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ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex.

Zhang Q, Yang Z, Jia Z, Liu C, Guo C, Lu H, Chen P, Ma K, Wang W, Zhou C - Mol. Cancer (2014)

Bottom Line: Recently, ISL-1 has been found in some types of human cancers.Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples.Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, 38 Xueyuan Road, 100191 Beijing, China. wwp@bjmu.edu.cn.

ABSTRACT

Background: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, is essential for the heart, motor neuron and pancreas development. Recently, ISL-1 has been found in some types of human cancers. However, how ISL-1 exerts the role in tumor development is not clear.

Methods and results: The expression of ISL-1 was assessed in 211 human lymphoma samples and 23 normal lymph node samples. Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples. CCK-8 analysis, cell cycle assay and xenograft model were performed to characterize the association between ISL-1 expression level and biological functions in NHL. The results showed that ISL-1 overexpression obviously promoted NHL cells proliferation, changed the cell cycle distribution in vitro and significantly enhanced xenografted lymphoma development in vivo. Real-time PCR, Western blot, luciferase assay and ChIP assay were used to explore the potential regulatory targets of ISL-1 and the results demonstrated that ISL-1 activated the c-Myc expression in NHL by direct binding to a conserved binding site on the c-Myc enhancer. Further results revealed that ISL-1 could be positively regulated by the c-Jun N-terminal kinase (JNK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Both the JNK and JAK/STAT signaling inhibitors could significantly suppressed the growth of NHL cells through the down-regulation of ISL-1 as demonstrated by CCK-8 and Western blot assays. Bioinformatic analysis and luciferase assay exhibited that ISL-1 was a novel target of p-STAT3 and p-c-jun. ChIP, Co-IP and ChIP-re-IP analysis revealed that ISL-1 could participate with p-STAT3 and p-c-Jun to form a p-STAT3/p-c-Jun/ISL-1 transcriptional complex that binds directly on the ISL-1 promoter, demonstrating a positive feedback regulatory mechanism for ISL-1 expression in NHL.

Conclusions: Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

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The expression of ISL-1 is positively correlated to the expression of p-STAT3, p-c-Jun and c-Myc. (A) NHL cell lines were analyzed by Western blot with indicated antibodies. (B) Immunohistochemistry for ISL-1, p-STAT3, p-c-Jun and c-Myc expression were performed in multiple specimens of normal lymph nodes (top panel) and NHL patients (bottom panel). Representative images of ISL-1, p-STAT3, p-c-Jun and c-Myc expression levels and cellular distributions in different samples are shown (200 ×). Scale bars = 100 μm
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Figure 4: The expression of ISL-1 is positively correlated to the expression of p-STAT3, p-c-Jun and c-Myc. (A) NHL cell lines were analyzed by Western blot with indicated antibodies. (B) Immunohistochemistry for ISL-1, p-STAT3, p-c-Jun and c-Myc expression were performed in multiple specimens of normal lymph nodes (top panel) and NHL patients (bottom panel). Representative images of ISL-1, p-STAT3, p-c-Jun and c-Myc expression levels and cellular distributions in different samples are shown (200 ×). Scale bars = 100 μm

Mentions: To explore the mechanism of ISL-1-stimulated NHL cell proliferation, bioinformatic analysis was performed with professional MatInspector software and refFlat Database to identify the downstream target genes of ISL-1. Several putative genes, including CyclinD1, BCL-6 and c-Myc were identified for further investigation, as these genes contain conserved ISL-1 binding sequences (YTAATGR) on the upstream of the ATG translation start site[16-18]. More importantly, they are remarkably related to the pathogenesis of NHL as previously reported[16-18]. However, the expression of CyclinD1 and BCL-6 did not show a predicted correlation with ISL-1 in NHL cells (data not shown). Therefore, we focused on c-Myc in the rest investigations.Western blot results showed that the basal expression level of c-Myc was positively correlated with the expression level of ISL-1 in NHL cell lines (will be discussed later in Figure 4A). Moreover, further results indicated that the overexpression of ISL-1 increased the expression of c-Myc at both mRNA and protein levels in Raji cells (Figure 5A, B left panel). Whereas, the significant decrease of c-Myc expression was associated with the knockdown of ISL-1 as compared with those in the control Ly3 cells (Figure 5A,B right panel). These results show that ISL-1 could act as a transcriptional activator of c-Myc.


ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex.

