NLK functions to maintain proliferation and stemness of NSCLC and is a target of metformin.
OBJECTIVE
Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine kinase that regulates the activity of a wide range of signal transduction pathways. Metformin, an oral antidiabetic drug, is used for cancer prevention. However, the significance and underlying mechanism of NLK and metformin in oncogenesis has not been fully elucidated. Here, we investigate a novel role of NLK and metformin in human non-small cell lung cancer (NSCLC).


METHODS
NLK expression was analyzed in 121 NSCLCs and 92 normal lung tissue samples from benign pulmonary disease. Lentivirus vectors with NLK-shRNA were used to examine the effect of NLK on cell proliferation and tumorigenesis in vitro. Then, tumor xenograft mouse models revealed that NLK knockdown cells had a reduced ability for tumor formation compared with the control group in vivo. Multiple cell cycle regulator expression patterns induced by NLK silencing were examined by western blots in A549 cells. We also employed metformin to study its anti-cancer effects and mechanisms. Cancer stem cell property was checked by tumor sphere formation and markers including CD133, Nanog, c-Myc, and KLF4.


RESULTS
Immunohistochemical (IHC) analysis revealed that NLK expression was up-regulated in NSCLC cases (p < 0.001) and correlated with tumor T stage (p < 0.05). Silencing of NLK suppressed cell proliferation and tumorigenicity significantly in vitro and in vivo, which might be modulated by JUN family proteins. Furthermore, metformin selectively inhibits NLK expression and proliferation in NSCLC cells, but not immortalized noncancerous lung bronchial epithelial cells. In addition, both NLK knockdown and metformin treatment reduced the tumor sphere formation capacity and percentage of CD133+ cells. Accordingly, the expression level of stem cell markers (Nanog, c-Myc, and KLF4) were decreased significantly [corrected].


CONCLUSIONS
NLK is critical for cancer cell cycle progression, and tumorigenesis in NSCLC, NLK knockdown, and metformin treatment inhibit cancer cell proliferation and stemness. Metformin inhibits NLK expression and might be a potential treatment strategy for NSCLC.
Cancer Research Institute of Southern Medical University, Guangzhou, Guangdong, People's Republic of China. dong.suwei@163.com.|Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. dong.suwei@163.com.
Dong
D
Suwei
Department of Pathology, Hunan Tumor Hospital, Changsha, Hunan, People's Republic of China. liangzenglz@163.com.
Zeng
Z
Liang
Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. liuzhiminlzm@yeah.net.
Liu
L
Zhimin
Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. lruileil@163.com.
Li
L
Ruilei
Department of Pathology and Pathophysiology, Kunming Medical University, Kunming, Yunnan, People's Republic of China. zouyingyingz@163.com.
Zou
Z
Yingying
Cancer Research Institute of Southern Medical University, Guangzhou, Guangdong, People's Republic of China. lizhendbao@163.com.|Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. lizhendbao@163.com.
Li
L
Zhen
Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. gechunleiz@163.com.
Ge
G
Chunlei
Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. laizhanghaom@163.com.
Lai
L
Zhangchao
Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. xueyuanbod@163.com.
Xue
X
Yuanbo
Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. yangjinyanyy@163.com.
Yang
Y
Jinyan
Department of Thoracic Surgery, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. ligaofenghl@126.com.
Li
L
Gaofeng
Cancer Research Institute of Southern Medical University, Guangzhou, Guangdong, People's Republic of China. songxin68@126.com.|Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China. songxin68@126.com.
Song
S
Xin
D000328:Adult; D000368:Aged; D000818:Animals; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects* / Q000235:genetics; D005260:Female; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D008297:Male; D008687:Metformin / Q000494:pharmacology*; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D023041:Xenograft Model Antitumor Assays
D007004:Hypoglycemic Agents; D047908:Intracellular Signaling Peptides and Proteins; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D008687:Metformin; C470551:NLK protein, human; D017346:Protein Serine-Threonine Kinases