以磷酸化蛋白質體學技術探討乳癌幹細胞之特性

Abstract

蛋白質磷酸化在真核細胞中是一個重要的調控機制，分析其動態變化可幫助了解磷酸化在生物體中的功能。儘管近年來將質譜技術應用於磷酸化蛋白質體的研究已有長足的進步，有效率的純化磷酸化胜肽仍是全面性分析磷酸化蛋白質體研究時不可或缺的步驟。基於磷酸根對三價鐵離子(Fe3+)以及4價鈦離子(Ti4+)具有不同程度的親和力和空間配位，我們在論文的第一部分新開發一互補式金屬離子親和層析法（IMAC）來增加磷酸化胜肽的鑑定數量。結果顯示藉由選擇有機酸結構以及濃度並添加有機溶劑能夠有效抑制非磷酸化胜肽之吸附，而且純化專一性可高達80%以上。透過分析最佳化的Fe3+-IMAC和Ti4+-IMAC方法從Raji B細胞樣品中所純化到的不同磷酸化胜肽，我們最大的發現是兩種方法所純化到的2905段磷酸化胜肽種類具有非常低的重複性(10%)。由於Fe3+-IMAC和Ti4+IMAC方法對偏酸性和偏鹼性磷酸化胜肽的親和力不同，並具有互補性的純化效果，可有效地的提高磷酸化蛋白的鑑定數量。此外這方法具有高重複性和低樣品損失率，我們認為此方法結合定量方法可應用於定量磷酸化蛋白質體的研究。 在開發出高效率的磷酸化胜肽純化方法後，我們想進一步在生物系統中驗證其應用性。因此我們利用此新開發的磷酸化胜肽純化方法結合無標記定量分析技術來研究醫學上的重要課題。人類乳癌幹細胞（BCSC）為一群具有新生腫瘤能力，起始細胞分化，並且對於放射治療和化療相對耐受性較高的細胞。在論文的第二部分，為從磷酸化蛋白質體的角度了解乳癌幹細胞獨特之性質，我們比較從同一乳癌組織(雌激素受體為陰性反應)中分出的乳癌幹細胞和非幹細胞之其他細胞的磷酸化蛋白質體差異。經由磷酸化蛋白質體之結果分析，我們得以描繪出可能參與細胞更新和能維護幹細胞特性的訊息傳遞路線，包括Notch，CDK/ Erk和JAK-STAT等訊息傳遞路線。透過生化及細胞實驗的驗證，我們證明了G protein-coupled estrogen receptor 1（GPER）於雌激素受體為陰性的乳癌患者中，在其過度表現或受其配體刺激時，會透過PKA造成BAD於Ser118位點上的磷酸化。透過使用RNAi來阻斷GPER的功能或進行BAD上Ser118位點突變，也同樣能夠降低乳癌幹細胞活性和乳腺球細胞的生成能力。這意味著GPER和其下游的PKA訊息傳遞路線為主要維持乳癌幹細胞特質的主角，而我們的研究結果顯示GPER具有高潛力成為新一代治療乳癌幹細胞之目標蛋白。

Protein phosphorylation is an important regulatory mechanism in eukaryotic cells, and the study of its dynamic change has become a major contributor to understand its functionalities. Despite advancement in mass spectrometry-based phosphoproteomics, effective phosphopeptide enrichment is a perquisite towards comprehensive mapping of the site-specific change of phosphorylation in the spatial and temporal domain. Based on the different binding affinity and coordination geometry of Fe3+ and Ti4+ ion with phosphate group, in the first part of the thesis, we developed a complementary immobilized metal ion affinity chromatography (IMAC) to increase the identification coverage of phosphoproteome. Based on phosphopeptides from standard phosphoproteins and Raji B cell, optimal enrichment specificity (>80%) was achieved by the selection of acid structure/concentration and organic solvent to compete with non-phosphopeptides. The remarkable discovery is that optimal Fe3+-IMAC and Ti4+-IMAC methods have low overlapping percentage (10%) among the 2905 enriched phosphopeptides from Raji B cell. The reported Fe3+-IMAC and Ti4+-IMAC can complementarily enrich acidic and basic phosphopeptides to effectively increase the identification coverage of the heterogeneous phosphoproteome. Given the reproducibility and low sample loss, the combination of our enrichment strategy with quantitative technique could be feasible for quantitative phosphoproteomics. Following the development of our enrichment strategy for phosphopeptides, we would like to further validate its capability by applying our enrichment method in combination with the label-free quantitation technique to study a medically relevant topic. Human breast cancer stem cells (BCSC) have the ability to initiate neoplastic growth with differentiation and are relatively resistant to radiation and chemotherapy. To identify novel targets for breast cancer stem cells, in the second part of the thesis, we quantitatively compared the phosphoproteomes between BCSC and non-BCSC derived from a xenograft of estrogen receptor negative human breast cancer. The phosphoproteomic mapping revealed signaling proteins involved in the Notch, CDK/Erk and JAK-STAT pathways, which may potentially orchestrate the self-renewal and stemness of BCSC. We demonstrated the overexpression of G-protein coupled estrogen receptor 1 (GPER) in ER-negative patients and its activation, stimulated by its ligands, induced protein kinase A (PKA)-mediated Ser118 phosphorylation of Bcl-2-associated death promoter (BAD). GPER silencing via RNAi or the dominant negative mutation of BAD Ser118 led to reduced survival and mammosphere forming capacity of BCSC, which implied GPER and its downstream PKA pathway as central to maintain BCSC characteristics. These in vivo findings provide new insights into GPER as a potential therapeutic target for BCSCs.