The Histone Demethylase KDM3A, Increased in Human Pancreatic Tumors, Regulates Expression of DCLK1 and Promotes Tumorigenesis in Mice
The Histone Demethylase KDM3A, Increased in Human Pancreatic Tumors, Regulates Expression of DCLK1 and Promotes Tumorigenesis in Mice
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DOI：
10.1053/j.gastro.2019.08.018
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发表时间：
2019-12-01
期刊：
影响因子：
29.4
通讯作者：
Dhar, Animesh
中科院分区：
文献类型：
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作者：
Dandawate, Prasad;Ghosh, Chandrayee;Dhar, Animesh
背景与目的：组蛋白赖氨酸去甲基化酶3A（KDM 3A）使H3 K9 me 1和H3 K9 Me 2去甲基化以增加基因转录，并且在肿瘤（包括胰腺肿瘤）中上调。我们研究了它在胰腺癌细胞系中的活性及其对编码双皮质素钙调素样激酶1（DCLK 1）（癌症干细胞的标志物）的基因的调节。方法：我们敲低了MiaPaCa-2和S2-007胰腺癌细胞系中的KDM 3A，并在HPNE细胞（人非癌胰腺导管细胞系）中过表达KDM 3A;我们评估了低氧和常氧条件下的细胞迁移、侵袭和球体形成。对裸鼠原位注射S2-007细胞（有或没有（对照）KDM 3A敲减）和HPNE细胞（有或没有（对照）KDM 3A过表达）;评估肿瘤生长。我们通过免疫组织化学和免疫印迹分析了小鼠胰腺肿瘤组织和胰腺癌细胞系。我们对敲低KDM 3A的MiaPaCa-2和S2-007细胞进行RNA测序分析，并通过免疫荧光评估DCLK 1和KDM 3A的定位。我们分析了癌症基因组图谱中人胰腺导管腺癌（PDAC）组织中KDM 3A和DCLK 1信使RNA的水平及其与患者生存时间的相关性。研究结果：与邻近的非肿瘤胰腺组织如胰岛和腺泡细胞相比，KDM 3A在人胰腺肿瘤组织和细胞系中的水平增加。与对照细胞相比，S2-007细胞中KDM 3A的敲低显著减少了集落形成、侵袭、迁移和球体形成，并减缓了小鼠原位肿瘤的生长。我们确定了DCLK 1启动子中的KDM 3A结合位点;敲低KDM 3A的S2-007细胞的DCLK 1水平降低。过表达KDM 3A的HPNE细胞在培养物中形成病灶和球体，并在小鼠中形成肿瘤和转移，而对照HPNE细胞则没有。缺氧诱导球体形成，并增加了S2-007细胞和过表达DCLK 1的HPNE细胞中KDM 3A的水平，但不包括对照HPNE细胞。人PDAC中KDM 3A和DCLK 1信使RNA的水平高于非肿瘤胰腺组织，并且与患者的生存时间较短相关。结论：我们发现人PDAC样品和胰腺癌细胞系过表达KDM 3A。KDM 3A增加DCLK 1的表达，并且在人PDAC样品中两种蛋白质的水平都增加。胰腺癌细胞系中KDM 3A的敲低降低了它们在培养中的侵袭性和球体形成活性以及小鼠原位肿瘤的形成。缺氧增加胰腺癌细胞KDM 3A的表达。破坏这一通路的策略可能会被开发用于胰腺癌的治疗。
BACKGROUND & AIMS: The histone lysine demethylase 3A (KDM3A) demethylates H3K9me1 and H3K9Me2 to increase gene transcription and is upregulated in tumors, including pancreatic tumors. We investigated its activities in pancreatic cancer cell lines and its regulation of the gene encoding doublecortin calmodulin-like kinase 1 (DCLK1), a marker of cancer stem cells. METHODS: We knocked down KDM3A in MiaPaCa-2 and S2-007 pancreatic cancer cell lines and overexpressed KDM3A in HPNE cells (human noncancerous pancreatic ductal cell line); we evaluated cell migration, invasion, and spheroid formation under hypoxic and normoxic conditions. Nude mice were given orthotopic injections of S2-007 cells, with or without (control) knockdown of KDM3A, and HPNE cells, with or without (control) overexpression of KDM3A; tumor growth was assessed. We analyzed pancreatic tumor tissues from mice and pancreatic cancer cell lines by immunohistochemistry and immunoblotting. We performed RNA-sequencing analysis of MiaPaCa-2 and S2-007 cells with knockdown of KDM3A and evaluated localization of DCLK1 and KDM3A by immunofluorescence. We analyzed the cancer genome atlas for levels of KDM3A and DCLK1 messenger RNA in human pancreatic ductal adenocarcinoma (PDAC) tissues and association with patient survival time. RESULTS: Levels of KDM3A were increased in human pancreatic tumor tissues and cell lines, compared with adjacent nontumor pancreatic tissues, such as islet and acinar cells. Knockdown of KDM3A in S2-007 cells significantly reduced colony formation, invasion, migration, and spheroid formation, compared with control cells, and slowed growth of orthotopic tumors in mice. We identified KDM3A-binding sites in the DCLK1 promoter; S2-007 cells with knockdown of KDM3A had reduced levels of DCLK1. HPNE cells that overexpressed KDM3A formed foci and spheres in culture and formed tumors and metastases in mice, whereas control HPNE cells did not. Hypoxia induced sphere formation and increased levels of KDM3A in S2-007 cells and in HPNE cells that overexpressed DCLK1, but not control HPNE cells. Levels of KDM3A and DCLK1 messenger RNA were higher in human PDAC than nontumor pancreatic tissues and correlated with shorter survival times of patients. CONCLUSIONS: We found human PDAC samples and pancreatic cancer cell lines to overexpress KDM3A. KDM3A increases expression of DCLK1, and levels of both proteins are increased in human PDAC samples. Knockdown of KDM3A in pancreatic cancer cell lines reduced their invasive and sphere-forming activities in culture and formation of orthotopic tumors in mice. Hypoxia increased expression of KDM3A in pancreatic cancer cells. Strategies to disrupt this pathway might be developed for treatment of pancreatic cancer.