行政院國家科學委員會補助專題研究計畫期中進度報告:細胞凋亡調控基因(sFRP2)在犬乳腺腫瘤之細胞凋亡調控角色及相關調節因子的研究(2/3)

乳腺腫瘤對動物與人類皆是重要且常見的 腫瘤，其產生的病因是複雜為多因子牽涉的結 果。Secreted frizzled related proteins (sFRPs)是近年的報告發現與Wnt-Frizzled 訊 息傳遞傳導路徑的調節和細胞凋亡(apoptosis) 的調節中扮演著雙重角色的蛋白質。我們實驗 室最近發現sFRP2 基因在人類與犬隻乳腺腫瘤 中有大量的表現及活化，但是在正常犬乳腺組 織中則沒有表現(前NSC project, 已發表於期 刊Breast Cancer Research and Treatment)。 我們為了有系統地進一步研究犬隻乳腺腫瘤中 的sFRP2 在功能上的角色與腫瘤分子生物學上 的機制，擬定了下列的幾項研究策略。 這個計畫包括了六個主要的部分，需要3 年的研究時間：第一年首先主要的工作在於建 立並分析多種新犬隻乳腺腫瘤組織的初代培養 (primary culture)細胞株並且純化、分析乳腺 上皮細胞。在第一年我們已成功地建立並分析 數個本地病例之犬乳腺腫瘤細胞株。這些細胞 利用下列技術分析其特性，包括增殖速度(by MTT assay)、反轉錄鏈聚合脢反應(RT-PCR)、 原位雜交法( in situ hybridization)與免疫化 學染色(immunohistochemistry)及西方墨點法 偵測sFRP2 的表現。結果發現sFRP2 基因之mRNA 及蛋白質在犬乳腺腫瘤細胞株有大量之表現， 然而在犬正常乳腺細胞及其他非MGT 細胞株則 無表現，第一年的成果已發表刊登。在第二階 段，犬sFRP2 被轉殖入含有GFP 基因與CMV 啟 動子的哺乳類細胞表現載體，藉由lipofection 方式將GFP-sFRP2 穩定地轉染入(transfect)犬 隻乳腺腫瘤初代培養與商品化乳腺腫瘤的細胞 株，在本年度之計畫中進行更進一步的sFRP2 調控細胞凋亡的功能分析。 在本年度之研究成果及進度方面，我們已 非常辛苦地分析確認sFRP2 基因確具有抗細胞 凋亡之功能，且分析複雜的調控細胞凋亡之相 關訊息傳遞途徑為fibronectin-integrin signal transduction pathway，非過去傳統所知之Wnt signaling，此重要發現為此新基因族之首篇調 控細胞凋亡功能訊息傳遞途徑之新發現，此階 段之研究成果也獲期刊Journal of Biological Chemistry (impact factor:7.0)接受刊登。本 階段之研究結果，預期將提供重要之學術資 訊，以了解sFRP 基因族在犬乳腺腫瘤細胞之 調控情形。此外，此計劃也為未來下一階段進 一步研究sFRP 基因族不同成員之各種功能， 及了解犬乳腺腫瘤複雜之病因，提供進一步研 究分析之基礎。 雖然夲階段的努力有獲得重大之進展及新 發現，然而調控細胞凋亡功能之訊息傳遞途徑 為十分複雜的研究領域，我們還須持續努力， 以解開更多的謎團。Mammary neoplasms are important and common tumors in both animals and humans and the etiology is complex. The secreted frizzled related proteins (sFRPs) are newly identified proteins and implicated to have dual roles of modulation of Wnt-Frizzled signal transduction pathway and regulation of apoptosis. We have recently found that sFRP2 was expressed abundantly in human and canine mammary gland tumors (MGT) tissues but was undetectable in normal canine mammary gland. To systematically 3 investigate the functional roles and molecular mechanisms of sFRP2 in canine MGT, several strategies are to be carried out as described below. The project is comprised of six major parts for a period of 3 years: In the 1st year, new primary cell cultures from native canine MGT tissues has been established and purified for mammary epithelial cells. We have successfully established and analyzed more native primary MGT cell lines from surgically excised MGT specimens. The cells are characterized for their cell origins, proliferation rate (by MTT assay), expression of sFRP2 by RT-PCR, in situ hybridization, and immunohistochemistry, and Western blotting. Expression experiments revealed the sFRP2 was abundantly expressed in canine MGT cell lines, but not expressed in normal canine MG cells nor other commercial non-MGT cell lines (previous NSC project, published in the Breast Cancer Research and Treatment). Canine sFRP2 is cloned into a mammalian expression vector with GFP reporter gene and CMV promoter. The GFP-sFRP2 is stably transfected into primary canine MGT and commercial MGT cell lines by lipofection for further analysis from the next stage of the project. To elucidate the role of SFRP2 in the tumorigenesis of MGTs, apoptosis regulation mediated by SFRP2 was investigated by overexpression of SFRP2 in MGT and MCF7 cells. DNA fragmentation and caspase 3 activity analyses showed that the susceptibility of the cells to UV-induced apoptosis decreased in the context of SFRP2 overexpression. To analyze the pathways through which SFRP2 transduces anti-apoptosis signals, co-immunoprecipitation and cell adhesion assays were carried out. SFRP2 was found secreted from cells and associated with the fibronectin-integrin protein complex and could promote cell adhesion. Moreover, by using heparin to block the SFRP2-fibronectin interaction or anti-integrin α5β1 antibody to interrupt the fibronectin-integrin connection, the anti-apoptosis activity of SFRP2 was decreased. Taken together, these results suggest that SFRP2 exert its anti-apoptotic function in mammary cancer cells through association with the fibronectin-integrin signal transduction pathway, not the Wnt signaling as previous thought. The important data has been published and accepted by the Journal of Biological Chemistry (impact factor: 7.0). The results of this stage of the project should offer important scientific basis and information to understand the roles of sFRP gene family in canine neoplastic cells. It also provides a basis for further analysis of functions of different members of the sFRP gene family and elucidation of the complex etiology and signaling pathways of mammary tumors