Wang Limengmeng, Xiao Haowen, Zhang Xing, Liao Weichao, Fu Shan, Huang He
Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, P.R. China.
Int J Oncol. 2015 Nov;47(5):1685-95. doi: 10.3892/ijo.2015.3163. Epub 2015 Sep 14.
All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance is a major problem influencing the efficacy of ATRA. Identification of mechanisms of ATRA resistance are urgenly needed. In the present study, we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Moreover, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cells, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. We restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells, respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence of myeloid differentiation. Further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensitivity of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, the present study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensitivity of APL cells. Restoring C/EBPα P42 is an attractive approach for differentiation therapy in ATRA resistant APL.
全反式维甲酸（ATRA）是急性早幼粒细胞白血病（APL）分化治疗的一线药物之一。然而，耐药性是影响ATRA疗效的主要问题。迫切需要确定ATRA耐药的机制。在本研究中，我们发现，与对ATRA敏感的NB4细胞相比，在对ATRA耐药的APL细胞系NB4-R1中，髓系分化的重要转录因子C/EBPα的表达显著受到抑制。此外，在NB4-R1细胞中，C/EBPα的两种形式受到的抑制程度不同。全长形式P42受到的抑制比截短形式P30更明显。在NB4-R1细胞中还观察到PI3K/Akt/mTOR信号通路的抑制。此外，PI3K抑制剂LY294002和mTOR抑制剂RAD001可降低NB4细胞中C/EBPα的表达，这表明PI3K/Akt/mTOR信号通路的失活是导致APL细胞中C/EBPα受抑制的原因。我们分别通过慢病毒载体在NB4-R1细胞中恢复了C/EBPα P42和P30的表达，发现C/EBPα P42而非P30可增加CD11b、CD14、G-CSFR和GM-CSFR的表达，这表明发生了髓系分化。在经ATRA处理后，恢复了C/EBPα P42的NB4-R1细胞中，发现CD11b表达进一步上调且出现了不同的形态学变化。然而，在感染了表达P30的载体或对照载体的NB4-R1细胞中，ATRA无法诱导CD11b表达及不同的形态学变化。因此，我们推断恢复C/EBPα P42可增强NB4-R1细胞对ATRA的敏感性。此外我们使用组蛋白去乙酰化酶抑制剂曲古抑菌素（TSA）来恢复NB4-R1细胞中C/EBPα的表达。检测到了类似的髓系分化增强和细胞生长停滞现象。总之，本研究表明，PI3K/Akt/mTOR抑制诱导的C/EBPα P42的抑制损害了APL细胞的分化和对ATRA的敏感性。恢复C/EBPα P42是对耐药性APL进行分化治疗的一种有吸引力的方法。
