PO.ET09.02 · 实验与分子治疗
p300溴结构域抑制剂通过诱导衰老抑制HPV阳性头颈部鳞状细胞癌的细胞增殖
p300 bromodomain inhibitors impair cell proliferation by inducing senescence in HPV positive head and neck squamous cell carcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
近几十年来,人乳头瘤病毒(HPV)相关头颈部鳞状细胞癌(HNSCC)的发病率急剧上升,目前已占美国新诊断口咽癌的大多数。标准治疗虽然有效,但会引起显著的毒性和长期并发症,凸显了对替代治疗策略的迫切需求。包括口咽肿瘤在内的HPV相关癌症为靶向治疗干预提供了独特的机会,因为其发病机制依赖于病毒癌蛋白E6和E7对主要抑癌通路的失活。一个关键的致癌机制是HPV E6能够结合转录共激活因子p300/CBP,从而阻断p53的乙酰化和激活。因此,我们假设p300/CBP溴结构域抑制剂(BDIs)可能破坏HPV E6-p300/CBP相互作用,并恢复p53抑癌程序。我们评估了两种p300/CBP BDIs(CCS1477和GNE-781)对HPV16+ HNSCC细胞系UM-SCC-47的影响。通过Incucyte®活细胞成像评估细胞增殖,通过集落形成实验评估克隆存活,通过碘化丙啶流式细胞术评估细胞周期分布。通过β-半乳糖苷酶染色测定衰老,并通过免疫印迹分析关键p53靶点的蛋白水平。两种p300/CBP BDIs均显著降低了细胞增殖和集落形成。增殖以剂量依赖方式下降,CCS1477的IC50为23.66 nM,GNE-781的IC50为9.06 nM。在100 nM时,CCS1477和GNE-781分别使集落形成减少94%(p<0.0001)和97.9%(p<0.0001)。p300/CBP BDIs诱导G1期阻滞,表现为G1期细胞比例增加15%(p<0.0001),S期细胞减少30%(p<0.0001)。两种抑制剂均使衰老细胞数量增加>8倍(p<0.0001)。免疫印迹分析显示p53、乙酰化p53(K382Ac)和p21水平升高,而caspase 3无变化,支持衰老而非凋亡是抗增殖作用的主要机制。总之,p300/CBP BDIs(CCS1477和GNE-781)在HPV16+ UM-SCC-47细胞中显示出抗肿瘤活性,可重新激活p53程序并诱导衰老。这些发现支持在其他HPV+癌症模型系统中进一步评估p300/CBP BDIs。
查看英文原文 English abstract
The incidence of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has risen sharply in recent decades and now accounts for most new oropharyngeal cancers diagnosed in the United States. Standard of care therapies are effective but cause significant toxicities and long-term morbidities, highlighting a critical need for alternative treatment strategies. HPV-associated cancers, including oropharyngeal tumors, present a unique opportunity for targeted therapeutic intervention since their pathogenesis depends on the inactivation of major tumor suppressor pathways by the viral oncoproteins E6 and E7. One key oncogenic mechanism is the ability of HPVE6 to bind the transcriptional co-activator p300/CBP, thereby blocking p53 acetylation and activation. Therefore, we hypothesized that p300/CBP bromodomain inhibitors (BDIs) may disrupt the HPVE6-p300/CBP interaction and restore the p53 tumor suppressor program. We evaluated the effect of two p300/CBP BDIs, CCS1477 and GNE-781, on the HPV16+ HNSCC cell line, UM-SCC-47. Cell proliferation was assessed by Incucyte® live cell imaging, clonogenic survival by colony formation assays, and cell-cycle distribution by flow cytometry with propidium iodide. Senescence was measured by beta-galactosidase staining, and protein levels of key p53 targets were analyzed by immunoblot. Both p300/CBP BDIs markedly reduced cell proliferation and colony formation. Proliferation decreased in a dose-dependent manner, with an IC50 of 23.66 nM for CCS1477 and 9.06 nM for GNE-781. At 100 nM, CCS1477 and GNE-781 reduced colony formation by 94% (p<0.0001) and 97.9% (p<0.0001), respectively. p300/CBP BDIs induced G1 arrest, reflected by a 15% (p<0.0001) increase in the proportion of cells in G1 and a 30% (p<0.0001) reduction in S phase. Both inhibitors triggered a >8-fold (p<0.0001) increase in the number of senescent cells. Immunoblot analysis showed increased levels of p53, acetylated p53 (K382Ac), and p21 with no change in caspase 3, supporting senescence, not apoptosis, as the main mechanism responsible for the anti-proliferative effects. In summary, the p300/CBP BDIs, CCS1477 and GNE-781, show anti-tumor activity in HPV16+ UM-SCC-47 cells, reactivating the p53 program and inducing senescence. These findings support further evaluation of p300/CBP BDIs in other HPV+ cancer model systems.
利益披露 Disclosure
J. Frutuoso do Nascimento, None..
W. O'Neill, None..
Q. Pan, None.