血癌細胞之CXCR4的表觀遺傳研究

Abstract

基因表觀修飾調控（epigenetic regulation）泛指DNA methylation與histone modification，負責調控基因的表現。目前有許多研究發現CpG 島的甲基化會抑制基因轉錄。在癌細胞方面，許多腫瘤抑制基因的CpG島亦有高度甲基化的現象，使chromatin形成較緊密的結構，進而抑制基因的表現。DNA 甲基酶（DNMTs）的抑制物與組織蛋白去乙烯酶（HDACs）的抑制物可有效回復基因的表現，故epigenetic therapy已成為當今癌症治療的熱門話題。 近年來許多研究發現諸多癌細胞亦表現CXCR4，且會受到CXCL12的趨化作用而進行轉移。對於AML細胞而言， CXCR4高表現的血癌細胞可能較易受到骨髓中的CXCL12吸引而滯留於骨髓中，躲開化療藥物的攻擊，而與minimal residue disease有關，目前已有研究指出CXCR4低表現的AML病人預後較CXCR4高表現的病人好。 本實驗室以甲基化特異聚合酶鏈鎖反應（MSP）的方法發現K562細胞的CXCR4基因有高度甲基化的現象。接著我們分別以1、2、或5 μM的DNMT抑制物DAC連續處理K562四天，並在最後一天合併加入0.5 μM的HDAC抑制物TSA，由MSP的結果確實可見到去甲基化效果，且real-time PCR的結果亦顯示mRNA的表現量與藥物濃度呈現正相關，免疫細胞化學染色與flow cytometry的結果亦可見到CXCR4蛋白質的表現量與藥物濃度成正相關。目前的實驗結果顯示CXCR4確實是受到表觀修飾調控。 此外，我們也利用MSP分析91位AML病人其CXCR4基因的甲基化狀態，結果發現約30%的病人呈現異合子的甲基化。另一方面，結合本實驗室之前的研究結果，我們也發現在帶有Flt3-ITD突變的病人之中，有高達60%的人其CXCR4基因呈現未甲基化，故我們推測Flt3-ITD突變可能影響CXCR4基因的表觀修飾。

DNA methylation and chromatin modifications are two ways that regulate gene expression without DNA sequences changes, the so-called epigenetic regulation. Methylation of CpG islands in promoter results in transcriptional repression by recruiting transcriptional repression complexes that interact with histone deacetylases (HDACs). HDACs remove the acetyl groups of histone proteins, which leads to a compact, transcriptionally inactive chromatin structure. In tumor cells, several tumor suppressor genes, or genes involved in metastasis, such as p15INK4b, E-cadherin, have been reported to be inactivated by hypermethylation. Hypermethylated genes can be re-activated by DNA methyltransferase (DNMT) inhibitors and HDAC inhibitors, such as DAC (5-aza-2’deoxycytidine) and trichostatin A (TSA) respectively. The CXCR4-CXCL12 axis plays an important role in hematopoietic progenitors homing, B-lymphocyte development, and tissue repair. In addition, more and more evidences indicate that CXCR4 involves in angiogenesis and metastasis of tumors. In acute myeloid leukemia (AML), CXCR4+ leukemia cells tend to be retained in bone marrow and avoid to be killed by chemotherapy agents, which may be associated with minimal residue disease. Recently, it has been reported that patients with low CXCR4 expression have better prognosis, comparing to those with high CXCR4 expression. Here, we demonstrated that the CXCR4 gene in leukemia cells was under epigenetic regulation. Analyzed by methylation-specific PCR (MSP), the CXCR4 in K562 cells showed hypermethylation in promoter region. After treatment of 1, 2, or 5 μM DAC for four days and combined with 0.5 μM TSA for the last day, the CXCR4 mRNA increased significantly. And the expression of CXCR4 protein, assayed by immunocytochemical staining and flow cytometry, restored and increased in a dose-dependant manner. Furthermore, we examined the CXCR4 methylation status in 91 AML patients. We observed that about 30% patients had heterogeneously methylated CXCR4, and up to 60% patients with Fms-like tyrosine kinase 3 (Flt3)-ITD mutation showed unmethylated CXCR4. It remains to be investigated further whether Flt3-ITD mutation has impacts on epigenetic regulation of CXCR4.