Al-Jamal Hamid Ali Nagi, Mat Jusoh Siti Asmaa, Hassan Rosline, Johan Muhammad Farid
Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia.
BMC Cancer. 2015 Nov 7;15:869. doi: 10.1186/s12885-015-1695-x.
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.
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.
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).
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.
在包括急性髓系白血病（AML）在内的多种癌症中，肿瘤抑制基因会因甲基化而失活。含Src同源2（SH2）结构域的蛋白酪氨酸磷酸酶1（SHP-1）是JAK/STAT信号通路的负调控因子。SHP-1的转录沉默通过激活STAT3在癌症的发生和发展中起关键作用。5-氮杂胞苷（5-Aza）是一种DNA甲基转移酶抑制剂，可导致DNA去甲基化，从而使沉默的SHP-1重新表达。来他替尼（CEP-701）是一种多靶点酪氨酸激酶抑制剂，能有效抑制FLT3酪氨酸激酶，并诱导携带FLT3基因内部串联重复（FLT3-ITD）的AML患者出现血液学缓解。然而，临床试验中的大多数患者对CEP-701产生了耐药性。因此，本研究的目的是评估SHP-1重新表达对耐药AML细胞中CEP-701敏感性的影响。
通过将MV4-11过度暴露于CEP-701来培养携带FLT3-ITD的耐药细胞，并研究5-Aza处理的效果。分别使用膜联蛋白V和MTS法测定CEP-701的凋亡和细胞毒性。通过定量实时PCR进行基因表达分析。通过蛋白质免疫印迹法检测STATs的活性，并使用甲基化特异性PCR（MS-PCR）和焦磷酸测序分析研究SHP-1的甲基化图谱。采用重复测量方差分析和Kruskal-Wallis检验进行统计分析。
与亲本细胞和MV4-11R-cep + 5-Aza细胞相比，CEP-701对耐药细胞的细胞毒性剂量显著更高（p = 0.004）。与其他细胞相比，耐药细胞显示出显著更高的活力和更低的凋亡率（p < 0.001）。与亲本细胞和耐药细胞相比，MV4-11R-cep + 5-Aza细胞中SHP-1的表达高7倍（p = 0.011）。耐药细胞中的STAT3被激活。MV4-11R-cep + 5-Aza细胞中SHP-1的甲基化显著降低（p = 0.002）。
SHP-1表达的恢复可诱导对CEP-701的敏感性，并可作为AML治疗的一个靶点。我们的研究结果支持以下假设，即SHP-1的肿瘤抑制作用因表观遗传沉默而丧失，其重新表达可能在重新诱导对酪氨酸激酶抑制剂（TKIs）的敏感性中起重要作用。因此，SHP-1可能是FLT3-ITD + AML患者对CEP-701产生耐药性过程中的一个合理候选因素。
