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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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Related in: MedlinePlus

ITLN1 is under-expressed in NB tissues and cell lines. (A) Immunohistochemical staining showing the expression of ITLN1 and NDRG2 in tumor specimens from 42 NB cases (arrowheads, brown). Scale bars: 100 μm. (B) Western blot showing the endogenous protein levels of ITLN1 and NDRG2 in lysate from NB tissues (n = 30), NB cell lines, and normal dorsal ganglia (DG). (C) Endogenous transcript levels of ITLN1 and NDRG2 in NB tissues (n = 30), NB cell lines, and DG as detected by real-time quantitative RT-PCR. (D) Pearson’s coefficient correlation analysis for the relationship between ITLN1 and NDRG2 transcript levels in NB tissues (n = 30). (E) Kaplan–Meier survival plots of well-defined NB patients with low and high expression of ITLN1 or NDRG2, as derived from R2 microarray analysis and visualization platform (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi). *P < 0.01 vs. DG.
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Fig6: ITLN1 is under-expressed in NB tissues and cell lines. (A) Immunohistochemical staining showing the expression of ITLN1 and NDRG2 in tumor specimens from 42 NB cases (arrowheads, brown). Scale bars: 100 μm. (B) Western blot showing the endogenous protein levels of ITLN1 and NDRG2 in lysate from NB tissues (n = 30), NB cell lines, and normal dorsal ganglia (DG). (C) Endogenous transcript levels of ITLN1 and NDRG2 in NB tissues (n = 30), NB cell lines, and DG as detected by real-time quantitative RT-PCR. (D) Pearson’s coefficient correlation analysis for the relationship between ITLN1 and NDRG2 transcript levels in NB tissues (n = 30). (E) Kaplan–Meier survival plots of well-defined NB patients with low and high expression of ITLN1 or NDRG2, as derived from R2 microarray analysis and visualization platform (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi). *P < 0.01 vs. DG.

Mentions: To investigate the ITLN1 and NDRG2 expression in NB, paraffin-embedded sections from 42 well-established primary cases were collected [20,21]. Immunohistochemical staining revealed that ITLN1 was expressed in the tumor cells of NB tissues (Figure 6A). ITLN1 expression was detected in 14/42 (33.3%) cases and the staining was weak in 6, moderate in 5, and intense in 3 (Additional file 7: Table S1). The ITLN1 immunoreactivity was significantly higher in NB cases with age less than 1 year (P = 0.03), good differentiation (P < 0.001), lower mitosis karyorrhexis index (MKI) (P = 0.002), and early INSS stages (P = 0.003) (Additional file 7: Table S1). Notably, the immunostaining of ITLN1 was associated with NDRG2 immunoreactivity in NB cases (correlation coefficient R = 0.676, P < 0.001; Figure 6A and Additional file 8: Table S2). Moreover, western blot and real-time quantitative RT-PCR indicated lower expression levels of ITLN1 and NDRG2 in subtotal 30 NB specimens and cultured SK-N-SH, SK-N-AS, SH-SY5Y, and SK-N-BE(2) cell lines, than those in normal dorsal ganglia (Figure 6B and C). There was a positive correlation between ITLN1 and NDRG2 transcript levels in NB tissues (correlation coefficient R = 0.827, P < 0.001, Figure 6D). Administration of DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR) or pan histone deacetylase inhibitor trichostatin A (TSA) resulted in slightly increased ITLN1 transcript levels in NB cells (Additional file 9: Figure S7). Kaplan–Meier survival plots of well-defined NB cases derived from R2 microarray analysis and visualization platform revealed that patients with high ITLN1 (P = 0.025) or NDRG2 (P = 0.0015) expression had greater survival probability than those with low expression (Figure 6E). These results indicate that ITLN1 is under-expressed and correlated with the NDRG2 expression in NB tissues and cell lines.Figure 6


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 is under-expressed in NB tissues and cell lines. (A) Immunohistochemical staining showing the expression of ITLN1 and NDRG2 in tumor specimens from 42 NB cases (arrowheads, brown). Scale bars: 100 μm. (B) Western blot showing the endogenous protein levels of ITLN1 and NDRG2 in lysate from NB tissues (n = 30), NB cell lines, and normal dorsal ganglia (DG). (C) Endogenous transcript levels of ITLN1 and NDRG2 in NB tissues (n = 30), NB cell lines, and DG as detected by real-time quantitative RT-PCR. (D) Pearson’s coefficient correlation analysis for the relationship between ITLN1 and NDRG2 transcript levels in NB tissues (n = 30). (E) Kaplan–Meier survival plots of well-defined NB patients with low and high expression of ITLN1 or NDRG2, as derived from R2 microarray analysis and visualization platform (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi). *P < 0.01 vs. DG.
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Related In: Results  -  Collection

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Fig6: ITLN1 is under-expressed in NB tissues and cell lines. (A) Immunohistochemical staining showing the expression of ITLN1 and NDRG2 in tumor specimens from 42 NB cases (arrowheads, brown). Scale bars: 100 μm. (B) Western blot showing the endogenous protein levels of ITLN1 and NDRG2 in lysate from NB tissues (n = 30), NB cell lines, and normal dorsal ganglia (DG). (C) Endogenous transcript levels of ITLN1 and NDRG2 in NB tissues (n = 30), NB cell lines, and DG as detected by real-time quantitative RT-PCR. (D) Pearson’s coefficient correlation analysis for the relationship between ITLN1 and NDRG2 transcript levels in NB tissues (n = 30). (E) Kaplan–Meier survival plots of well-defined NB patients with low and high expression of ITLN1 or NDRG2, as derived from R2 microarray analysis and visualization platform (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi). *P < 0.01 vs. DG.
Mentions: To investigate the ITLN1 and NDRG2 expression in NB, paraffin-embedded sections from 42 well-established primary cases were collected [20,21]. Immunohistochemical staining revealed that ITLN1 was expressed in the tumor cells of NB tissues (Figure 6A). ITLN1 expression was detected in 14/42 (33.3%) cases and the staining was weak in 6, moderate in 5, and intense in 3 (Additional file 7: Table S1). The ITLN1 immunoreactivity was significantly higher in NB cases with age less than 1 year (P = 0.03), good differentiation (P < 0.001), lower mitosis karyorrhexis index (MKI) (P = 0.002), and early INSS stages (P = 0.003) (Additional file 7: Table S1). Notably, the immunostaining of ITLN1 was associated with NDRG2 immunoreactivity in NB cases (correlation coefficient R = 0.676, P < 0.001; Figure 6A and Additional file 8: Table S2). Moreover, western blot and real-time quantitative RT-PCR indicated lower expression levels of ITLN1 and NDRG2 in subtotal 30 NB specimens and cultured SK-N-SH, SK-N-AS, SH-SY5Y, and SK-N-BE(2) cell lines, than those in normal dorsal ganglia (Figure 6B and C). There was a positive correlation between ITLN1 and NDRG2 transcript levels in NB tissues (correlation coefficient R = 0.827, P < 0.001, Figure 6D). Administration of DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR) or pan histone deacetylase inhibitor trichostatin A (TSA) resulted in slightly increased ITLN1 transcript levels in NB cells (Additional file 9: Figure S7). Kaplan–Meier survival plots of well-defined NB cases derived from R2 microarray analysis and visualization platform revealed that patients with high ITLN1 (P = 0.025) or NDRG2 (P = 0.0015) expression had greater survival probability than those with low expression (Figure 6E). These results indicate that ITLN1 is under-expressed and correlated with the NDRG2 expression in NB tissues and cell lines.Figure 6

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