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，這些特別的細胞有 CD24-CD44+的表面分子標記或帶有細胞內高度乙醛去氫酶(aldehyde dehydrogenase)活 性 (ALDH+)。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)。 基於過去的研究成果，我們認為 Hsp27與其磷酸化對於乳癌幹細胞的維持以及致癌性 具有重要的影響。 本研究計畫的第一項研究特定目標為了解細胞表面 Hsp27的表現是否為新穎乳癌幹細 胞標記。我們計畫利用流式細胞分選儀分選出表現表面 Hsp27的細胞，利用免疫不全 小鼠的異種腫瘤生長模式、體外自我更新培養以及幹細胞相關基因表現，檢測細胞表 面 Hsp27是否為乳癌幹細胞之特異性分子標記。 本研究計畫的第二項研究特定目標則是研究 Hsp27的磷酸化在乳癌幹細胞致癌性的角 色。我們將利用基因重組技術，在乳癌細胞中表現 Hsp27之磷酸化突變型蛋白，利用 免疫不全小鼠的異種腫瘤生長模式、體外自我更新培養與上皮-間質轉換特性分析，來 檢測 Hsp27磷酸化對於維持乳癌幹細胞的重要性。 本計畫第三項研究特定目標則是研究磷酸化態 Hsp27在乳癌幹細胞中如何調節與其作 用之客戶蛋白，並尋找新穎的磷酸化 Hsp27之客戶蛋白。 此部分將著重於三個訊息傳 遞路徑: (a) insulin-like growth factor-1 receptor (IGF-1R)； -catenin/Wnt；(c) c-Myc/Bmi1 。藉由 cycloheximide處裡，瞭解 Hsp27是否影響 IGF-1R的蛋白穩定性； 在 Hsp27特異性干擾 RNA處理下，檢測 focal adhesion kinase (FAK) 的活化，以釐清 Hsp27是否藉由調節 FAK的活性，進而影響 IGF-1R的表現；透過冷光報導基因測試， 了解缺少Hsp27磷酸化的乳癌幹細胞中-catenin的活性是否受影響；藉由提高-catenin 活性，觀察是否能反轉因降低 Hsp27所抑制的癌症幹細胞特性；藉由免疫沉澱法研究 Hsp27是否能與乳癌幹細胞中的 c-Myc進行交互作用，並進一步影響乳癌幹細胞中 Bmi1的表現，並了解過度表現 c-Myc是否可以逆轉因降低 Hsp27所抑制的癌症幹細胞 特性。我們也將利用質譜法尋找磷酸化 Hsp27在乳癌幹細胞中的新穎客戶蛋白，並驗 證它們對於乳癌幹細胞自我更新的影響。 透過本研究計畫的成果，我們認為將能對於以 Hsp27做為標的的治療方式提供基礎性 以及臨床性的訊息，對於未來乳癌治療有所貢獻。 Heat shock proteins (Hsps) are agroup 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) have been identified as a subpopulation of breast cancer cells with markers of CD24-CD44+ or aldehyde dehydrogenase activity high (ALDH+) and 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 (Wei L, et al. Breast Cancer Res. 2011. 13:R101.; Lee CH, et al. Biochimie. 2012. 94:1382). We also found that Hsp27 phosphorylation was increased in ALDH+ BCSCs. Here we plan to further investigate the role of Hsp27 and its phosphorylation in the maintenance of BCSCs. There are three specific aims in this project: Specific Aim 1: Investigate whether surface Hsp27 (sHsp27) could be a unique marker of breast cancer stem cells. We will further investigate if surface Hsp27 (sHsp27) could serve as a unique marker for BCSCs. The CSC properties of sHsp27+ cells will be examined by (1) xenotransplantation assay in NOD/SCID mice (2) in vitro self-renewal capability (3) the expression of stemness signatures. Specific Aim 2: Determine the role of Hsp27 phosphorylation in the maintenance of breast cancer stem cells. The involvement of Hsp27 phosphorylation in the maintenance of BCSCs will be investigated by overexpression of phorphor mimic and phorphor dead mutant form in breast cancer cells. The mammosphere formation capability, stemness signatures and in vivo tumorigenicity will be determined in Hsp27 mutant expressed mammosphere cells. Specific Aim 3: Determine molecules regulated by Hsp27 phosphorylation in breast cancer stem cells. The effect of Hsp27 and its phosphorylation in the signal transduction molecules which involved in the maintenance of BCSCs will be explored. We will focus on three targets (1) insulin-like growth factor-1 receptor (IGF-1R) (2) -catenin and (3) c-Myc which have been reported to play roles in breast cancer progression. The effect of Hsp27 in the stability of IGF-1R protein will be examined by cycloheximide assay under Hsp27 knockdown cells or Hsp27 mutant expressed mammosphere cells. The involvement of focal adhesion kinase in Hsp27 regulated IGF-1R expression will also be explored. By overexpression of GFP-based reporter, the activation of -catenin in Hsp27 mutant expressed mammosphere cells will be investigated to demonstrate the role of Hsp27 phosphorylation in -catenin activity of BCSCs. With immunoprecipitation method, the interaction between Hsp27 and c-Myc in breast cancer cells will be examined. The reverse effect of c-Myc in BCSC characters of Hsp27 knockdown or Hsp27 mutant expressed breast cancer cells will also be investigated. Finally, we will apply the immunoprecipitation and mass spectrometer analysis to identify novel clients of phosphorylated Hsp27. The identified candidates will confirm by co-immunoprecipitation analysis and siRNA mediated gene silence to prove their participation in self-renewal capability of BCSCs. We believe that the results of this project will provide new insights in the development of Hsp27-based targeting therapy in breast cancer.
