Division of Endocrinology, Metabolism, and Diabetes, Department of Medicine, Colorado Anschutz Medical Campus, Aurora, Colorado.
Research Service Veterans Affairs Medical Center, Denver, Colorado.
Endocrinology. 2018 Jul 1;159(7):2532-2544. doi: 10.1210/en.2018-00310.
Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5-year survival rate <35%. Mortality remains high due to lack of targeted therapies. Using bioinformatic analyses, we identified maternal embryonic leucine zipper kinase (MELK) as 4.1-fold overexpressed in ACC compared with normal adrenal samples. High MELK expression in human tumors correlated with shorter survival and with increased expression of genes involved in cell division and growth. We investigated the functional effects of MELK inhibition using newly developed ACC cell lines with variable MELK expression, CU-ACC1 and CU-ACC2, compared with H295R cells. In vitro treatment with the MELK inhibitor, OTSSP167, resulted in a dose-dependent decrease in rates of cell proliferation, colony formation, and cell survival, with relative sensitivity of each ACC cell line based upon the level of MELK overexpression. To confirm a MELK-specific antitumorigenic effect, MELK was inhibited in H295R cells via multiple short hairpin RNAs. MELK silencing resulted in 1.9-fold decrease in proliferation, and 3- to 10-fold decrease in colony formation in soft agar and clonogenicity assays, respectively. In addition, although MELK silencing had no effect on survival in normoxia, exposure to a hypoxia resulted in a sixfold and eightfold increase in apoptosis as assessed by caspase-3 activation and TUNEL, respectively. Together these data suggest that MELK is a modulator of tumor cell growth and survival in a hypoxic microenvironment in adrenal cancer cells and support future investigation of its role as a therapeutic kinase target in patients with ACC.
肾上腺皮质癌 (ACC) 是一种侵袭性癌症，5 年生存率<35%。由于缺乏靶向治疗，死亡率仍然很高。通过生物信息学分析，我们发现与正常肾上腺样本相比，母系胚胎亮氨酸拉链激酶 (MELK) 在 ACC 中表达上调了 4.1 倍。人肿瘤中高 MELK 表达与生存率降低以及与细胞分裂和生长相关的基因表达增加相关。我们使用新开发的具有不同 MELK 表达水平的 ACC 细胞系 CU-ACC1 和 CU-ACC2 与 H295R 细胞进行比较，研究了 MELK 抑制的功能影响。用 MELK 抑制剂 OTSSP167 进行体外处理导致细胞增殖、集落形成和细胞存活的速率呈剂量依赖性下降，每种 ACC 细胞系的相对敏感性基于 MELK 过表达的水平。为了确认 MELK 特异性抗肿瘤作用，通过多个短发夹 RNA 抑制 H295R 细胞中的 MELK。MELK 沉默导致增殖减少 1.9 倍，在软琼脂和集落形成测定中分别减少 3 到 10 倍的集落形成。此外，尽管 MELK 沉默对常氧条件下的存活没有影响，但在缺氧条件下，用 caspase-3 激活和 TUNEL 分别评估的凋亡增加了六倍和八倍。这些数据表明，MELK 是肾上腺皮质癌细胞中缺氧微环境中肿瘤细胞生长和存活的调节剂，并支持进一步研究其作为 ACC 患者治疗性激酶靶标的作用。
