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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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p-c-Jun and p-STAT3 contribute to ISL-1 overexpression in NHL cells. (A to B) real-time RT-PCR (A) and Western blot (B) show the expression changes of ISL-1 in Raji and Ly3 cells after treated with JNK signaling pathway activator (Anisomycin, 15 ng/ml) or inhibitor (SP60012, 10 μM) for 6 h. (C to D) real-time RT-PCR (C) and Western blot (D) show the expression changes of ISL-1 in Ly3 cells after treated with JAK/STAT signaling pathway activator (IL-6, 4 ng/ml) or inhibitor (STATTIC, 6 μM) for 24 h. Each bar represents mean ± SD from three samples. p values were calculated using a Student t-test (*p < 0.05, vs. the control).
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Figure 6: p-c-Jun and p-STAT3 contribute to ISL-1 overexpression in NHL cells. (A to B) real-time RT-PCR (A) and Western blot (B) show the expression changes of ISL-1 in Raji and Ly3 cells after treated with JNK signaling pathway activator (Anisomycin, 15 ng/ml) or inhibitor (SP60012, 10 μM) for 6 h. (C to D) real-time RT-PCR (C) and Western blot (D) show the expression changes of ISL-1 in Ly3 cells after treated with JAK/STAT signaling pathway activator (IL-6, 4 ng/ml) or inhibitor (STATTIC, 6 μM) for 24 h. Each bar represents mean ± SD from three samples. p values were calculated using a Student t-test (*p < 0.05, vs. the control).

Mentions: As we known, p-c-Jun and p-STAT3 belong to JNK and JAK/STAT signal pathways, respectively. They are the most important functional activators for the signaling transduction and closely link to lymphoma cell survival, proliferation and transformation[21,24,26-29]. To verify how ISL-1 is regulated by JNK and JAK/STAT signal pathways, we first analyzed the basal expression levels and correlations of p-c-Jun, p-STAT3, along with ISL-1 and the prominent oncogenic protein c-Myc in a series of NHL cell lines and numbers of human NHL tissue specimens. The results of Western blot showed that the p-c-Jun and p-STAT3 were readily detectable and positive consistent with the expression level of ISL-1 and c-Myc in all these cell lines (Figure 4A). We then analyzed the expression of ISL-1, p-c-Jun, p-STAT3 and c-Myc at protein level on 35 cases of human NHL and 10 cases of human normal lymph node by immunohistochemical staining. As shown in Figure 4B, p-c-Jun, p-STAT3 and c-Myc staining were considerably stronger in NHL than in normal lymph node, in parallel with the pattern observed for ISL-1 in NHL. Pearson correlation analysis revealed that the expression level of ISL-1 protein is strongly correlated with p-STAT3, p-c-Jun and c-Myc protein levels in human NHL samples surveyed (Pearson correlation coefficient r = 0.737, 0.501, 0.803 respectively, all p < 0.001). These data indicate that increased coexpression of ISL-1, p-STAT3, p-c-Jun and c-Myc may be associated with the development of NHL. The above results indicate that JNK and JAK/STAT signaling pathways are likely to promote ISL-1 expression through the constitutively activated p-c-Jun and p-STAT3.Further analysis showed that the significantly increased ISL-1 expression was positively associated with the activation of p-c-Jun or p-STAT3, after treated with JNK or JAK/STAT activator (Anisomycin or IL-6). Conversely, after treated with JNK or JAK/STAT inhibitor (SP600125 or STATTIC), the expression of ISL-1 was obviously decreased (Figure 6). These results show that persistent activation of p-c-Jun and p-STAT3 lead to the aberrant transcription of ISL-1 in NHL cells.


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)

p-c-Jun and p-STAT3 contribute to ISL-1 overexpression in NHL cells. (A to B) real-time RT-PCR (A) and Western blot (B) show the expression changes of ISL-1 in Raji and Ly3 cells after treated with JNK signaling pathway activator (Anisomycin, 15 ng/ml) or inhibitor (SP60012, 10 μM) for 6 h. (C to D) real-time RT-PCR (C) and Western blot (D) show the expression changes of ISL-1 in Ly3 cells after treated with JAK/STAT signaling pathway activator (IL-6, 4 ng/ml) or inhibitor (STATTIC, 6 μM) for 24 h. Each bar represents mean ± SD from three samples. p values were calculated using a Student t-test (*p < 0.05, vs. the control).
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Figure 6: p-c-Jun and p-STAT3 contribute to ISL-1 overexpression in NHL cells. (A to B) real-time RT-PCR (A) and Western blot (B) show the expression changes of ISL-1 in Raji and Ly3 cells after treated with JNK signaling pathway activator (Anisomycin, 15 ng/ml) or inhibitor (SP60012, 10 μM) for 6 h. (C to D) real-time RT-PCR (C) and Western blot (D) show the expression changes of ISL-1 in Ly3 cells after treated with JAK/STAT signaling pathway activator (IL-6, 4 ng/ml) or inhibitor (STATTIC, 6 μM) for 24 h. Each bar represents mean ± SD from three samples. p values were calculated using a Student t-test (*p < 0.05, vs. the control).
Mentions: As we known, p-c-Jun and p-STAT3 belong to JNK and JAK/STAT signal pathways, respectively. They are the most important functional activators for the signaling transduction and closely link to lymphoma cell survival, proliferation and transformation[21,24,26-29]. To verify how ISL-1 is regulated by JNK and JAK/STAT signal pathways, we first analyzed the basal expression levels and correlations of p-c-Jun, p-STAT3, along with ISL-1 and the prominent oncogenic protein c-Myc in a series of NHL cell lines and numbers of human NHL tissue specimens. The results of Western blot showed that the p-c-Jun and p-STAT3 were readily detectable and positive consistent with the expression level of ISL-1 and c-Myc in all these cell lines (Figure 4A). We then analyzed the expression of ISL-1, p-c-Jun, p-STAT3 and c-Myc at protein level on 35 cases of human NHL and 10 cases of human normal lymph node by immunohistochemical staining. As shown in Figure 4B, p-c-Jun, p-STAT3 and c-Myc staining were considerably stronger in NHL than in normal lymph node, in parallel with the pattern observed for ISL-1 in NHL. Pearson correlation analysis revealed that the expression level of ISL-1 protein is strongly correlated with p-STAT3, p-c-Jun and c-Myc protein levels in human NHL samples surveyed (Pearson correlation coefficient r = 0.737, 0.501, 0.803 respectively, all p < 0.001). These data indicate that increased coexpression of ISL-1, p-STAT3, p-c-Jun and c-Myc may be associated with the development of NHL. The above results indicate that JNK and JAK/STAT signaling pathways are likely to promote ISL-1 expression through the constitutively activated p-c-Jun and p-STAT3.Further analysis showed that the significantly increased ISL-1 expression was positively associated with the activation of p-c-Jun or p-STAT3, after treated with JNK or JAK/STAT activator (Anisomycin or IL-6). Conversely, after treated with JNK or JAK/STAT inhibitor (SP600125 or STATTIC), the expression of ISL-1 was obviously decreased (Figure 6). These results show that persistent activation of p-c-Jun and p-STAT3 lead to the aberrant transcription of ISL-1 in NHL cells.

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