Doxycycline down-regulates DNA-PK and radiosensitizes tumor initiating cells: Implications for more effective radiation therapy.
Doxycycline down-regulates DNA-PK and radiosensitizes tumor initiating cells: Implications for more effective radiation therapy.
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DOI：
10.18632/oncotarget.4159
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发表时间：
2015-06-10
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影响因子：
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通讯作者：
Lisanti MP
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作者：
Lamb R;Fiorillo M;Chadwick A;Ozsvari B;Reeves KJ;Smith DL;Clarke RB;Howell SJ;Cappello AR;Martinez-Outschoorn UE;Peiris-Pagès M;Sotgia F;Lisanti MP
DNA-PK是一种DNA修复所需的酶，被认为可以赋予癌细胞放射抗性。因此，它是新药开发的一个备受瞩目的验证目标。然而，没有FDA批准的DNA-PK抑制剂出现，尽管多年的药物发现和铅优化。这主要是因为现有的DNA-PK抑制剂的药代动力学较差。它们不能很好地吸收和/或不稳定，血浆半衰期短。在这里，我们通过“化学蛋白质组学”鉴定了第一个FDA批准的DNA-PK抑制剂。为了了解多西环素如何靶向癌症干细胞（CSC），我们偶然发现多西环素使DNA-PK蛋白表达降低了近15倍（> 90%）。根据这些观察结果，我们发现强力霉素在功能上使乳腺CSC放射增敏高达4.5倍。此外，我们证明DNA-PK在MCF 7和T47 D衍生的乳腺球中高度过表达。有趣的是，MCF 7细胞中DNA-PK的遗传或药理学抑制足以在功能上阻断乳腺球形成。因此，看起来主动DNA修复是CSC克隆扩增所需的。从机制上讲，多西环素治疗显著降低了癌细胞的氧化线粒体能力和糖酵解活性，这与先前将DNA-PK表达与线粒体DNA完整性和拷贝数的适当维持联系起来的研究一致。使用基于腺苷酸酶的测定，我们观察到多西环素处理定量地降低了抗氧化反应（NRF 1/2），并有效地阻断了通常与干细胞相关的多个独立途径的信号传导，包括STAT 1/3、Sonic Hedgehog（Shh）、Notch、WNT和TGF-β信号传导。总之，我们建议，多西环素作为DNA-PK抑制剂的疗效应在II期临床试验中进行测试，与放射治疗相结合。强力霉素具有良好的药代动力学，在每天200 mg的标准剂量下，几乎100%口服吸收和长血清半衰期（18-22小时）。为了进一步支持这一观点，我们表明，强力霉素有效地抑制原发性乳腺癌样本的乳腺球形成活性，来自转移性疾病部位（胸腔积液或腹水）。我们的研究结果也可能对脑肿瘤和/或脑转移瘤的放射治疗产生影响，因为已知强力霉素可以有效地穿过血脑屏障。还需要进一步的研究来确定其他四环素家族成员是否也具有放射敏感性。
DNA-PK is an enzyme that is required for proper DNA-repair and is thought to confer radio-resistance in cancer cells. As a consequence, it is a high-profile validated target for new pharmaceutical development. However, no FDA-approved DNA-PK inhibitors have emerged, despite many years of drug discovery and lead optimization. This is largely because existing DNA-PK inhibitors suffer from poor pharmacokinetics. They are not well absorbed and/or are unstable, with a short plasma half-life. Here, we identified the first FDA-approved DNA-PK inhibitor by “chemical proteomics”. In an effort to understand how doxycycline targets cancer stem-like cells (CSCs), we serendipitously discovered that doxycycline reduces DNA-PK protein expression by nearly 15-fold (> 90%). In accordance with these observations, we show that doxycycline functionally radio-sensitizes breast CSCs, by up to 4.5-fold. Moreover, we demonstrate that DNA-PK is highly over-expressed in both MCF7- and T47D-derived mammospheres. Interestingly, genetic or pharmacological inhibition of DNA-PK in MCF7 cells is sufficient to functionally block mammosphere formation. Thus, it appears that active DNA-repair is required for the clonal expansion of CSCs. Mechanistically, doxycycline treatment dramatically reduced the oxidative mitochondrial capacity and the glycolytic activity of cancer cells, consistent with previous studies linking DNA-PK expression to the proper maintenance of mitochondrial DNA integrity and copy number. Using a luciferase-based assay, we observed that doxycycline treatment quantitatively reduces the anti-oxidant response (NRF1/2) and effectively blocks signaling along multiple independent pathways normally associated with stem cells, including STAT1/3, Sonic Hedgehog (Shh), Notch, WNT and TGF-beta signaling. In conclusion, we propose that the efficacy of doxycycline as a DNA-PK inhibitor should be tested in Phase-II clinical trials, in combination with radio-therapy. Doxycycline has excellent pharmacokinetics, with nearly 100% oral absorption and a long serum half-life (18–22 hours), at a standard dose of 200-mg per day. In further support of this idea, we show that doxycycline effectively inhibits the mammosphere-forming activity of primary breast cancer samples, derived from metastatic disease sites (pleural effusions or ascites fluid). Our results also have possible implications for the radio-therapy of brain tumors and/or brain metastases, as doxycycline is known to effectively cross the blood-brain barrier. Further studies will be needed to determine if other tetracycline family members also confer radio-sensitivity.