新型Azaaryl HDAC6抑制劑在三重陰性乳癌中抑制細胞移行且增強Taxol抗轉移效果之研究

Abstract

實驗目的：三重陰性乳癌相較其他乳癌更具侵略性及轉移性，臨床上病人預後較差且容易復發與轉移，可能與三重陰性乳癌大量表現Aurora-A (極光激酶A)有關，而此蛋白表現受到其上游HDAC6 (組蛋白去乙醯酶6)所調控，因此，抑制HDAC6可能可以抑制三重陰性乳癌轉移。本研究將探討一新型化合物──專一性HDAC6抑制劑Azaaryl衍生物MPT0G211針對三重陰性乳癌在細胞移行能力的影響，並結合目前普遍治療三重陰性乳癌之化療藥物──微管穩定劑Taxol (紫杉醇)在實驗動物模型中抗癌細胞轉移之加成效果探討。 實驗方法：利用wound healing assay評估MPT0G211以及結合Taxol對三重陰性乳癌細胞移動能力之抑制效用；以免疫沉澱法觀察MPT0G211影響Hsp90 (熱休克蛋白90)乙醯化程度與Aurora-A之間交互作用；mRNA及蛋白質之表現由即時定量聚合酶鏈鎖反應與西方墨點法來觀察；細胞中球型肌動蛋白與絲肌動蛋白比例由G-actin/F-actin assay kit測定；利用phalloidin染色法觀察細胞肌動蛋白的聚集現象。另外，藉由在乳癌細胞中過量表現HDAC6來確認MPT0G211是否透過抑制HDAC6影響Aurora-A及其下游蛋白降低細胞移動能力。而在動物模式我們評估MPT0G211結合Taxol在活體內抑制癌細胞轉移之效果。 實驗結果：在本研究中，相較目前普遍已知HDAC6抑制劑Tubastatin A，MPT0G211有更好的抑制三重陰性乳癌細胞移動能力，且結合Taxol效果更為顯著。MPT0G211能藉由抑制HDAC6，促使Aurora-A與Hsp90分離，失去Hsp90保護的Aurora-A會被proteasome (蛋白酶體)降解，降低其下游去磷酸酶SSH1的基因轉錄，使能促進肌動蛋白聚集之cofilin (肌動蛋白解聚因子)非活化態增多，肌動蛋白無法聚集的情況下，細胞移動的第一步驟──偽足形成則受到抑制；而在荷爾蒙受體陽性之乳癌細胞SSH1與活化態之cofilin的原本表現較三重陰性乳癌低，受此影響不大，說明cofilin-F-actin路徑在三重陰性乳癌相對重要，MPT0G211可針對於此有效治療易轉移的三重陰性乳癌。在動物實驗中，MPT0G211在25 mg/kg的腹腔注射給予之下，可抑制三重陰性乳癌之轉移，結合Taxol則有更顯著抑制效果。 結論：實驗結果顯示，在三重陰性乳癌當中，MPT0G211可藉由抑制HDAC6促使Aurora-A降解，有效抑制cofilin-F-actin路徑，降低細胞移行能力，於動物實驗中可抑制癌細胞轉移，並增強Taxol治療效果。目前在市面上或是臨床試驗並無專一性的HDAC6抑制劑用於治療三重陰性乳癌，因此，MPT0G211提供了轉移性高的三重陰性乳癌療效好、與傳統化療藥物合併可降低毒性的絕佳治療選擇。

Objective. Triple-negative breast cancer (TNBC) is more invasive and has higher metastatic rate compared to other breast cancers. Patients with TNBC have poor prognosis with high risk of relapse and metastasis. It may be related to Aurora-A overexpression in TNBC, which is dysregulated by histone deacetylase 6 (HDAC6). Therefore, HDAC6 inhibitor may act as a treatment for metastatic TNBC. The aim of this study is to investigate the impact of azaaryl derivative MPT0G211, a novel HDAC6 inhibitor, on cell migration of triple-negative cell line and hormone receptor positive cell line as comparison, by in vitro and in vivo models. We also examined the combinatorial effect of MPT0G211 with a microtubule stabilizing agent, Taxol, which is commonly used as chemotherapy in TNBC. Methods. Cell migration was evaluated by wound healing assay. Protein-protein interaction was studied by immunoprecipitation. Messenger RNA and protein expression level were determined by QPCR and western blot. Filamentous actin (F-actin) and globular actin (G-actin) fractions were obtained using an F-actin/G-actin assay kit. F-actin polymerization was observed by phalloidin staining. Animal study was evaluated by MDA-MB-231 tumor metastatic assay and IHC stain. Results. In this study, MPT0G211 exhibited higher selectivity and lower IC50 against HDAC6 compared to a well-known HDAC6 selective inhibitor Tubastatin A. MPT0G211 not only decreased triple-negative breast cancer cell migration, but also enhanced the inhibitory effect of Taxol. We observed that MPT0G211 increased Hsp90 acetylation resulting in disassociation with Aurora-A, the unprotected Aurora-A then was degraded by proteasome. Furthermore, MPT0G211 significantly disrupted F-actin polymerization via decreasing the expression of slingshot protein phosphatase 1 (SSH1) and cofilin active form. Consequently, formation of lamellipodia was inhibited by MPT0G211. In addition, hormone receptor positive MCF-7 cell line has lower SSH1 and cofilin expression compared to triple-negative MDA-MB-231 cell line, suggesting that cofilin-F-actin-mediated cell migration may has more important role in invasive TNBC cells. In animal study, intraperitoneal injection of MPT0G211 (25 mg/kg) significantly ameliorated TNBC metastasis, and enhanced inhibitory effect of Taxol. Conclusion. Our results showed that MPT0G211 promotes Aurora-A degradation and inhibits cofilin-F-actin pathway by inhibiting HDAC6 activity, then decreases cell motility. Therefore, HDAC6 inhibitor MPT0G211 may provide a better treatment option when combined with traditional chemotherapy for invasive TNBC.