| Abstract: | 國際抗癌聯盟(Union for International Cancer Control, UICC)調查統計，全球常見前五大癌症依序為肺癌、乳癌、大腸直腸癌、攝護腺癌與胃癌，乳癌排名第二位。乳癌是台灣女性十大癌症發生率之首，故乳癌治療成為當前社會關注的重要議題。乳癌中以三陰性乳腺癌最難治癒，因其轉移能力強且無標靶藥物治療，造成乳腺癌患者死亡率增加及預後較差。上皮間質轉化(Epithelial-mesenchymal transition, EMT)是啟動癌細胞轉移的重要步驟，與增加癌細胞移行及侵襲能力，提高腫瘤惡性程度有關。Twist1是調控EMT相關基因表現之重要轉錄因子，Twist1大量表現有助於啟動EMT。本研究以MDA-MB-231三陰性乳癌細胞模式，探討次亞麻油酸(α-linolenic acid, ALA )調控乳癌細胞Twist1表現、EMT相關蛋白表現及移行能力之機制。實驗結果顯示，在三種乳腺癌細胞中，惡性度最高的MDA-MB-231細胞具有較高程度EMT標誌蛋白表現。以100 μM ALA及Twist1 siRNA處理可顯著抑制癌細胞Twist1表現及移行能力，並可顯著增加E-cadherin而下調N-cadherin等EMT相關蛋白質表現。本研究進一步釐清ALA減少Twist1表現是否與其促進Twist1蛋白質降解有關。結果顯示，ALA可降低Twist1 serine-68位點產生磷酸化、增加Twist1泛素化進而加速Twist1被蛋白?體降解。此外，本研究也觀察ALA降低Twist1蛋白質表現是否與其抑制mRNA表現有關。結果顯示，ALA處理細胞4小時後可顯著降低Twist1 mRNA表現，處理ALA 60分鐘可顯著降低STAT3α磷酸化表現，而ALA於處理細胞1 小時即可減少與上調Twist1表現之轉錄因子如SP1、STAT3α的核內累積。以STAT3α si-RNA及ALA處理可顯著抑制STAT3α、N-cadherin及Twist1表現。綜合實驗結果，ALA可透過抑制STAT3α磷酸化，降低Twist1蛋白質生成，也可藉由減少Twist1 serine 68磷酸化，增加Twist1泛素化進而加速Twist1被蛋白?體降解，下調EMT相關蛋白質表現，達到抑制MDA-MB-231人類乳腺癌細胞移行。
Union for International Cancer Control indicates that the most common cancers diagnosed in the world are lung cancer, breast cancer, colorectal cancer, prostate cancer and gastric cancer. Breast cancer’s incidence rate is the first of the top ten cancer in Taiwanese women, so currently, breast cancer treatment has become an important topic in social concern. Triple-negative breast cancer is the most difficult breast cancer subtype to be cured, due to the absence of effective target therapies and strong migration ability, which leads to a poor clinical outcome and high mortality rate. Epithelial-mesenchymal transition (EMT) has been known as a critical process of the tumor metastatic and is associated with enhanced migration, invasion and malignant tumor progression. Twist1, a transcription factor, has been reported to be a regulator of EMT-related gene. High level of Twist1 expression can induce EMT program. In this study, we investigated the effect of α-linolenic acid on Twist1 protein level, EMT-related protein expression and cell migration on TNBC cell line MDA-MB-231, as well as explored the underlying molecular mechanisms. We found a much higher EMT markers protein level when compare malignantly MDA-MB-231 cell with the other two cells. Treatment with 100 μM ALA and si-Twist significantly inhibited Twist1 expression and breast cancer cell migration. Using ALA for 24 hours significantly increased E-cadherin and decreased N-cadherin and other EMT-related proteins expression. To identify whether ALA inhibits Twist1 protein expression is due to promote Twist1 degradation. The result showed that ALA downregulated the phosphorylation of Serine 68 on Twist1 and increase Twist1 degradation by ubiquitin proteasome system. In addition, we also wondered that ALA suppressed Twist1 protein expression is concerned with inhibition of Twist1 mRNA level. Result showed treatment with ALA for 4 hours significantly decreased mRNA level. Treatment with ALA for 60 minutes significantly inhibited the phosphorylation of STAT3α. Furthermore, treatment ALA for 1 hours significantly reduced the nuclear accumulation of transcription factor such as SP1 and STATαwhich are associated with Twist1 expression. Treatment with STAT3α siRNA and ALA suppressed protein expression of STAT3α、N-cadherin and Twist1. In conclusion, ALA downregulates Twist1 protein production through inhibition of STAT3α phosphorylation. Moreover, ALA also reduced the phosphorylation of Serine 68 on Twist1 and increased Twist1 degradation by ubiquitin proteasome system. These demonstrated that ALA inhibited MDA-MB-231 human triple-negative breast cancer cell migration via decreasing Twist1 and EMT-related protein expression. |
