行政院國家科學委員會專題研究計畫成果報告: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(3/3)

行政院國家科學委員會專題研究計畫 成果報告

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(3/3)

計畫類別： 個別型計畫 計畫編號： NSC93-2313-B-002-022- 執行期間： 93 年 08 月 01 日至 94 年 07 月 31 日 執行單位： 國立臺灣大學獸醫學系暨研究所 計畫主持人： 林中天 計畫參與人員： 林念儀、謝岱儒、李佳霖 報告類型： 完整報告 處理方式： 本計畫可公開查詢

中 華 民 國 94 年 9 月 26 日

行政院國家科學委員會補助專題研究計畫

成果報告

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 (3/3)

計畫類別： 個別型計畫

計畫編號：NSC 93－2313－B002－022－

執行期間：

93 年

8

月

1

日至

94

年

7

月

31

日

計畫主持人：

林中天

計畫參與人員：研究生 李佳霖、林念儀、謝岱儒

成果報告類型(依經費核定清單規定繳交)：完整報告

執行單位：

國立台灣大學獸醫學系

中

華

民

國

94

年

9

月

6

日

行政院國家科學委員會專題研究計

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 (3/3)

計畫編號：NSC 93-2313-B-002-022 執行期限：93 年 8 月 1 日至 94 年 7 月 31 日 主持人：林中天 國立台灣大學獸醫學系 計畫參與人員: 李佳霖、林念儀、謝岱儒 等研究生 一、中文摘要 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於期 刊 In vitro cellular and developmental biology-Animal(2003)。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。 在第二年度之研究方面，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，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，研究成 果也已發表。 在第三年度之研究方面，研究 sFRP2 之抗 細胞凋亡與其他訊息因子或轉錄因子之關聯 性。實驗結果發現，表現 sFRP2 基因之細胞受 到紫外光誘發細胞凋亡後，FAK 之 tyrosine 磷 酸化程度會增加，且有 NF-κB 之活性增加及 JNK 之活性抑制的情況。 本研究之結果，預期提供重要之學術資 訊，以了解 sFRP 基因族在犬乳腺腫瘤細胞之 細胞凋亡調控之訊息傳遞情形。此外，此計劃 也為未來進一步研究 sFRP 基因族不同成員之 各種功能，及了解乳腺腫瘤複雜之病因，提供 進一步研究分析之基礎。 關 鍵 詞 ： 分 泌 性 細 胞 凋 亡 基 因 ， 分 泌 性 frizzled 蛋白基因，細胞凋亡，訊息傳遞，乳 腺腫瘤 二、計畫英文摘要 (Abstract)

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 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 1styear, 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.

In the 2nd year, 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 51 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. In the 3rd year, analysis of the relation of sFRP2 transduced

anti-apoptosis with other signaling and

transcription factors, multiple-color

immunofluorescence staining, immunoprecipita-tion, and immunoblotting were carried out. SFRP2 was found co-localized in the extracellular matrix of MGTs and the tyrosine phosphorylation of FAK was enhanced. Moreover, JNK was suppressed and NF-kB was activated in the cells expressing SFRP2 after UV-induced apoptosis analyzed by immunoblotting and electrophoretic mobility shift assay (EMSA). Taken together, these results suggest that SFRP2 exerts its anti-apoptotic function in mammary cancer cells with NF-κB activation orJNK suppression.

The results of this project should offer important scientific basis and information to understand the roles of sFRP-mediated signaling in canine mammary tumor 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.

Keywords: secreted apoptosis related protein

secreted frizzled related protein, apoptosis, signal transduction, gene transfection, mammary

neoplasia

二、緣由與目的

The frizzled and secreted frizzled related

protein family is thought to modulate

Wnt-Frizzled signal transduction pathway which plays an important role in normal development

and oncogenesis, particularly in mammary

neoplasia. More recently it has been reported that

the sARP1 (also named sFRP2) possesses

anti-apoptosis activity while sARP2 (also named sFRP1) induces pro-apoptosis in the breast tumor

cells. In our previous NSC project (NSC

91-2313-B-002-404), sFRP2 was found to be expressed abundantly in 31 different canine MGT tissues, but not expressed in normal MG tissues. This striking finding stimulated our interest in further investigation of the gene family. The roles of the gene family in tumor tissues remain to be

determined.

Canine mammary gland tumor (MGT) is the canine counterpart of human breast cancer that shares significant similarities in several aspects. MGT is the most common tumor type in female dogs comprising of 52% of all neoplasms in the bitches. However, the etiology of MGT is mostly unknown and surprisingly very few advanced molecular studies regarding MGTs have been done to date.

The aim of the study is to understand the mechanism or/and signaling pathways associated with the sFRP2 mediated anti-apoptosis.

三、結果

Because it is the full report of the 3-year project, summary of the data from the study are presented on a year-by-year basis.

I. Year 1 (1 Aug. 2002-31 July 2003)

1. Establishment of canine MGT primary cell cultures from fresh MGT tissue specimens Freshly excised MGT tumors were rinsed by PBS and maintained in Dulbecco’s modified Eagle’s

medium (DMEM) supplemented with

streptomycin (1g/ml) and 10% fetal calf serum Primary cell culture and cell lines from canine MGT specimens were established by more than 90 to 100 passages of the MGT cells. The cell

types were confirmed by morphology and

immunostaining of cytokeratin (Figure 1). 2. Characterization of MPG and NMG cells

To characterize the difference between

primary canine MGT cells (MPG) and normal mammary gland (NMG) cells, colony-forming efficiency and MTT assay were performed. Colony-forming efficiency of MPG cells was significantly higher than NMG cells (Fig. 2A). Additionally, proliferation rate of MPG cells was also higher than NMG cells by MTT assay (Fig. 2B). Therefore, we suggested that MPG cells were tumor-like not normal epithelial cells.

3. RNA purification from canine MGT and normal MG primary cell cultures

Canine normal MG and MGT cells were prepared for RNA extraction using Trizol reagent. