Department of Therapeutic Radiology, Yale University, New Haven, CT.
Transl Oncol. 2013 Jun 1;6(3):382-91. doi: 10.1593/tlo.13232. Print 2013 Jun.
The endoplasmic reticulum (ER) provides a specialized environment for the folding and modification of trans-membrane proteins, including receptor tyrosine kinases (RTKs), which are vital for the growth and survival of malignancies. To identify compounds which disrupt the function of the ER and thus could potentially impair cancer cell survival signaling, we adapted a set of glycosylation-sensitive luciferase reporters for the development and optimization of a cell-based high-throughput screen (HTS). Secondary screens for false-positive luciferase activation and tertiary lectin-based and biochemical analyses were also devised for compound triage. Through a pilot screen of 2802 compounds from the National Cancer Institute (NCI) chemical libraries, we identified aclacinomycin (Acm) as a compound that preferentially affects ER function. We report that Acm reduces plasma membrane expression of glycoproteins including epidermal growth factor receptor (EGFR) and Met but does not inhibit N-linked glycosylation or generalized protein translation. Fluorescence microscopy co-localization experiments were also performed and demonstrated Acm accumulation in the ER in further support of the overall HTS design. The consequences of Acm treatment on cell survival were analyzed through clonogenic survival analysis. Consistent with the reduction of EGFR levels, pretreatment with Acm sensitizes the EGFR-mutant non-small cell lung cancer (NSCLC) cell lines HCC827 and HCC2935 to ionizing radiation and did not affect the sensitivity of the RTK-independent and KRAS-mutant A549 NSCLC cell line. Thus, Acm and similar compounds targeting the ER may represent a novel approach for radiosensitizing tumor cells dependent on RTK function.
内质网（ER）为跨膜蛋白的折叠和修饰提供了一个特殊的环境，包括受体酪氨酸激酶（RTKs），它们对恶性肿瘤的生长和存活至关重要。为了鉴定破坏 ER 功能的化合物，从而可能损害癌细胞存活信号，我们适应了一组糖基化敏感的荧光素酶报告基因，用于开发和优化基于细胞的高通量筛选（HTS）。还设计了用于化合物分类的二次筛选，以排除假阳性荧光素酶激活，以及基于三级凝集素的和生化分析。通过对来自国家癌症研究所（NCI）化学文库的 2802 种化合物进行初步筛选，我们发现阿克拉霉素（Acm）是一种优先影响 ER 功能的化合物。我们报告说，Acm 降低了包括表皮生长因子受体（EGFR）和 Met 在内的糖蛋白的质膜表达，但不抑制 N 连接糖基化或普遍的蛋白质翻译。荧光显微镜共定位实验也进行了，并证明 Acm 在 ER 中的积累进一步支持了整个 HTS 设计。通过集落形成生存分析分析了 Acm 处理对细胞存活的影响。与 EGFR 水平降低一致，Acm 预处理使 EGFR 突变型非小细胞肺癌（NSCLC）细胞系 HCC827 和 HCC2935 对电离辐射敏感，并且不影响 RTK 非依赖性和 KRAS 突变型 A549 NSCLC 细胞系的敏感性。因此，针对 ER 的 Acm 类似物可能代表一种针对依赖 RTK 功能的肿瘤细胞的新型放射增敏方法。