Zhang Q, Yang Z, Jia Z, Liu C, Guo C, Lu H, Chen P, Ma K, Wang W, Zhou C - Mol. Cancer (2014)

The expression of ISL-1 is positively correlated to the expression of p-STAT3, p-c-Jun and c-Myc. (A) NHL cell lines were analyzed by Western blot with indicated antibodies. (B) Immunohistochemistry for ISL-1, p-STAT3, p-c-Jun and c-Myc expression were performed in multiple specimens of normal lymph nodes (top panel) and NHL patients (bottom panel). Representative images of ISL-1, p-STAT3, p-c-Jun and c-Myc expression levels and cellular distributions in different samples are shown (200 ×). Scale bars = 100 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4125377&req=5

Figure 4: The expression of ISL-1 is positively correlated to the expression of p-STAT3, p-c-Jun and c-Myc. (A) NHL cell lines were analyzed by Western blot with indicated antibodies. (B) Immunohistochemistry for ISL-1, p-STAT3, p-c-Jun and c-Myc expression were performed in multiple specimens of normal lymph nodes (top panel) and NHL patients (bottom panel). Representative images of ISL-1, p-STAT3, p-c-Jun and c-Myc expression levels and cellular distributions in different samples are shown (200 ×). Scale bars = 100 μm
Mentions: To explore the mechanism of ISL-1-stimulated NHL cell proliferation, bioinformatic analysis was performed with professional MatInspector software and refFlat Database to identify the downstream target genes of ISL-1. Several putative genes, including CyclinD1, BCL-6 and c-Myc were identified for further investigation, as these genes contain conserved ISL-1 binding sequences (YTAATGR) on the upstream of the ATG translation start site[16-18]. More importantly, they are remarkably related to the pathogenesis of NHL as previously reported[16-18]. However, the expression of CyclinD1 and BCL-6 did not show a predicted correlation with ISL-1 in NHL cells (data not shown). Therefore, we focused on c-Myc in the rest investigations.Western blot results showed that the basal expression level of c-Myc was positively correlated with the expression level of ISL-1 in NHL cell lines (will be discussed later in Figure 4A). Moreover, further results indicated that the overexpression of ISL-1 increased the expression of c-Myc at both mRNA and protein levels in Raji cells (Figure 5A, B left panel). Whereas, the significant decrease of c-Myc expression was associated with the knockdown of ISL-1 as compared with those in the control Ly3 cells (Figure 5A,B right panel). These results show that ISL-1 could act as a transcriptional activator of c-Myc.

Bottom Line: Recently, ISL-1 has been found in some types of human cancers.Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples.Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, 38 Xueyuan Road, 100191 Beijing, China. wwp@bjmu.edu.cn.

ABSTRACT

Background: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, is essential for the heart, motor neuron and pancreas development. Recently, ISL-1 has been found in some types of human cancers. However, how ISL-1 exerts the role in tumor development is not clear.

Methods and results: The expression of ISL-1 was assessed in 211 human lymphoma samples and 23 normal lymph node samples. Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples. CCK-8 analysis, cell cycle assay and xenograft model were performed to characterize the association between ISL-1 expression level and biological functions in NHL. The results showed that ISL-1 overexpression obviously promoted NHL cells proliferation, changed the cell cycle distribution in vitro and significantly enhanced xenografted lymphoma development in vivo. Real-time PCR, Western blot, luciferase assay and ChIP assay were used to explore the potential regulatory targets of ISL-1 and the results demonstrated that ISL-1 activated the c-Myc expression in NHL by direct binding to a conserved binding site on the c-Myc enhancer. Further results revealed that ISL-1 could be positively regulated by the c-Jun N-terminal kinase (JNK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Both the JNK and JAK/STAT signaling inhibitors could significantly suppressed the growth of NHL cells through the down-regulation of ISL-1 as demonstrated by CCK-8 and Western blot assays. Bioinformatic analysis and luciferase assay exhibited that ISL-1 was a novel target of p-STAT3 and p-c-jun. ChIP, Co-IP and ChIP-re-IP analysis revealed that ISL-1 could participate with p-STAT3 and p-c-Jun to form a p-STAT3/p-c-Jun/ISL-1 transcriptional complex that binds directly on the ISL-1 promoter, demonstrating a positive feedback regulatory mechanism for ISL-1 expression in NHL.

Conclusions: Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

Show MeSH
Related in: MedlinePlus