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Intelectin 1 suppresses the growth, invasion and metastasis of neuroblastoma cells through up-regulation of N-myc downstream regulated gene 2.

Li D, Mei H, Pu J, Xiang X, Zhao X, Qu H, Huang K, Zheng L, Tong Q - Mol. Cancer (2015)

Bottom Line: 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.Patients with high ITLN1 or NDRG2 expression had greater survival probability.These findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. d.li26@hotmail.com.

ABSTRACT

Background: Recent 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.

Methods: Human 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.

Results: Mining 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.

Conclusions: These findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.

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ITLN1 facilitates the expression of KLF4 via inactivation of PI3K/AKT signaling. (A) Western blot showing the phosphorylation of AKT (T308 and S473) and expression of KLF4 in solvent (mock)- or recombinant ITLN1-treated SH-SY5Y (1 and 2 μg/ml for 24 hrs) and SK-N-BE(2) (1 μg/ml for 24 and 36 hrs) cells. (B) Western blot showing the phosphorylation of AKT (T308 and S473) and expression of KLF4 in NB cells stably transfected with sh-Scb or sh-ITLN1, and those pre-treated with LY294002 (10 μmol/L).
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Fig3: ITLN1 facilitates the expression of KLF4 via inactivation of PI3K/AKT signaling. (A) Western blot showing the phosphorylation of AKT (T308 and S473) and expression of KLF4 in solvent (mock)- or recombinant ITLN1-treated SH-SY5Y (1 and 2 μg/ml for 24 hrs) and SK-N-BE(2) (1 μg/ml for 24 and 36 hrs) cells. (B) Western blot showing the phosphorylation of AKT (T308 and S473) and expression of KLF4 in NB cells stably transfected with sh-Scb or sh-ITLN1, and those pre-treated with LY294002 (10 μmol/L).

Mentions: To further explore the mechanisms for ITLN1-induced KLF4 expression, we observed the changes in phosphoinositide 3-kinase (PI3K)/AKT signaling that regulates the KLF4 expression [19]. Administration of recombinant ITLN1 protein (1 and 2 μg/ml) into SH-SY5Y and SK-N-BE(2) cells reduced the phosphorylation of AKT (T308 and S473), and up-regulated the expression of KLF4 at 24 and 36 hrs post-administration, than those treated with solvent control (mock) (Figure 3A). In contrast, stable knockdown of ITLN1 induced the phosphorylation of AKT (T308 and S473), and down-regulated the KLF4 expression in SH-SY5Y and SK-N-SH cells, which was abolished by administration of PI3K activity inhibitor LY294002 (Figure 3B). These results suggest that ITLN1 facilitates the KLF4 expression through attenuating PI3K/AKT signaling in NB cells.Figure 3


Intelectin 1 suppresses the growth, invasion and metastasis of neuroblastoma cells through up-regulation of N-myc downstream regulated gene 2.

Li D, Mei H, Pu J, Xiang X, Zhao X, Qu H, Huang K, Zheng L, Tong Q - Mol. Cancer (2015)

ITLN1 facilitates the expression of KLF4 via inactivation of PI3K/AKT signaling. (A) Western blot showing the phosphorylation of AKT (T308 and S473) and expression of KLF4 in solvent (mock)- or recombinant ITLN1-treated SH-SY5Y (1 and 2 μg/ml for 24 hrs) and SK-N-BE(2) (1 μg/ml for 24 and 36 hrs) cells. (B) Western blot showing the phosphorylation of AKT (T308 and S473) and expression of KLF4 in NB cells stably transfected with sh-Scb or sh-ITLN1, and those pre-treated with LY294002 (10 μmol/L).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359454&req=5

Fig3: ITLN1 facilitates the expression of KLF4 via inactivation of PI3K/AKT signaling. (A) Western blot showing the phosphorylation of AKT (T308 and S473) and expression of KLF4 in solvent (mock)- or recombinant ITLN1-treated SH-SY5Y (1 and 2 μg/ml for 24 hrs) and SK-N-BE(2) (1 μg/ml for 24 and 36 hrs) cells. (B) Western blot showing the phosphorylation of AKT (T308 and S473) and expression of KLF4 in NB cells stably transfected with sh-Scb or sh-ITLN1, and those pre-treated with LY294002 (10 μmol/L).
Mentions: To further explore the mechanisms for ITLN1-induced KLF4 expression, we observed the changes in phosphoinositide 3-kinase (PI3K)/AKT signaling that regulates the KLF4 expression [19]. Administration of recombinant ITLN1 protein (1 and 2 μg/ml) into SH-SY5Y and SK-N-BE(2) cells reduced the phosphorylation of AKT (T308 and S473), and up-regulated the expression of KLF4 at 24 and 36 hrs post-administration, than those treated with solvent control (mock) (Figure 3A). In contrast, stable knockdown of ITLN1 induced the phosphorylation of AKT (T308 and S473), and down-regulated the KLF4 expression in SH-SY5Y and SK-N-SH cells, which was abolished by administration of PI3K activity inhibitor LY294002 (Figure 3B). These results suggest that ITLN1 facilitates the KLF4 expression through attenuating PI3K/AKT signaling in NB cells.Figure 3

Bottom Line: 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.Patients with high ITLN1 or NDRG2 expression had greater survival probability.These findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. d.li26@hotmail.com.

ABSTRACT

Background: Recent 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.

Methods: Human 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.

Results: Mining 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.

Conclusions: These findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.

Show MeSH
Related in: MedlinePlus