一个涉及Wnt/β-连环蛋白/MYC/ Sox2轴的正反馈回路定义了ALK阳性间变性大细胞淋巴瘤中一个高度致瘤性的细胞亚群。
A positive feedback loop involving the Wnt/β-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subpopulation in ALK-positive anaplastic large cell lymphoma.
作者信息
Wu Chengsheng, Zhang Hai-Feng, Gupta Nidhi, Alshareef Abdulraheem, Wang Qian, Huang Yung-Hsing, Lewis Jamie T, Douglas Donna N, Kneteman Norman M, Lai Raymond
机构信息
Department of Laboratory Medicine and Pathology, 5142J Katz Group Centre for Pharmacy and Health Research, University of Alberta, Edmonton, Alberta, T6G 1Z2, Canada.
Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou, China.
出版信息
J Hematol Oncol. 2016 Nov 8;9(1):120. doi: 10.1186/s13045-016-0349-z.
BACKGROUND
We have previously described the existence of two phenotypically distinct cell subsets in ALK-positive anaplastic large cell lymphoma (ALK + ALCL) based on their differential responsiveness to a Sox2 reporter (SRR2), with reporter-responsive (RR) cells being more tumorigenic and chemoresistant than reporter-unresponsive (RU) cells. However, the regulator(s) of RU/RR dichotomy are not identified. In this study, we aim to delineate the key regulator(s) of RU/RR dichotomy.
METHODS
JASPER motif match analysis was used to identify the putative factors binding to SRR2 sequence. SRR2 probe pull-down assay and quantitate real-time PCR were performed to analyze the regulation of Sox2 transcriptional activity by MYC. Methylcellulose colony formation assay, chemoresistance to doxorubicin and mouse xenograft study were performed to investigate the biological functions of MYC. PCR array and western blotting were executed to study related signaling pathways that regulate MYC expression. Immunofluorescence and immunohistochemistry assay were initiated to evaluate the expression of MYC and its correlation with its regulator by chi-square test analysis in human primary tumor cells.
RESULTS
We identified MYC as a potential regulator of RU/RR dichotomy. In support of its role, MYC was highly expressed in RR cells compared to RU cells, and inhibition of MYC substantially decreased the Sox2/SRR2 binding, Sox2 transcriptional activity, chemoresistance, and methylcellulose colony formation. In contrast, enforced expression of MYC in RU cells conferred the RR phenotype. The Wnt/β-catenin pathway, a positive regulator of MYC, was highly active in RR but not RU cells. While inhibition of this pathway in RR cells substantially decreased MYC expression and SRR2 reporter activity, experimental activation of this pathway led to the opposite effects in RU cells. Collectively, our results support a model in which a positive feedback loop involving Wnt/β-catenin/MYC and Sox2 contributes to the RR phenotype. In a mouse xenograft model, RU cells stably transfected with MYC showed upregulation of the Wnt/β-catenin/MYC/Sox2 axis and increased tumorigenecity. Correlating with these findings, there was a significant correlation between the expression of active β-catenin and MYC in ALK + ALCL primary tumor cells.
CONCLUSIONS
A positive feedback loop involving the Wnt/β-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subset in ALK + ALCL.
背景
我们之前曾描述过，在ALK阳性间变性大细胞淋巴瘤（ALK+ALCL）中存在两种表型不同的细胞亚群，这基于它们对Sox2报告基因（SRR2）的不同反应，其中报告基因反应性（RR）细胞比报告基因无反应性（RU）细胞更具致瘤性和化疗抗性。然而，RU/RR二分法的调节因子尚未确定。在本研究中，我们旨在确定RU/RR二分法的关键调节因子。
方法
使用JASPER基序匹配分析来识别与SRR2序列结合的假定因子。进行SRR2探针下拉试验和定量实时PCR以分析MYC对Sox2转录活性的调节。进行甲基纤维素集落形成试验、对阿霉素的化疗抗性和小鼠异种移植研究以研究MYC的生物学功能。执行PCR阵列和蛋白质印迹以研究调节MYC表达的相关信号通路。启动免疫荧光和免疫组织化学试验以评估MYC的表达及其在人原发性肿瘤细胞中与其调节因子的相关性，并通过卡方检验分析。
结果
我们确定MYC是RU/RR二分法的潜在调节因子。为支持其作用，与RU细胞相比，MYC在RR细胞中高表达，并且抑制MYC可显著降低Sox2/SRR2结合、Sox2转录活性、化疗抗性和甲基纤维素集落形成。相反，在RU细胞中强制表达MYC赋予RR表型。Wnt/β-连环蛋白通路是MYC的正调节因子，在RR细胞中高度活跃，但在RU细胞中不活跃。虽然在RR细胞中抑制该通路可显著降低MYC表达和SRR2报告基因活性，但在RU细胞中实验性激活该通路则产生相反的效果。总体而言，我们的结果支持一种模型，即涉及Wnt/β-连环蛋白/MYC和Sox2的正反馈环促成RR表型。在小鼠异种移植模型中，稳定转染MYC的RU细胞显示Wnt/β-连环蛋白/MYC/Sox2轴上调且致瘤性增加。与这些发现相关的是，在ALK+ALCL原发性肿瘤细胞中，活性β-连环蛋白和MYC的表达之间存在显著相关性。
结论
涉及Wnt/β-连环蛋白/MYC/Sox2轴的正反馈环定义了ALK+ALCL中一个高度致瘤的细胞亚群。
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