腸道上皮肌凝蛋白輕鏈激酶透過調節TEAD4/CD44表現抑制腫瘤進程

Abstract

背景：肌凝蛋白輕鏈激酶（Myosin Light Chain Kinase, MLCK）由短型與長型組成，其編碼在同一個基因MYLK的蛋白，其在肌肉或非肌肉細胞中會磷酸化肌凝蛋白輕鏈（Myosin Light Chain, MLC）調控肌動凝蛋白（Actinomysin）收縮。腸道上皮細胞表現長型MLCK調節屏障功能。在大腸直腸癌（Colorectal Cancer, CRC）的檢體中發現MYLK 的低表現量。大腸直腸癌是一種固態腫瘤由高表現CD44與CD133分子的癌幹細胞（Cancer Stem Cell, CSC）所維持。目的：為了探討長型MLCK在大腸癌腫瘤生成與癌症幹性的分子機制。方法材料：來自台大醫院病患檢體被納入研究分析。三維初代大腸類器官（Organoid）從長型MLCK-210缺失（MLCK(-/-)）小鼠與野生型（Wild-type, WT）小鼠培養於基質膠（Matrigel Matrix）。人類大腸腺瘤Caco-2細胞株處理ML-7（MLCK藥物抑制劑）與由慢病毒小髮夾RNA（Short Hairpin RNA, shRNA）進行MLCK基因減弱（Knockdown, KD）技術。在人類大腸腺瘤細胞株Caco-2經由CRISPR/Cas9系統進行MYLK基因剔除（Knockout, KO）後，藉由流式細胞儀（Flow Cytometry）染上Ki-67與碘化丙啶（Propidium Iodide, PI）來評估細胞週期速率。結果：在同觀察到其較低的長型MYLK表現在腫瘤組織中相較周邊非腫瘤組織。從MLCK剔除的大腸類器官顯示尺寸較大且較高的Cd44表現於野生型小鼠的。我們使用生物資訊工具：基因轉錄調控資料庫（Gene Transcription Regulation Database, GTRD）根據染色質免疫沉澱測序實驗（Chromatin Immunoprecipitation-sequencing, ChIP-seq）建立的資料庫，發現了潛在CD44轉錄因子（Transcription Factor, TF）--TEAD4，並且使用螢光素酶測定確認TEAD4會結合在CD44啟動子上。利用兩株大腸癌細胞株Caco-2與HT-29藉由Verteporfin抑制TEAD4的功能後，其CD44表現量下降。在ML-7藥物與慢病毒載體基因減弱MLCK促使CD44與TEAD4表現提升，而不改變CD133與LGR5表現或是其他假定的轉錄因子（像是SP1, TP63 , TP53）MLCK基因剔除細胞加速細胞週期（Cell Cycle）速率，然而轉染質體MLCK於KO細胞過表現後抵銷了細胞過度增生與CD44和TEAD4表現下降。利用西方墨點法觀察到MLCK-KO細胞的細胞核會出現YAP與VGLL3蛋白量趨勢上升相較於野生型Caco-2細胞，然而轉染轉染質體MLCK1或2後，發現分別降低VGLL3與YAP於核轉移蛋白量。結論：長型MYLK是個新穎的腫瘤抑制基因，其不同的同功型（isoform）MLCK1/2藉由不同TEAD4的輔助因子(cofactor) VGLL3/YAP調控CD44表現。

Background: Myosin light chain kinase (MLCK), comprised of short and long isoforms encoded by a single MYLK gene, phosphorylates myosin light chain (MLC) to regulate actinomyosin contraction in muscles and nonmuscle cells. The intestinal epithelial cells express long MLCK to regulate barrier functions. Reduced MYLK transcripts were found in human colon carcinoma (CRC) specimens. CRC is a solid tumor clonally sustained by cancer stem cells (CSCs) highly expressed with CD44 and CD133 molecules. Aim: To investigate the mechanistic role of long MLCK in colon tumorigenesis and cancer stemness. Methods: Human CRC specimens collected from National Taiwan University Hospital (NTUH) were analyzed. Three-dimensional primary colonic organoids were developed from mice deficient of long MLCK-210 (MLCK(-/-)) and wild type (WT) mice by Matrigel culturing. Human colonic adenocarcinoma Caco-2 cells were administered ML-7 (an MLCK inhibitor) or gene knockdown (KD) by lentiviral shRNA. CRISPR/Cas9-based MYLK gene knockout (KO) in Caco-2 cells were assessed for cell cycle rates by flow cytometry with Ki-67 and propidium iodide (PI) staining. Results: Lower expression of long MYLK mRNA was observed in human CRC tissues than adjacent non-tumor tissues. Colonic organoids from MLCK(-/-) mice showed larger sizes and higher Cd44 expression compared to those from WT mice. We found a potential CD44 transcription factor, TEAD4, by bioinformatics analysis using Gene Transcription Regulation Database (GTRD) based on ChIP-seq experiments. We further confirmed TEAD4 as a transcription factor of CD44 using a luciferase reporter assay. Inhibition of TEAD4 function by verteporfin reduced CD44 expression in Caco-2 and HT29 cells. Administration of ML-7 and lentiviral KD of MLCK induced an elevation of CD44 and TEAD4 expression in Caco-2 cells, without changes in the CD133 and LGR5 levels or other putative transcription factors (e.g. SP1, TP63, TP53). Caco-2 cells with MLCK-KO showed accelerated cell cycle rates, whereas transfection with MLCK-encoding plasmids in the KO cells counterbalanced the cell hyperproliferation and decreased the expression of CD44 and TEAD4. Densitometric analysis of Western blots showed significantly increased YAP and a trend to increased VGLL3 in the nuclear fraction of MLCK-KO cells compared to wild type Caco-2 cells; whereas transfection with human MLCK1/2- encoding plasmids reduced the nuclear translocation of VGLL3 and YAP, respectively. Conclusion: Long MYLK is novel tumor suppressor gene of which the splice variants MLCK1/2 reduced CD44 expression through differential regulation of TEAD4 transcriptional cofactors.