶（PI3 kinase）家族成员，在紫外线损伤信号通路（UV damage signaling）中发挥关键作用；而在表达无法介导p300降解的突变型HPV 8型E6的细胞中，上述分子水平未受影响。我们通过实验验证了p300降解可导致ATR蛋白水平下调：当将抵抗HPV 8型介导降解的p300突变体与HPV 8型E6共转染时，ATR的蛋白水平可恢复。反之，当通过小干扰RNA（siRNA）靶向降解p300时，ATR的蛋白水平会降低。此外，我们发现，表达HPV 5和8型E6的细胞中ATR水平降低，会导致ATR激活延迟，以及细胞在UVB暴露后磷酸化p53并积累p53的能力减弱，最终造成显著的细胞周期阻滞（cell cycle arrest）缺陷。综上，本研究数据表明，β-HPV的E6表达可通过促进p300降解、下调ATR水平，进而延长胸腺嘧啶二聚体存留时间并增加UVB诱导的DSB发生，最终增强UVB暴露的致癌潜能。