Abstract
BackgroundRecent studies have revealed the potential roles of intelectin 1 (ITLN1) in tumorigenesis. However, its functions and underlying mechanisms in neuroblastoma (NB), the most common extracranial solid tumor in childhood, still remain largely unknown.MethodsHuman neuroblastoma cell lines were treated with recombinant ITLN1 protein or stably transfected with ITLN1 expression and short hairpin RNA vectors. Gene expression and signaling pathway were detected by western blot and real-time quantitative RT-PCR. Gene promoter activity and transcription factor binding were detected by luciferase reporter and chromatin immunoprecipitation assays. Growth and aggressiveness of tumor cells were measured by MTT colorimetry, colony formation, scratch assay, matrigel invasion assay, and nude mice model.ResultsMining of public microarray databases revealed that N-myc downstream regulated gene 2 (NDRG2) was significantly correlated with ITLN1 in NB. Gain- and loss-of-function studies indicated that secretory ITLN1 facilitated the NDRG2 expression, resulting in down-regulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9), in NB cell lines SH-SY5Y, SK-N-BE(2), and SK-N-SH. Krüppel-like factor 4 (KLF4), a transcription factor crucial for NDRG2 expression, was up-regulated by ITLN1 in NB cells via inactivation of phosphoinositide 3-kinase (PI3K)/AKT signaling. Ectopic expression of ITLN1 suppressed the growth, invasion and metastasis of NB cells in vitro and in vivo. Conversely, knockdown of ITLN1 promoted the growth, invasion, and metastasis of NB cells. In addition, rescue experiments in ITLN1 over-expressed or silenced NB cells showed that restoration of NDRG2 expression prevented the tumor cells from ITLN1-mediated changes in these biological features. In clinical NB tissues, ITLN1 was down-regulated and positively correlated with NDRG2 expression. Patients with high ITLN1 or NDRG2 expression had greater survival probability.ConclusionsThese findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0320-6) contains supplementary material, which is available to authorized users.
Highlights
Recent studies have revealed the potential roles of intelectin 1 (ITLN1) in tumorigenesis
Further analysis revealed six over-lapping genes significantly correlated with ITLN1 in these cancers (Additional file 1: Figure S1B), including N-myc downstream regulated gene 2 (NDRG2), chaperonin containing TCP1 subunit 3 (CCT3), defective in cullin neddylation 1 domain containing 5 (DCUN1D5), enolase 1 (ENO1), microtubuleactin crosslinking factor 1 (MACF1), and Mg2+/Mn2+ dependent protein phosphatase 1G (PPM1G)
ITLN1 vector was stably transfected into SHSY5Y and SK-N-BE(2) cells, resulting in enhanced ITLN1 expression and secretion into culture supernatant and increased NDRG2 expression levels, than those stably transfected with empty vector (Figure 1B and C)
Summary
Recent studies have revealed the potential roles of intelectin 1 (ITLN1) in tumorigenesis. Its functions and underlying mechanisms in neuroblastoma (NB), the most common extracranial solid tumor in childhood, still remain largely unknown. Neuroblastoma (NB), the most common extracranial solid tumor in childhood, accounts for 15% of all pediatric cancer deaths [1]. Recent evidence indicates that galectins, a family of animal lectins, are aberrantly expressed in tumor tissues and play crucial roles in neoplastic transformation and the growth, migration, invasion, and metastasis of tumor cells [2]. Inhibition of galectin-1 expression significantly suppresses the transformed phenotypes of human glioma cells [3]. Previous evidence indicates that both galectin-1 and galectin-7 inhibit the growth of NB cells [6,7], while galectin-3 is broadly expressed in NB cells to impair the apoptosis-sensitive phenotype induced by MYCN [8]. The roles of other lectins in the progression and aggressiveness of NB still remain largely unknown and warrant further investigation
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