BackgroundLipocalin2 (LCN2) is a secretory protein that is aberrantly expressed in several types of cancer and has been involved in metastatic progression. However, neither mechanisms nor the role that LCN2 plays in the metastasis of colorectal cancer are clear.MethodsLCN2 expression in colorectal cancer was detected by immunohistochemistry in 400 tissue specimens and Kaplan-Meier survival analysis was performed. In vitro, real-time PCR, western blot, colony formation assay, immunofluorescence assay, wound healing assay, migration and invasion experiment were performed to investigate the effects of LCN2 in epithelial mesenchymal transition (EMT), migration and invasion, respectively. In vivo mouse xenograft and metastasis models were utilized to determine tumorigenicity and metastasis ability, and immunohistochemistry, real-time PCR, western blot were used to evaluate the related protein expression. Luciferase reporter assay was used to explore the role of LCN2 on NF-ĸB promoter.ResultsLCN2 was highly expressed in 66.5% of the specimens, and significantly correlated with positive E-cadherin in the membrane and negative nuclear β-catenin. Higher expression of LCN2 together with negative NF-κB expression was negatively related to nuclear accumulation of snail and predicted favorable prognosis. LCN2 blocked cell proliferation, migration and invasion in vitro and in vivo, and inhibited translocation of NF-κB into nucleus. NF-κB could reverse the effect of LCN2 on EMT and promote snail expression. Rescued snail expression had similar effect without influencing NF-κB activity.ConclusionLCN2 may be an important negative regulator in EMT, invasion and metastasis of CRC via acting as upstream of NF-κB/snail signaling pathway. Thereby combinative manipulation of LCN2 and NF-κB/snail pathway may represent a novel and promising therapeutic approach for the patients with CRC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0564-9) contains supplementary material, which is available to authorized users.
Background
Cancer cell is generally characterized by enhanced glycolysis. Inflammasome activation is interaction with glycolysis. The concentration of lipopolysaccharide (LPS), a classic inflammasome activator, is significantly higher in colorectal cancer tissue than in normal intestinal mucosa. However, the mechanism of LPS on glycolysis and metastasis has not been fully elucidated. This study aimed to investigate the roles of LPS on inflammasome activation, glycolysis, and metastasis, and unravel metformin’s potential in treatment of CRC.
Methods
We detected inflammasome activation and cell motility following LPS exposure in CRC cell lines. Glycolysis analysis was performed, and the key glycolytic rate-limiting enzymes were detected. Dual-luciferase reporter gene assay, co-immunoprecipitation, chromatin immunoprecipitation (ChIP) analysis, and ChIP-reChIP assay were performed to identify the specific mechanisms of LPS on glycolysis. Mouse metastasis models were used to determine the effects of LPS and metformin on metastasis. Correlation analysis of the expression of various molecules was performed in 635 CRC samples from The Cancer Genome Atlas and 83 CRC samples from our lab.
Results
LPS activates caspase-1 through NF-κB and upregulates the expression of Snail and HK3 depending on caspase-1 activation. LPS potentiates migration and invasion depending on accelerated glycolysis, which could be reversed by knockdown of glycolytic rate-limiting enzyme HK3. Nuclear Snail is upregulated by NF-κB under LPS treatment and then forms a complex with NF-κB, then directly binds to the HK3 promoter region to upregulate the expression of HK3. Metformin suppresses the NF-κB/Snail/HK3 signaling axis that is activated by LPS and then inhibits LPS-induced metastasis. In vivo, LPS-treated cells form more metastasis in the lungs of mice, and metformin completely reverses this effect of LPS.
Conclusion
LPS activates inflammasomes in cancer cells through NF-κB and promotes metastasis through glycolysis enhanced by the NF-κB/Snail/HK3 signaling pathway in CRC. Metformin could prevent this effect of LPS.
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