Down-Regulation of the Oncogene PTTG1 via the KLF6 Tumor Suppressor during Induction of Myeloid Differentiation

The aberrant expression of proto-oncogenes is involved in processes that are responsible for cellular proliferation and the inhibition of myeloid differentiation in acute myeloid leukemia (AML). Pituitary Tumor-Transforming gene 1 (PTTG1), an oncogenic transcription factor, is abundantly expressed in various human cancers and hematopoietic malignancies. However, its expression in normal leukocytes and most normal tissues is very low or undetectable. The mechanism by which PTTG1 overexpression modifies myeloid cell development and promotes leukemogenesis remain unclear. To investigate the mechanistic links between PTTG1 overexpression and leukemia cell differentiation, we utilized phorbol 12-myristate 13-acetate (PMA), a well-known agent that triggers monocyte/macrophage differentiation, to analyze the expression patterns of PTTG1 in PMA-induced myeloid differentiation. We found that PTTG1 is down-regulated at the transcriptional level in PMA-treated HL-60 and THP1 cells. In addition, we identified a binding site for a tumor suppressor protein, Kruppel-like factor 6 (KLF6), in the PTTG1 promoter. We found that KLF6 could directly bind and repress PTTG1 expression. In HL-60 and THP1 cells, KLF6 mRNA and protein levels are up-regulated with a concordant reduction of PTTG1 expression upon treatment with PMA. Furthermore, KLF6 knockdown by shRNA abolished the suppression of PTTG1 and reduced the activation of the differentiation marker CD11b in PMA-primed cells. The protein kinase C (PKC) inhibitor and the MAPK/ERK kinase (MEK) inhibitor significantly blocked the potentiation of PMA-mediated KLF6 induction and the down-regulation of PTTG1, indicating that PTTG1 is suppressed via the activation of PKC/ERK/KLF6 pathway. Our findings suggest that drugs that increase the KLF6 inhibition of PTTG1 may have a therapeutic application in AML treatment strategies.

原癌基因的异常表达参与了急性髓系白血病（AML）中细胞增殖与髓系分化抑制相关的病理过程。垂体瘤转化基因1（PTTG1）作为一种致癌性转录因子，在多种人类癌症及造血系统恶性肿瘤中呈高表达，但该基因在正常白细胞及多数正常组织中的表达水平极低，甚至无法被检测到。然而，PTTG1过表达调控髓系细胞发育并促进白血病发生的具体分子机制仍未阐明。为探究PTTG1过表达与白血病细胞分化之间的机制关联，本研究采用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）——一种经典的单核细胞/巨噬细胞分化诱导剂——来分析PTTG1在PMA诱导的髓系分化过程中的表达模式。研究发现，经PMA处理的HL-60与THP1细胞中，PTTG1在转录水平呈现下调表达。此外，本研究在PTTG1的启动子区域中，发现了肿瘤抑制蛋白Kruppel样因子6（KLF6）的结合位点，实验证实KLF6可直接结合并抑制PTTG1的基因表达。在HL-60与THP1细胞中，经PMA处理后，KLF6的mRNA及蛋白水平均呈上调表达，同时伴随PTTG1表达的同步降低。进一步实验显示，通过短发夹RNA（shRNA）敲低KLF6后，PMA诱导的细胞中PTTG1的表达抑制被解除，且分化标志物CD11b的激活水平也出现下降。蛋白激酶C（PKC）抑制剂与丝裂原活化蛋白激酶/细胞外调节蛋白激酶激酶（MAPK/ERK kinase, MEK）抑制剂可显著阻断PMA介导的KLF6诱导效应及PTTG1的下调过程，这表明PTTG1的表达抑制是通过PKC/ERK/KLF6信号通路的激活实现的。本研究结果提示，能够增强KLF6对PTTG1抑制作用的药物，有望在急性髓系白血病的治疗策略中得到应用。