細胞週期調控蛋白stathmin在泌尿上皮癌的表現

Abstract

研究背景： Stathmin是一種存在於細胞質內的ubiquitous磷酸化蛋白，stathmin對細胞週期的調控是藉由對微小管（microtubule）聚合（polymerization）的調控，已知stathmin在許多惡性腫瘤會高度表現且與其預後有關， stathmin的表現也與taxanes類化學治療的反應有關。目前對於侵犯性泌尿上皮癌的化學治療中，paclitaxel為一重要藥物且stathmin在泌尿上皮癌的表現在過去並無相關研究，故設計此實驗。

研究目的： 探討stathmin在不同抗藥性的泌尿上皮癌細胞株的表現情形。另外，stathmin在泌尿上皮癌腫瘤組織上的表現情形。其表現是否與病人之臨床、病理參數及預後有關？是否與化學治療的反應有關？s-16 stathmin (Ser16 phosphorylated stathmin) 的表現是否與total stathmin不同？

研究方法： 利用不同抗藥性泌尿上皮癌的細胞株 (NTU-B1, NTU-B1/P, NTU-B1/T)，利用兩株stathmin的抗體（s-16 stathmin及total stathmin）進行免疫螢光染色及西方點墨法偵測其表現。為確定其細胞內染色的位置 (intracellular localization)及在不同細胞週期中的表現，吾人利用細胞核質分離及nocodazole synchronized cell cycle來研究。並利用臨床上收集之泌尿上皮癌之組織，以此兩種抗體進行免疫組織化學染色，與病人之年齡，性別，臨床資料（如是否合併慢性腎衰竭及末期腎病變、是否抽煙、是否居住於烏腳病盛行區、腫瘤原發部位、病理分級，臨床病理分期、預後及化學治療的反應，以chi-square test及Fischer’s exact test作雙尾檢定，p<0.05為具有統計上之顯著意義。

結果： 在泌尿上皮癌細胞株的total stathmin及s-16 stathmin表現，可見total stathmin及s-16 stathmin在三株不同的細胞株中均有高度表現。特別的是，S-16 stathmin除了在細胞質內，在細胞核內也有高度表現，且在各個不同的細胞週期內均有表現，並不侷限於G2-M期。而total stathmin的表現則主要細胞質中，在細胞核中也有表現，也可見表現於不同的細胞週期。 Stathmin在泌尿上皮癌組織中之表現及其與臨床病理參數的關係顯示： 1.在85位泌尿上皮癌病人中： Total stathmin在所有泌尿上皮癌組織中均有表現（100％），其中弱陽性為10.6％，中度陽性為16.5％，高度陽性為72.9％，total stathmin的表現與腫瘤之病理期別 (pT staging) 有統計上的相關（p=0.002）。在S-16 stathmin方面，在泌尿上皮癌組織中均有表現（100％），其中弱陽性為21.4％，中度陽性為34.6％，高度陽性為44.0％，其表現強度除了與腫瘤之病理期別 (staging)有關，也與腫瘤細胞惡性度分級(grading)有關。 2.在44位侵犯性泌尿上皮癌病人中： 僅有s-16 stathmin表現與腫瘤病理期別有關（p=0.065）也與化學治療的反應有關（p=0.03），而total stathmin的表現與化學治療的反應無關（p=0.26）。 3.在41位表淺性泌尿上皮癌腫瘤中： 僅有total stathmin的表現與表淺性的腫瘤進行（progression）成侵犯性的病變有關（p=0.014）。

結論： S-16 stathmin的表現在泌尿上皮癌與腫瘤侵犯病理期別及病理組織分級有關。侵犯性泌尿上皮癌的比表淺性泌尿上皮癌表現要強，高惡性度（high grade）比低惡性度（low grade）的表現更強。而在侵犯性泌尿上皮癌的化學治療（以TP-HDFL處方治療）上，表現較強者，似乎其化學治療的反應較差，與過去在乳癌的研究相符。Total stathmin僅與腫瘤侵犯病理期別與表淺性的腫瘤進行（progression）成侵入性的病變有關。而在泌尿上皮癌細胞株的實驗上，是否s-16 stathmin在細胞週期的不同階段，有除了調控紡錘絲的形成及分解之外的不同的功能，而能表現在細胞核內，仍須進一步研究。

Purpose: Stathmin is a ubiquitous cytosolic phosphoprotein that regulates dynamics of microtubule polymerization. The activity of stathmin is regulated via phosphorylation and dephosphrylation by various protein kinase. Stathmin has been proved to play a role in human cancers. including lung, breast, ovary, leukemia, lymphoma, neuroblastoma etc. However, no relevant data in urothelial carcinoma was reported. In this way, we hope to analyze the expression of stathmin in human urothelial carcinoma (UC) cell lines and tissue samples. We also hope to study the potential of stathmin as predictor in tumor behavior and response to chemotherapy. The differential expression of Ser-16 phosphorylated stathmin (s-16 stathmin) and total stathmin was also studied. Materials and methods: We collected 3 urothelial cell lines NTUB1 (high grade urothelial carcinoma), NTUB1/P (cisplatin resistant), NTUB1/T (taxol resistant) and checked the total stathmin and s-16 stathmin expression by immunofluorescence staining and western blot analysis. Besides, we collected 85 urothelial carcinoma surgical specimens and analyzed the stathmin expression by immunohistochemical staining in paraffin-embedded cancer tissue samples. Correlation between stathmin expression and clinicopathologic features, such as age, sex, primary tumor site, black foot disease endemic area residency, chronic renal insufficiency, tumor grading and pathological staging, overall survival, disease free survival and response to TP-HDFL regimen (paclitaxel, cisplatin, high dose fluorouracil, and leucovorin) were examined. Results: Total stathmin and s-16 stathmin were both highly expressed in 3 cell lines without significant quantitative diffenence. The expression present in both cytoplasm and nucleus was proved by nuclear cytoplasmic fractionation and western blot analysis. Stathmin was also expressed in all stages of cell cycle. In urothelial carcinoma tissue, s-16 stathmin expression was significantly associated with tumor grading( p=0.022) and pathological staging (p=0.006). The response to TP-HDFL in metastatic invasive urothelial carcinoma was significantly correlated with s-16 stathmin expression. There was no difference in overall and progression free survival in reference to the expression of stathmin. Total stathmin expression was significantly associated with pathological staging. Total stathmin expressin is significantly related to the risk of superficial urothelial carcinoma progression to invasive disease. Conclusions: In our preliminary data, we observed high expression rate of stathmin in urothelial carcinoma cell lines and tumor tissue. Stathmin expression is related to disease status and tumor behavior of urothelial carcinoma. Further study will be necessary to determine the role of stathmin in urothelial carcinoma and the potential application for prediction of chemotherapy response.