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Enhancing SHP-1 expression with 5-azacytidine may inhibit STAT3 activation and confer sensitivity in lestaurtinib (CEP-701)-resistant FLT3-ITD positive acute myeloid leukemia.

Al-Jamal HA, Mat Jusoh SA, Hassan R, Johan MF - BMC Cancer (2015)

Bottom Line: The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p < 0.001).Methylation of SHP-1 was significantly decreased in MV4-11R-cep + 5-Aza cells (p = 0.002).Our findings support the hypothesis that, the tumor-suppressor effect of SHP-1 is lost due to epigenetic silencing and its re-expression might play an important role in re-inducing sensitivity to TKIs.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia. aljamalhamid08@gmail.com.

ABSTRACT

Background: Tumor-suppressor genes are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Src homology-2 (SH2)-containing protein-tyrosine phosphatase 1 (SHP-1) is a negative regulator of the JAK/STAT pathway. Transcriptional silencing of SHP-1 plays a critical role in the development and progression of cancers through STAT3 activation. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA demethylation resulting in re-expression of silenced SHP-1. Lestaurtinib (CEP-701) is a multi-targeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in AML patients harboring the internal tandem duplication of the FLT3 gene (FLT3-ITD). However, the majority of patients in clinical trials developed resistance to CEP-701. Therefore, the aim of this study, was to assess the effect of re-expression of SHP-1 on sensitivity to CEP-701 in resistant AML cells.

Methods: Resistant cells harboring the FLT3-ITD were developed by overexposure of MV4-11 to CEP-701, and the effects of 5-Aza treatment were investigated. Apoptosis and cytotoxicity of CEP-701 were determined using Annexin V and MTS assays, respectively. Gene expression was performed by quantitative real-time PCR. STATs activity was examined by western blotting and the methylation profile of SHP-1 was studied using MS-PCR and pyrosequencing analysis. Repeated-measures ANOVA and Kruskal-Wallis tests were used for statistical analysis.

Results: The cytotoxic dose of CEP-701 on resistant cells was significantly higher in comparison with parental and MV4-11R-cep + 5-Aza cells (p = 0.004). The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p < 0.001). Expression of SHP-1 was 7-fold higher in MV4-11R-cep + 5-Aza cells compared to parental and resistant cells (p = 0.011). STAT3 was activated in resistant cells. Methylation of SHP-1 was significantly decreased in MV4-11R-cep + 5-Aza cells (p = 0.002).

Conclusions: The restoration of SHP-1 expression induces sensitivity towards CEP-701 and could serve as a target in the treatment of AML. Our findings support the hypothesis that, the tumor-suppressor effect of SHP-1 is lost due to epigenetic silencing and its re-expression might play an important role in re-inducing sensitivity to TKIs. Thus, SHP-1 is a plausible candidate for a role in the development of CEP-701 resistance in FLT3-ITD+ AML patients.

No MeSH data available.


Related in: MedlinePlus

Real-time quantitative PCR (RQ-PCR) and methylation status of SHP-1 in all cells. a The relative normalized ratio of RQ-PCR revealed that, SHP-1 is re-expressed in MV4-11R-cep + 5-Aza cells 7-fold higher compared with that in MV4-11 and MV4-11R-cep cells (p = 0.011). b Methylation-specific polymerase chain reaction (MS-PCR) showed methylation of SHP-1 in MV4-11 and MV4-11R-cep cells but not in MV4-11R-cep + 5-Aza cells. c Pyrosequencing analysis revealed low methylation levels of the CpG islands in the promoter region of SHP-1 in MV4-11R-cep + 5-Aza cells. The Kruskal–Wallis test was applied followed by the Multiple Mann–Whitney Test with Bonferroni correction. The box blot showed a significant lower (p = 0.023) of methylation in the CpG islands of SHP-1 gene in MV4-11R-cep + 5-Aza cells compared with that in MV4-11 and MV4-11R-cep cells. However, there was no significant difference in the methylation levels of CpG islands in the same region of SHP-1 genes between MV4-11 and MV4-11R-cep cells (p = 0.200)
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Fig4: Real-time quantitative PCR (RQ-PCR) and methylation status of SHP-1 in all cells. a The relative normalized ratio of RQ-PCR revealed that, SHP-1 is re-expressed in MV4-11R-cep + 5-Aza cells 7-fold higher compared with that in MV4-11 and MV4-11R-cep cells (p = 0.011). b Methylation-specific polymerase chain reaction (MS-PCR) showed methylation of SHP-1 in MV4-11 and MV4-11R-cep cells but not in MV4-11R-cep + 5-Aza cells. c Pyrosequencing analysis revealed low methylation levels of the CpG islands in the promoter region of SHP-1 in MV4-11R-cep + 5-Aza cells. The Kruskal–Wallis test was applied followed by the Multiple Mann–Whitney Test with Bonferroni correction. The box blot showed a significant lower (p = 0.023) of methylation in the CpG islands of SHP-1 gene in MV4-11R-cep + 5-Aza cells compared with that in MV4-11 and MV4-11R-cep cells. However, there was no significant difference in the methylation levels of CpG islands in the same region of SHP-1 genes between MV4-11 and MV4-11R-cep cells (p = 0.200)

Mentions: To investigate the correlation between re-expression of SHP-1 and demethylation, gene expression by RT-qPCR was performed on MV4-11, MV4-11R-cep, and MV4-11R-cep + 5-Aza cells. The results showed a significant up-regulation of SHP-1 in MV4-11R-cep + 5-Aza cells compared with MV4-11 and MV4-11R-cep cells (p = 0.011 and p = 0.002, respectively; Fig. 4-a).Fig 4


Enhancing SHP-1 expression with 5-azacytidine may inhibit STAT3 activation and confer sensitivity in lestaurtinib (CEP-701)-resistant FLT3-ITD positive acute myeloid leukemia.

