Olfactomedin 1 negatively regulates NF‐κB signalling and suppresses the growth and metastasis of colorectal cancer cells

Abstract

Uncontrolled growth and distant metastasis are hallmarks of colorectal cancer (CRC), but the mechanisms are poorly understood. Olfactomedin 1 (OLFM1), a member of the olfactomedin domain-containing protein family, plays an important role in the development of neurogenic tissues. Recently, OLFM1 deregulation was frequently observed in several cancers, and it was induced in colon cell lines after treatment with the demethylating agent 5-aza-2'-deoxycytidine. However, the function of OLFM1 in CRC remains unknown. In this study, we reanalysed published microarray data and found that OLFM1 was significantly down-regulated in primary CRC samples compared to adjacent non-cancerous tissues. The results of immunohistochemistry indicated that decreased OLFM1 expression was significantly associated with lymph node status (p = 0.023), distant metastasis (p < 0.001), and AJCC/TNM stage (p = 0.013), and CRC patients with low OLFM1 expression had consistently poor overall survival (OS; p < 0.001) and progression-free survival (PFS; p < 0.001). Further analysis demonstrated that OLFM1 was epigenetically silenced in CRC tissues and cell lines via promoter hypermethylation. Overexpression and knockdown of OLFM1 attenuated and increased, respectively, CRC cells' proliferation, migration, and invasion in vitro and metastasis to the lung and liver in vivo. Mechanistically, the promotion of growth and metastasis of CRC cells by silencing of OLFM1 was associated with the activation of the non-canonical NF-κB signalling pathway. OLFM1 interacted with NF-κB-inducing kinase (NIK; MAP3K14) and repressed the phosphorylation of its downstream substrate Ikappa B kinase alpha (IKKα). OLFM1 expression was negatively correlated with the phosphorylation level of IKKα in CRC tissue samples. Knockdown of NIK impaired the ability of OLFM1 to repress NF-κB signalling, cell growth or migration. Thus, OLFM1 may be a valuable biomarker and therapeutic target for CRC patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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“…49, 50 Briefly, cells in 24-well plates were co-transfected with 500 ng of luciferase reporter plasmids consisting of a promoter containing the indicated transcription factor binding sequence or response element (Addgene, Cambridge, MA, USA) and 10 ng of internal control pRL-TK Renilla plasmid (Promega). After 24 h, the cells were lysed, and the luciferase and Renilla signals were measured using Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instructions.…”

“…The tumor volume was evaluated using the following formula: tumor volume=4 π /3 × (width/2) 2 × (length/2). 49 …”

ARHGEF19 interacts with BRAF to activate MAPK signaling during the tumorigenesis of non‐small cell lung cancer

“…Plasmids or miRNAs were co‐transfected with a firefly luciferase reporter plasmid (pGL3‐WT 3′UTR, pGL3‐MUT 3′UTR or SRE reporter) into cancer cells; a Renilla luciferase reporter plasmid (pRL‐TK) was used as the internal reference. Luciferase activity was detected using the Dual‐Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions …”

“…Each group contained 10 mice. After 6 weeks, the mice were sacrificed, and the lungs were removed for pathological examination as described previously …”

“…After 6 weeks, the mice were sacrificed, and the lungs were removed for pathological examination as described previously. 32 For inguinal lymph node metastasis models, 2 3 10 6 cells were inoculated into the footpads of mice (n 5 10/group) at Day 0. All mice were sacrificed after 5 weeks, and all inguinal lymph nodes were enucleated and fixed in formalin, followed by pathological examination.…”