2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/651196摘要：腎細胞癌(renal cell carcinoma, RCC)是腎臟惡性腫瘤中最常見的疾病。腎細胞癌的治療目前以手術根除法為主，缺乏早期的症狀以及腫瘤標記，使得許多的腫瘤無法早期診斷，是造成腎細胞癌大量增生與轉移主要原因。轉移或復發的腎細胞癌對於傳統的化學抗癌藥物有很強地抗藥性，即使是接受免疫療法(immunotherapy)或是標靶治療(target therapy)，其效果亦不甚理想，因此許多病人死於術後復發或是轉移的抗藥性腎細胞癌。所以如何分類找出具潛力的腫瘤標記分子及探討其分子作用機制，因而達到早期診斷與治療效果，這對於腎細胞癌的治療是迫切需要的。趨化激素(chemokine) 為一群類細胞激素的小分子分泌蛋白，早期趨化激素的研究主要與免疫細胞發炎反應有關，最近研究發現趨化激素breast and kidney-expressed chemokine (BRAK/CXCL14)與癌細胞形成、癌細胞轉移(cell migration)及血管新生(angiogenesis)作用有密切關係。但是目前對於BRAK 在腎細胞癌之分子角色與作用機制並不是很清楚，所以本計畫欲探討BRAK 在腎細胞癌之表現意義與扮演之角色、分子作用及化學抗藥性機制。初步結果：1. 利用RT-PCR，real-time PCR 與Western blotting 分析發現，BRAK 基因與蛋白質層次表現量在腎癌細胞株與腎癌組織分別是上升的。2. 利用免疫組織染色技術分析發現，BRAK 蛋白質表現量在腎細胞癌組織是比正常的腎臟組織有意義地增加，同時也發現轉移的腎癌組織比其它未轉移組織表現更大量的BRAK 蛋白。3. 外加BRAK 蛋白可以促進腎細胞株(HK2)增生與對cisplatin 抗藥性增加。4. 利用外加BRAK 蛋白實驗，抑制劑與BRAK siRNA 實驗術發現，BRAK 可能經由活化EGFR 與integrin 相關路徑；使得下游MAPK 訊息傳遞活化，進而活化細胞轉移相關分子造成腎癌細胞的轉移。此三年研究計畫主要有四大研究方向：1. 研究BRAK在腎細胞癌形成過程中，所扮演之分子角色與機制。2. 探討BRAK如何調控與作用在腎細胞癌進展（progression）之訊息傳遞過程。3. 探討BRAK在腎細胞癌調控細胞轉移角色與機制。4. 探討BRAK在腎細胞癌之化學抗藥性角色與機制。未來三年的研究：(1)利用基因轉殖表現、siRNA，專一性抑制劑，MTT, median effect analysis, migrationassay, wound healing assay, flow cytometry, 免疫沈澱法與Western blotting，探討BRAK 在腎細胞癌調控那些化學抗藥性分子，細胞增生與細胞轉移相關途徑，並找出其訊息傳遞過程。(2) 大量收集病人檢體，利用RT-PCR，real-time PCR，Western blotting 與免疫組織染色(immunohistochemistry)方法，分析BRAK 及相關訊息傳遞分子在病人組織表現量，評估以BRAK 作為偵測腎細胞癌之標記分子。(3) 以裸鼠腫瘤移植實驗進行體內研究，探討BRAK 調控腎細胞癌抗藥性機轉，細胞增生與細胞轉移機制，進而嘗試發展出臨床上對腎細胞癌有效治療模式。這些研究結果對於未來在臨床上偵測腎細胞癌與評估以BRAK 作為一種新的抗藥性標的分子治療具化學抗藥性腎細胞癌也許是有幫助的。<br> Abstract: Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney.Surgical resection of the diseased tissue has been considered as the only curativetreatment. Delayed diagnosis may result in progression and metastasis of RCC.Metastatic RCC is highly resistant to systemic chemotherapy. However, the responserates of RCC to immunotherapy or target therapy are still disappointing. Despite thetreatments available many patients die of the metastatic and chemoresistant RCC. Itseems desirable to identify and characterize potential molecular markers and theirmechanisms appearing during the course of tumorigenesis which may provide rapidand effective possibilities for early detection and treatment of RCC. Chemokines are afamily of small, secreted chemotactic cytokines. They initially are described by theirability to stimulate leukocyte chemotaxis and activation into inflammatory tissues.Recent studies have showed the breast and kidney-expressed chemokine(BRAK/CXCL14) is a pivotal regulator associated with cell proliferation, migration andangiogenesis in tumorigenesis. However, little is known the molecular role andmechanism of BRAK in RCC. This study is to explore the expression and role ofBRAK and BRAK-mediated molecular events and mechanisms in renaltumorigenesis.Our preliminary results1. BRAK gene and protein levels are up-regulation in RCC cell lines and tissues,respectively, by RT-PCR, real-time PRC and Western blot.2. Expression of BRAK protein is significantly higher in RCC compared to normalkidney tissues by Immunohistochemical stain. Of note, BRAK is overexpression inmetastatic RCC tissues.3. BRAK can enhance the cell proliferation and cisplatin resistance in immortalizedkidney cell line (HK-2).4. BRAK-mediated migration of RCC cells are through the EGFR- andintegrin-related MAPK signal pathway by co-treated BRAK protein and BRAKsiRNA assays, respectively.There are four specific aims in the proposal:1. To characterize the molecular role of BRAK in renal tumorigenesis.2. To identify the action of BRAK in modulating the signaling pathway of RCCprogression.3. To examine the metastatic role and mechanism of BRAK in RCC.4. To examine the chemoresistant role and mechanism of BRAK in RCC.In the three-year study:(I) To identify and determine the molecular events and molecular signaling pathwaysof BRAK-regulated cell proliferation, chemoresistance and migration in RCC cellsusing MTT, median effect analysis, migration assay, wound healing assay, flowcytometry, immunoprecipitation, Western blotting, overexpression BRAK of RCCsubclone, BRAK siRNA and BRAK inhibitors.(II) To identify the BRAK-induced molecules based on previous findings of clinicaltissue samples by immunohistochemical stain and evaluate the BRAK as anpotential biomarker in RCC for clinical application.(III) To explore the molecular mechanism of BRAK-regulated cell proliferation,chemoresistance and migration on xenografted tumors on nude mice experimentsand set up the useful treatment for BRAK-mediated RCC in future clinical trial.These data may be useful for the clinical application of RCC detection and BRAKas a novel targeted molecule for RCC treatment in future.化學抗藥性腫瘤形成機制趨化激素腎細胞癌癌細胞轉移chemoresistancetumorigenesischemokinerenal cell carcinomametastasis