NLK functions to maintain proliferation and stemness of NSCLC and is a target of metformin

Abstract

ObjectiveNemo-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).Materials and methodsNLK 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 TLF4.ResultsImmunohistochemical (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 TLF4) were decreased significantly.ConclusionNLK 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.

Exosome‐transferred long non‐coding RNA ASMTL‐AS1 contributes to malignant phenotypes in residual hepatocellular carcinoma after insufficient radiofrequency ablation

“…Besides, NLK is reported to regulate various signalling pathways, such as pro‐tumour Wnt,36 Notch37 and mTOR signalling38 as well as anti‐tumour Hippo signalling 39. This might be the reason why NLK exerts diverse functions during different cancers 23,40,41. However, sometimes there are contradictions about NLK function when in different diseases.…”

Increased expression of the RNA‑binding motif protein 47�predicts poor prognosis in non‑small‑cell lung cancer

“…IHC was performed in order to determine RBM47 expression levels and distribution patterns of 175 pairs of NSCLC tissues. IHC was performed in accordance with previous studies (30,31). Tissue samples were cut into 4-µm-thick sections and incubated in Rabbit polyclonal RBM7 antibody (1:500; cat.…”

“…The intensity of immunoreactivity was scored as follows: 0, negative; 1, weak; 2, intermediate and 3, strong. The samples were grouped according to the sum of the immunoreactivity score as follows: 0, negative; 1-2, weak; 3, moderate and 4-6, strong (31). Final immunoreactivity scores >0 were considered positive, and those at 0 were considered negative.…”

Chemoresistance of Lung Cancer Cells: 2D and 3D In Vitro Models for Anticancer Drug Screening

“…against LTs have been published. In vivo studies have been performed to explore and extend observed drug efficacy against LTs in a more physiological setting [87,88]. Several studies have tried combination therapy approaches in LTs setting to develop effective drug combinations to alleviate therapy resistance-related concerns and improve efficacy of existing cancer drugs [68,[89][90][91][92].…”