Al-Jamal HA, Mat Jusoh SA, Hassan R, Johan MF - BMC Cancer (2015)

Real-time quantitative PCR (RQ-PCR) and methylation status of SHP-1 in all cells. a The relative normalized ratio of RQ-PCR revealed that, SHP-1 is re-expressed in MV4-11R-cep + 5-Aza cells 7-fold higher compared with that in MV4-11 and MV4-11R-cep cells (p = 0.011). b Methylation-specific polymerase chain reaction (MS-PCR) showed methylation of SHP-1 in MV4-11 and MV4-11R-cep cells but not in MV4-11R-cep + 5-Aza cells. c Pyrosequencing analysis revealed low methylation levels of the CpG islands in the promoter region of SHP-1 in MV4-11R-cep + 5-Aza cells. The Kruskal–Wallis test was applied followed by the Multiple Mann–Whitney Test with Bonferroni correction. The box blot showed a significant lower (p = 0.023) of methylation in the CpG islands of SHP-1 gene in MV4-11R-cep + 5-Aza cells compared with that in MV4-11 and MV4-11R-cep cells. However, there was no significant difference in the methylation levels of CpG islands in the same region of SHP-1 genes between MV4-11 and MV4-11R-cep cells (p = 0.200)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4637135&req=5

Fig4: Real-time quantitative PCR (RQ-PCR) and methylation status of SHP-1 in all cells. a The relative normalized ratio of RQ-PCR revealed that, SHP-1 is re-expressed in MV4-11R-cep + 5-Aza cells 7-fold higher compared with that in MV4-11 and MV4-11R-cep cells (p = 0.011). b Methylation-specific polymerase chain reaction (MS-PCR) showed methylation of SHP-1 in MV4-11 and MV4-11R-cep cells but not in MV4-11R-cep + 5-Aza cells. c Pyrosequencing analysis revealed low methylation levels of the CpG islands in the promoter region of SHP-1 in MV4-11R-cep + 5-Aza cells. The Kruskal–Wallis test was applied followed by the Multiple Mann–Whitney Test with Bonferroni correction. The box blot showed a significant lower (p = 0.023) of methylation in the CpG islands of SHP-1 gene in MV4-11R-cep + 5-Aza cells compared with that in MV4-11 and MV4-11R-cep cells. However, there was no significant difference in the methylation levels of CpG islands in the same region of SHP-1 genes between MV4-11 and MV4-11R-cep cells (p = 0.200)
Mentions: To investigate the correlation between re-expression of SHP-1 and demethylation, gene expression by RT-qPCR was performed on MV4-11, MV4-11R-cep, and MV4-11R-cep + 5-Aza cells. The results showed a significant up-regulation of SHP-1 in MV4-11R-cep + 5-Aza cells compared with MV4-11 and MV4-11R-cep cells (p = 0.011 and p = 0.002, respectively; Fig. 4-a).Fig 4

Bottom Line: The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p < 0.001).Methylation of SHP-1 was significantly decreased in MV4-11R-cep + 5-Aza cells (p = 0.002).Our findings support the hypothesis that, the tumor-suppressor effect of SHP-1 is lost due to epigenetic silencing and its re-expression might play an important role in re-inducing sensitivity to TKIs.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia. aljamalhamid08@gmail.com.

ABSTRACT

Background: Tumor-suppressor genes are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Src homology-2 (SH2)-containing protein-tyrosine phosphatase 1 (SHP-1) is a negative regulator of the JAK/STAT pathway. Transcriptional silencing of SHP-1 plays a critical role in the development and progression of cancers through STAT3 activation. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA demethylation resulting in re-expression of silenced SHP-1. Lestaurtinib (CEP-701) is a multi-targeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in AML patients harboring the internal tandem duplication of the FLT3 gene (FLT3-ITD). However, the majority of patients in clinical trials developed resistance to CEP-701. Therefore, the aim of this study, was to assess the effect of re-expression of SHP-1 on sensitivity to CEP-701 in resistant AML cells.

Methods: Resistant cells harboring the FLT3-ITD were developed by overexposure of MV4-11 to CEP-701, and the effects of 5-Aza treatment were investigated. Apoptosis and cytotoxicity of CEP-701 were determined using Annexin V and MTS assays, respectively. Gene expression was performed by quantitative real-time PCR. STATs activity was examined by western blotting and the methylation profile of SHP-1 was studied using MS-PCR and pyrosequencing analysis. Repeated-measures ANOVA and Kruskal-Wallis tests were used for statistical analysis.

Results: The cytotoxic dose of CEP-701 on resistant cells was significantly higher in comparison with parental and MV4-11R-cep + 5-Aza cells (p = 0.004). The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p < 0.001). Expression of SHP-1 was 7-fold higher in MV4-11R-cep + 5-Aza cells compared to parental and resistant cells (p = 0.011). STAT3 was activated in resistant cells. Methylation of SHP-1 was significantly decreased in MV4-11R-cep + 5-Aza cells (p = 0.002).

Conclusions: The restoration of SHP-1 expression induces sensitivity towards CEP-701 and could serve as a target in the treatment of AML. Our findings support the hypothesis that, the tumor-suppressor effect of SHP-1 is lost due to epigenetic silencing and its re-expression might play an important role in re-inducing sensitivity to TKIs. Thus, SHP-1 is a plausible candidate for a role in the development of CEP-701 resistance in FLT3-ITD+ AML patients.

No MeSH data available.


Related in: MedlinePlus