Fanconi anemia repair pathway dysfunction, a potential therapeutic target in lung cancer.
Fanconi anemia repair pathway dysfunction, a potential therapeutic target in lung cancer.
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DOI：
10.3389/fonc.2014.00368
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发表时间：
2014
影响因子：
4.7
通讯作者：
Villalona-Calero MA
中科院分区：
文献类型：
--
作者：
Duan W;Gao L;Aguila B;Kalvala A;Otterson GA;Villalona-Calero MA
Fanconi贫血（FA）途径是同源重组DNA修复的主要机制。该途径的功能解读是通过FANCD2的单泛素化激活，导致核病灶的修复。我们最近开发了一种基于FA三染色免疫荧光的方法（FATSI）来评估福尔马林固定石蜡包埋（FFPE）肿瘤样本中FANCD2病灶的形成。考虑到在这种环境下吸烟造成的损伤持续存在，以及考虑到dna损伤药物可能提高疗效，dna修复缺陷被认为是肺癌预防的兴趣所在。我们通过FATSI分析筛选了139例非小细胞肺癌（NSCLC） FFPE肿瘤的FANCD2灶形成。在104个可评估的肿瘤中，23个（22%）为FANCD2灶阴性，因此存在修复缺陷。为了评估和比较FA缺乏背景下的新型靶向药物，我们利用RNAi技术使几种肺癌细胞系FANCD2缺乏。FANCD2蛋白的减少证实了FANCD2蛋白的成功敲除。随后，我们用PARP抑制剂veliparib (ABT-888) （5 μM）和BMN673 （0.5 μM）以及CHK1抑制剂ry-575 （0.5 μM）处理FA缺陷的H1299D2-down和A549D2-down NSCLC细胞及其FA正常对照（空载体对照）。用2 μM剂量的BCL-2/XL抑制剂ABT-263治疗FA缺陷小细胞肺癌细胞株H719D2-down和H792D2-down及其对照。处理后的细胞在处理后24、48和72 h收获。MTT细胞活力分析显示，每种药物对FANCD2敲除细胞的细胞毒性更大。在所有的测试中，与对照组相比，FA缺陷肺癌细胞在治疗72小时后具有较少的活细胞。比较两种PARP抑制剂的MTT和克隆原分析显示，BMN673比veliparib更有效。鉴于FA通路在DNA损伤反应中起着重要作用，我们的研究结果表明，一部分肺癌患者可能更容易接受基于DNA交联的治疗，或者针对其他修复机制的治疗。这些受试者可以通过FATSI分析来识别。需要临床试验来评估这种治疗概念。
The Fanconi anemia (FA) pathway is a major mechanism of homologous recombination DNA repair. The functional readout of the pathway is activation through mono-ubiquitination of FANCD2 leading to nuclear foci of repair. We have recently developed an FA triple-staining immunofluorescence based method (FATSI) to evaluate FANCD2 foci formation in formalin fixed paraffin-embedded (FFPE) tumor samples. DNA-repair deficiencies have been considered of interest in lung cancer prevention, given the persistence of damage produced by cigarette smoke in this setting, as well as in treatment, given potential increased efficacy of DNA-damaging drugs. We screened 139 non-small cell lung cancer (NSCLC) FFPE tumors for FANCD2 foci formation by FATSI analysis. Among 104 evaluable tumors, 23 (22%) were FANCD2 foci negative, thus repair deficient. To evaluate and compare novel-targeted agents in the background of FA deficiency, we utilized RNAi technology to render several lung cancer cell lines FANCD2 deficient. Successful FANCD2 knockdown was confirmed by reduction in the FANCD2 protein. Subsequently, we treated the FA defective H1299D2-down and A549D2-down NSCLC cells and their FA competent counterparts (empty vector controls) with the PARP inhibitors veliparib (ABT-888) (5 μM) and BMN673 (0.5 μM), as well as the CHK1 inhibitor Arry-575 at a dose of 0.5 μM. We also treated the FA defective small cell lung cancer cell lines H719D2-down and H792D2-down and their controls with the BCL-2/XL inhibitor ABT-263 at a dose of 2 μM. The treated cells were harvested at 24, 48, and 72 h post treatment. MTT cell viability analysis showed that each agent was more cytotoxic to the FANCD2 knock-down cells. In all tests, the FA defective lung cancer cells had less viable cells as comparing to controls 72 h post treatment. Both MTT and clonogenic analyses comparing the two PARP inhibitors, showed that BMN673 was more potent compared to veliparib. Given that FA pathway plays essential roles in response to DNA damage, our results suggest that a subset of lung cancer patients are likely to be more susceptible to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. These subjects can be identified through FATSI analysis. Clinical trials to evaluate this therapeutic concept are needed.