Cheng Joyce Y, Brown Taylor C, Murtha Timothy D, Stenman Adam, Juhlin C Christofer, Larsson Catharina, Healy James M, Prasad Manju L, Knoefel Wolfram T, Krieg Andreas, Scholl Ute I, Korah Reju, Carling Tobias
Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT, USA.
Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital, CCK, Stockholm, Sweden.
BMC Cancer. 2017 Mar 1;17(1):164. doi: 10.1186/s12885-017-3152-5.
Dysregulated WNT signaling dominates adrenocortical malignancies. This study investigates whether silencing of the WNT negative regulator DKK3 (Dickkopf-related protein 3), an implicated adrenocortical differentiation marker and an established tumor suppressor in multiple cancers, allows dedifferentiation of the adrenal cortex.
We analyzed the expression and regulation of DKK3 in human adrenocortical carcinoma (ACC) by qRT-PCR, immunofluorescence, promoter methylation assay, and copy number analysis. We also conducted functional studies on ACC cell lines, NCI-H295R and SW-13, using siRNAs and enforced DKK3 expression to test DKK3's role in blocking dedifferentiation of adrenal cortex.
While robust expression was observed in normal adrenal cortex, DKK3 was down-regulated in the majority (>75%) of adrenocortical carcinomas (ACC) tested. Both genetic (gene copy loss) and epigenetic (promoter methylation) events were found to play significant roles in DKK3 down-regulation in ACCs. While NCI-H295R cells harboring β-catenin activating mutations failed to respond to DKK3 silencing, SW-13 cells showed increased motility and reduced clonal growth. Conversely, exogenously added DKK3 also increased motility of SW-13 cells without influencing their growth. Enforced over-expression of DKK3 in SW-13 cells resulted in slower cell growth by an extension of G1 phase, promoted survival of microcolonies, and resulted in significant impairment of migratory and invasive behaviors, largely attributable to modified cell adhesions and adhesion kinetics. DKK3-over-expressing cells also showed increased expression of Forkhead Box Protein O1 (FOXO1) transcription factor, RNAi silencing of which partially restored the migratory proficiency of cells without interfering with their viability.
DKK3 suppression observed in ACCs and the effects of manipulation of DKK3 expression in ACC cell lines suggest a FOXO1-mediated differentiation-promoting role for DKK3 in the adrenal cortex, silencing of which may allow adrenocortical dedifferentiation and malignancy.
WNT信号失调在肾上腺皮质恶性肿瘤中占主导地位。本研究调查WNT负调节因子DKK3（Dickkopf相关蛋白3）的沉默是否会导致肾上腺皮质去分化，DKK3是一种与肾上腺皮质分化相关的标志物，也是多种癌症中公认的肿瘤抑制因子。
我们通过qRT-PCR、免疫荧光、启动子甲基化分析和拷贝数分析，分析了DKK3在人肾上腺皮质癌（ACC）中的表达和调控。我们还使用小干扰RNA（siRNAs）和强制表达DKK3，对ACC细胞系NCI-H295R和SW-13进行功能研究，以测试DKK3在阻止肾上腺皮质去分化中的作用。
虽然在正常肾上腺皮质中观察到DKK3有较强表达，但在所检测的大多数（>75%）肾上腺皮质癌（ACC）中，DKK3表达下调。基因（基因拷贝数缺失）和表观遗传（启动子甲基化）事件在ACC中DKK3下调中均起重要作用。携带β-连环蛋白激活突变的NCI-H295R细胞对DKK3沉默无反应，而SW-13细胞的运动性增加，克隆生长减少。相反，外源性添加DKK3也增加了SW-13细胞的运动性，但不影响其生长。在SW-13细胞中强制过表达DKK3导致细胞生长减慢，G1期延长，促进微克隆存活，并导致迁移和侵袭行为显著受损，这主要归因于细胞黏附及黏附动力学的改变。过表达DKK3的细胞还显示叉头框蛋白O1（FOXO1）转录因子表达增加，RNA干扰沉默该转录因子可部分恢复细胞的迁移能力，且不影响细胞活力。
在ACC中观察到的DKK3抑制以及在ACC细胞系中操纵DKK3表达的效果表明，DKK3在肾上腺皮质中具有由FOXO1介导的促进分化作用，其沉默可能导致肾上腺皮质去分化和恶性肿瘤发生。
