熱休克蛋白(heat shock proteins, Hsps)的功能主要為維持細胞內蛋白質穩定正確結構，這些熱休克蛋白通常在細胞遭受環境壓力時會大量表現。Hsps也被發現在許多癌症組織以及癌細胞中有過度表現的現象，在乳癌細胞中，Hsp27的表現與細胞的移動侵襲以及抗藥性能力有關。近年來在乳癌研究中發現存在一群獨特的乳癌幹細胞，具有起始腫瘤生成以及分化的能力，並且具有抗藥性以及轉移的特性，這些特別的細胞可利用螢光分子標記以流式細胞儀進行細胞分選或以乳腺小球(mammosphere)培養取得。目前這群乳癌幹細胞被認為是重要的治療標的。在過去的研究中，我們先前實驗已證實乳癌幹細胞具有較高量的Hsp27以及其磷酸化態表現，並已證明Hsp27可透過調節上皮-間質轉換以及核因子B的活性，進而調節乳癌幹細胞的自我更新與致癌性(Wei L, et al. Breast Cancer Res. 2011. 13:R101)。我們也證明IGF-1R訊息路徑可調控乳癌幹細胞的自我更新(Chang WW. et al., Breast Cancer Res. 2013. 15:R39)。在本計畫第一年中，我們發現帶有表面Hsp27表現的乳癌細胞，其形成乳腺小球的能力高於表面Hsp27陰性的細胞，顯示表面Hsp27可能為新穎之乳癌幹細胞標記。利用乳腺小球培養取得乳癌幹細胞，並利用慢病毒攜帶Hsp27的磷酸化突變型，我們也發現當乳癌幹細胞表現Hsp27之磷酸化死亡型時，其細胞內c-Myc轉錄活性明顯減少，細胞內c-Myc之表現下降；當乳癌幹細胞表現Hsp27之模擬磷酸化突變型時，則可增加細胞內c-Myc轉錄活性，並保留與c-Myc之表現。過度表現Hsp27磷酸化死亡型之乳癌細胞，其形成乳腺小球之能力明顯下降，並形成之乳腺小球，其細胞內c-Myc或其他幹細胞相關基因(Bmi1, Oct4, Sox2, Nanog)的表現也明顯減少，顯示Hsp27之磷酸化是乳癌幹細胞維持自我更新的重要關鍵。在第二年計畫中，我們將進一步分析帶有表面Hsp27表現之乳癌細胞在免疫缺陷小鼠乳腺中形成腫瘤的能力是否優於表面Hsp27陰性之細胞，並分析此兩群細胞內幹細胞相關基因的表現，以確認表面Hsp27為新穎之乳癌幹細胞標記。我們也預計利用具Hsp27結合力之aptamer核酸分子，進行抗癌藥物之修飾，進行體外乳癌幹細胞存活及活體抗腫瘤活性測試，以評估抗癌藥物修飾之Hsp27專一性aptamer核酸分子的抗癌功效。我們也將利用免疫沉澱法，分析c-Myc與Hsp27磷酸化突變型之結合能力，並分析表現Hsp27磷酸化死亡型之乳癌細胞內c-Myc蛋白的穩定性是否下降，以確認Hsp27之磷酸化是影響c-Myc活性的重要因素。我們也預計讓乳癌細胞表現FAK穩定活化型或FAK顯性負活性突變型，分析Hsp27-IGF1R-FAK之間的相互調控關係。
Heat shock proteins (Hsps) are a group of proteins which express under environmental stress to serve as a chaperone for maintenance of corrected protein folding. Hsps have been reported to be overexpressed in many cancers including breast cancer. Breast cancer stem cells (BCSCs) could be enriched by fluorescence-based cell sorting or by mammosphere cultivation. These particular breast cancer cells have been proved to be associated with radiation resistance and metastasis. Recently, we have demonstrated that Hsp27 plays a role in the maintenance, tumorigenicity and drug resistance of BCSCs. We also found that Hsp27 phosphorylation was increased in ALDH+ BCSCs. We also have demonstrated that IGF-1R is a novel marker for BCSCs and its signal regulates self-renewal of BCSCs. In the first year of project, we discovered that breast cancer cells with surface Hsp27 expression displayed a great activity in formation of mammospheres and it suggests that surface Hsp27 might be a novel marker for BCSCs. Using lentivurs transduction to express Hsp27 phosphorylation mutants in mammosphere cells, we discovered that the transcriptional activity of c-Myc was decreased in cells with Hsp27 phosphor-dead (Hsp27A) overexpression but increased in those with Hsp27 phophor-mimic (Hsp27D) overexpression. Hsp27 A overexpression caused the reduction of mammosphere formation and decreased the expression of c-Myc and other stemness genes (Bmi1, Oct4, Sox2, Nanog). It suggests that the phosphorylation of Hsp27 is the key in the maintenance of BCSCs. In the second year of this project, we will continue to examine if surface Hsp27 is a novel marker for BCSCs by analyzing the difference of tumorigenicity between surface Hsp27 positive or negative breast cancer cells. The expression of stemness genes between these two populations of breast cancer cells will be also determined. We also plan to examine the anti-tumor efficacy of anti-cancer drug-modified Hsp27 specific aptamer in vitro and in vivo to evaluate its potential in breast cancer treatment. We will use immunoprecipitation to analyze the interaction between c-Myc and Hsp27 phosphorylation mutants to know if Hsp27 phosphorylation influences the binding between c-Myc and Hsp27. The protein stability of c-Myc in breast cancer cells with Hsp27 phosphorylation mutants overexpression will also be investigated. We will also use different FAK plasmids to investigate the regulation among Hsp27, FAK and IGF-1R.
