乳癌細胞內熱休克蛋白27之磷酸化對於其參與之訊息傳遞網路的影響

熱休克蛋白27( Heat Shock Protein 27,Hsp27 )的功能為幫助細胞內變性蛋白質再次摺疊以維持其客戶蛋白的穩定性，研究指出Hsp27能藉由與肌動蛋白結合、調節肌動蛋白的聚合作用達到調節細胞移動，並具有抗細胞凋亡的能力，此外Hsp27也具調節細胞內訊息傳導的功能。我們實驗室過去發現乳癌癌症幹細胞內Hsp27及p-Hsp27的表現較一般乳癌細胞增加，並且用RNA干擾技術抑制Hsp27的表現能抑制乳癌癌症幹細胞的致癌性，顯示Hsp27對於乳癌癌症幹細胞致癌性的維持相當重要。另外我們也發現乳癌癌症幹細胞具有血管擬態的活性，血管擬態的活性則會受到p-Hsp27的調控。本研究則進一步想了解Hsp27的磷酸化改變是否影響乳癌幹細胞的子我更新以及細胞內其交互作用蛋白的功能。在研究策略上，我們使用慢病毒載體來構築Hsp27之磷酸化突變型(磷酸化死亡型: Hsp27A與磷酸化模擬型:Hsp27D)使乳癌細胞表現，並利用免疫沉澱法分析Hsp27交互作用蛋白是否因為磷酸化位點突變有所變化。初步結果上，我們利用螢光顯微鏡及西方點墨法確認慢病毒感染成功讓乳癌細胞過度表現野生型Hsp27(WtHsp27)與兩種Hsp27磷酸化突變型，也使用免疫沉澱法確認可以成功沉澱過度表現的Hsp27與其磷酸化突變型蛋白。利用癌症球體分析，我們發現表現Hsp27A的乳癌細胞其自我更新能力明顯下降，同時表現Hsp27A之MDA-MB-231細胞其活體致癌力顯著較WtHsp27或Hsp27D者為低；透過即時定量PCR分析則發現，表現Hsp27A的乳癌細胞內與乳癌幹細胞自我更新的相關基因，如IGF1R(insulin-like growth factor receptor-1)或TWIST1(twist family bHLH transcription factor 1)的表現顯著下降。透過免疫沉澱法結合質譜儀分析，我們發現MECP2(methyl-CpG binding protein 2)與XRCC1(X-ray repair cross complementing 1)可以與WtHsp27或Hsp27D交互作用，但與Hsp27A的交互作用則減弱；利用西方點墨法，我們則發現MECP2或XRCC1的表現在Hsp27A有明顯的下降。本研究發現Hsp27的磷酸化能正向調控乳癌幹細胞的自我更新以及活體致癌力，而此作用可能與MECP2或XRCC1有關，但未來仍需要進一步研究Hsp27的磷酸化對MECP2或XRCC1活性的影響。

Heat Shock Protein 27(Hsp27) acts as a cellular chaperon to maintain stability of its client proteins. Hsp27 also functions in the cell migration and anti-apoptosis in cancer cells. We previously found that Hsp27 and its phosphorylation were increased in breast cancer stem cells (BCSCs) and inhibition of Hsp27 expression suppressed the tumorigeicity of these particular breast cancer cells. In addition, we also found that vasculogenic mimicry activity of BCSCs was mediated by Hsp27 phosphorylation. In this study, we try to understand impact of Hsp27 phosphorylation in the maintenance of BCSCs. By overexpression of the phosphorylation mutant forms of Hsp27 (Hsp27A for phosphor dead form and Hsp27D for phosphor mimic form) in MDA-MB-231 or Hs578t breast cancer cells, the mammosphere formation was increased in Hsp27D overexpressed cells than wildtype ones and was decreased when overexpressed Hsp27A. By quantitative RT-PCR method, the expression of insulin-like growth factor receptor-1, Nanog and Oct4 was decreased in mammospheres from Hsp27A overexpressed MDA-MB-231 sphere cells but upregulated in Hsp27D ones. Through immunoprecipitation and mass spectrometry analysis, we found that the interaction with MeCP2, XRCC1 or UBAP2L was increased in Hsp27D cells but decreased in Hsp27A ones. In addition, we also found that the expression of MeCP2 or XRCC1 in MDA-MB-231 cells was reduced by overexpression of Hsp27A. In conclusion, Hsp27 regulates the self-renewal and tumorignicity of BCSCs through its phosphorylation and may involve the regulation of stemness genes or the interaction with MeCP2 or XRCC1.