Department of Cancer Biology, University of Kansas Medical Center, Kansas City, Kansas.
Interdisciplinary Science and Technology Research Academy, Abeda Inamdar Senior College, Camp, Pune, India.
Gastroenterology. 2019 Dec;157(6):1646-1659.e11. doi: 10.1053/j.gastro.2019.08.018. Epub 2019 Aug 20.
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.
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.
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.
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.
组蛋白赖氨酸去甲基酶 3A（KDM3A）可使 H3K9me1 和 H3K9Me2 去甲基化，从而增加基因转录，并且在肿瘤中上调，包括胰腺肿瘤。我们研究了其在胰腺癌细胞系中的活性及其对双皮质醇钙调激酶 1（DCLK1）基因的调控，DCLK1 是癌症干细胞的标志物。
我们敲低了 MiaPaCa-2 和 S2-007 胰腺癌细胞系中的 KDM3A，并在 HPNE 细胞（人非癌性胰腺导管细胞系）中转染 KDM3A；我们评估了缺氧和常氧条件下细胞迁移、侵袭和球体形成。裸鼠接受 S2-007 细胞的原位注射，有无（对照）敲低 KDM3A，以及有无（对照）过表达 KDM3A 的 HPNE 细胞；评估肿瘤生长情况。我们通过免疫组化和免疫印迹分析了来自小鼠和胰腺癌细胞系的胰腺肿瘤组织。我们对敲低 KDM3A 的 MiaPaCa-2 和 S2-007 细胞进行了 RNA 测序分析，并通过免疫荧光评估了 DCLK1 和 KDM3A 的定位。我们分析了癌症基因组图谱中人类胰腺导管腺癌（PDAC）组织中 KDM3A 和 DCLK1 信使 RNA 的水平及其与患者生存时间的关系。
与胰岛和腺泡细胞等相邻非肿瘤胰腺组织相比，KDM3A 在人胰腺肿瘤组织和细胞系中的水平升高。与对照细胞相比，S2-007 细胞中 KDM3A 的敲低显著降低了集落形成、侵袭、迁移和球体形成，并且减缓了小鼠原位肿瘤的生长。我们在 DCLK1 启动子中鉴定了 KDM3A 结合位点；敲低 KDM3A 的 S2-007 细胞中 DCLK1 水平降低。过表达 KDM3A 的 HPNE 细胞在培养中形成焦点和球体，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 S2-007 细胞和过表达 DCLK1 的 HPNE 细胞的球体形成并增加 KDM3A 水平，但对照 HPNE 细胞则没有。与非肿瘤胰腺组织相比，人类 PDAC 样本中的 KDM3A 和 DCLK1 信使 RNA 水平更高，且与患者的生存时间较短相关。
我们发现人类 PDAC 样本和胰腺癌细胞系过度表达 KDM3A。KDM3A 增加了 DCLK1 的表达，并且在人类 PDAC 样本中两种蛋白质的水平都升高。胰腺癌细胞系中 KDM3A 的敲低降低了它们在培养中的侵袭和球体形成活性以及在小鼠中的原位肿瘤形成。缺氧增加了胰腺癌细胞中 KDM3A 的表达。破坏这种途径的策略可能被开发用于治疗胰腺癌。
