2016-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/692950摘要：乳癌是全球女性中最常見的癌症種類，其中骨轉移又是乳癌病患最容易復發與轉移之部位，然而對其分子機制及幫助診斷之生物標記仍所知甚少。四締素與脂締素近來發現為血漿中由脂肪細胞大量表現之分泌蛋白，最近本實驗室產製脂締素第一型受體（adiponectin receptor 1）之轉基因小鼠後發現其大量表現可抵抗肥胖所引起之代謝症候群 (Lin et al., 2013; Liu et al., 2013)，另一方面則利用蛋白質體首次證明四締素為脂肪分泌激素，可調控脂質基因表現並增加脂質生合成。最近初步試驗結果則發現脂締素第一型受體大量表現可增加造骨細胞分化而影響骨生成，另一方面之初步試驗則發現四締素可增加不同乳癌細胞株之侵略能力。這些都是前人所未知之功能，因此本計劃欲由這些表現型延伸探討其分子機制。由於乳房組織中乳腺周圍披有脂肪組織，顯示脂締素與四締素這些脂肪分泌激素可能影響乳癌生成微環境，進而於乳癌生成與乳癌骨轉移中扮演重要角色。因此本計畫第一部分（第一及二年）欲利用脂締素第一型受體大量表現小鼠動物模式進行活體及細胞試驗，細胞試驗欲利用轉基因小鼠所分離之骨髓幹細胞探討脂締素調控骨細胞分化之分子機制。我們第一年已利用脂締素第一型受體轉基因小鼠及骨髓幹細胞試驗證明脂締素可顯著改善老化所引起之骨質疏鬆以及脂締素第一型受體大量表現可增加造骨細胞分化，其中是透過GSK-3β/β-Catenin 訊息傳遞路徑。第二年試驗欲繼續探討（一）脂締素第一型受體是否藉由APPL1此一脂締素adaptor protein調控下游叉頭轉錄因子O1（FoxO1）及骨頭分泌激素骨鈣化素（osteocalcin）而增加骨質生成; （二）脂締素第一型受體是否藉由影響APPL1調控不同型骨形成蛋白（bone morphogenetic protein, BMP）及其下游訊息傳遞路徑BMP-SMAD-p38MAPK 影響骨生成; （三）脂締素第一型受體是否抑制receptor activator of nuclear factor kappa-B ligand （RANKL）此一蝕骨細胞分化之調控蛋白，由於最近已知RANKL另外影響乳癌轉移，然而RANKL由誰調控仍不知道，因此我們猜測脂締素第一型受體可能藉由抑制RANKL以減少乳癌生成與骨轉移; （四）進行小鼠活體試驗打入乳癌細胞於此轉基因鼠之脂肪墊（fat pad），證明脂締素及其受體可調節乳癌生成微環境，以抑制乳癌生成及乳癌骨轉移。本計畫第二部分（第二年）則欲證明四締素增加乳癌細胞株之侵略能力及其分子機制，試驗將證明四締素可能透過影響epidermal growth factor receptor （EGFR）與focal adhesion kinase （FAK）其訊息傳遞路徑影響乳癌細胞轉移之能力。本計畫第三部分（第三年）則欲分析不同時期之乳癌及有無骨轉移之病患血漿，證明脂締素、四締素、骨鈣化素、骨形成蛋白作為不同時期乳癌與是否有骨轉移之血漿生物標記之可能性。預期此研究將可釐清乳癌與乳癌骨轉移之分子機制，並利用血漿中脂肪分泌激素脂締素與四締素作為乳癌生成及乳癌轉移診斷之應用。<br> Abstract: Breast cancer is the most prominent cancer type in females. Bone metastasis is the most evident recurring and metastatic site for advanced breast cancer. However, the detailed mechanisms regulating this process and diagnostic biomarkers are poorly understood. Adiponectin and tetranectin are recently revealed as plasma proteins secreted by adipocytes (termed adipokine or adipocytokine). Our previous studies demonstrated that overexpressing adiponectin receptor 1 (AdipoR1) in mice could protect from diet-induced obesity and metabolic syndrome (Lin et al., 2013; Liu et al., 2013). Another our study showed tetranectin is a confirmed adipocyte-secreted adipocytokine identified by proteomics and upregulated fatty acid synthesis in adipocytes. Our preliminary data demonstrated that overexpressing AdipoR1 in mice can modulate bone formation and tetranectin increases invasion of multiple breast cancer cell lines. These results showed previously unknown functions. Therefore, this project would like to extend and reveal the detailed mechanisms. Because mammary glands are covered by adipose tissues, these adipocytokines could modulate breast cancer microenvironment to regulate breast cancer formation and breast cancer bone metastasis. This project would like to use mice overexpressing AdipoR1 as an invivo cancer animal model and an invitro primary cell culture system. Bone marrow-derived mesenchymal stem cells from mice overexpressing AdipoR1 (AdipoR1 BM-MSC) will be isolated and evaluated the detailed mechanisms involving bone formation. In the first year, we already demonstrated that AdipoR1 mice had higher bone volume and trabecular number than wild-type mice and AdipoR1 may be involved in bone metabolism through GSK-3β/β-Catenin signaling. In the following second year of project 1, we would like to extend this finding and demonstrate (1) adiponectin binds to AdipoR1 and recruit the adaptor protein APPL1 to regulate downstream signaling FoxO1 and osteocalcin in AdipoR1 BM-MSC differentiated osteoblasts; (2) adiponectin binds to AdipoR1 and recruit APPL1 to regulate BMP-SMAD-p38MAPK signaling in AdipoR1 BM-MSC differentiated osteoblasts; (3) adiponectin binds to AdipoR1 to inhibit receptor activator of nuclear factor kappa-B ligand (RANKL), a known osteoclast differentiation factor recently revealed to enhance invasive breast carcinoma, therefore, to reduce breast cancer metastasis. (4) AdipoR1 would modulate tumor microenvironment to reduce breast cancer formation when mice overexpressing AdipoR1 injected breast cancer cells in fat pads. The second part project is to further elucidate the role of tetranectin in breast cancer invasion. Tetranectin could modulate epidermal growth factor receptor（EGFR）and focal adhesion kinase（FAK） signaling in regulating invasiveness of breast cancer cells. The third part of this project is to analyze plasma concentrations of these adipocytokines or bone formation factors including adiponectin, tetranectin, osteocalcin, BMPs in different stages of clinical breast cancer subjects whether they have bone metastasis or not.To conclude, in this project, we could clarify the detailed mechanisms involving breast cancer formation and related bone metastasis and identified potential biomarker for breast cancer diagnosis by these adipocytokines.乳癌乳癌骨轉移脂締素脂締素第一型受體四締素生物標記breast cancerbone metastasisadiponectinAdipoR1tetranectinbiomarker