Myosin light chain kinase (MLCK) regulates actinomyosin contraction. Two splice variants of long MLCK are expressed in epithelial cells and divergently regulate gut barrier functions; reduced MLCK levels in human colorectal cancers (CRC) with unclarified significance have been reported. CRC are solid tumors clonally sustained by stem cells highly expressing CD44 and CD133. The aim was to investigate the role of MLCK splice variants in CRC tumorigenesis. We found lower MLCK1/2 and higher CD44 expression in human CRC, but no change in CD133 or LGR5. Large-scale bioinformatics showed an inverse relationship between MYLK and CD44 in human sample gene datasets. A 3-fold increased tumor burden was observed in MLCK(-/-) mice compared with wild-type (WT) mice in a chemical-induced CRC model. Primary tumorspheres derived from the MLCK(-/-) mice displayed larger sizes and higher CD44 transcript levels than those from the WT mice. Bioinformatics revealed binding of TEAD4 (a transcriptional enhancer factor family member in the Hippo pathway) to CD44 promoter, which was confirmed by luciferase reporter assay. Individually expressing MLCK1 and MLCK2 variants in the MLCK-knockout (KO) Caco-2 cells inhibited the nuclear localization of TEAD4 cofactors, VGLL3 and YAP1, respectively, and both variants reduced the CD44 transcription. Accelerated cell cycle transit was observed in the MLCK-KO cells, whereby expression of MLCK1/2 variants counterbalanced the cell hyperproliferation. In conclusion, MLCK1/2 variants are novel tumor suppressors by downregulating the TEAD4/CD44 axis via reducing nuclear translocation of distinct transcriptional coactivators. The reduction of epithelial MLCKs, especially isoform 2, may drive cancer stemness and tumorigenesis.

中文翻译：

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通过下调 TEAD4/CD44 轴，肌球蛋白轻链激酶剪接变体的新型肿瘤抑制作用。

肌球蛋白轻链激酶 (MLCK) 调节放线菌蛋白收缩。长 MLCK 的两种剪接变体在上皮细胞中表达并不同地调节肠道屏障功能；据报道，人类结直肠癌 (CRC) 中 MLCK 水平降低，但意义不明。CRC 是由高表达 CD44 和 CD133 的干细胞克隆维持的实体瘤。目的是研究 MLCK 剪接变体在 CRC 肿瘤发生中的作用。我们在人类 CRC 中发现较低的 MLCK1/2 和较高的 CD44 表达，但 CD133 或 LGR5 没有变化。大规模生物信息学显示人类样本基因数据集中 MYLK 和 CD44 之间存在反比关系。在化学诱导的 CRC 模型中，与野生型 (WT) 小鼠相比，MLCK(-/-) 小鼠的肿瘤负荷增加了 3 倍。来自 MLCK(-/-) 小鼠的原发性肿瘤球显示出比 WT 小鼠更大的尺寸和更高的 CD44 转录物水平。生物信息学揭示了 TEAD4（Hippo 通路中的转录增强因子家族成员）与 CD44 启动子的结合，这通过荧光素酶报告基因测定得到证实。在 MLCK 敲除 (KO) Caco-2 细胞中单独表达 MLCK1 和 MLCK2 变体分别抑制 TEAD4 辅因子 VGLL3 和 YAP1 的核定位，并且这两种变体都减少了 CD44 转录。在 MLCK-KO 细胞中观察到加速的细胞周期转运，由此 MLCK1/2 变体的表达抵消了细胞过度增殖。综上所述，MLCK1/2 变体是新型肿瘤抑制因子，通过减少不同转录共激活因子的核转位来下调 TEAD4/CD44 轴。上皮 MLCK 的减少，尤其是亚型 2，可能会促进癌症干性和肿瘤发生。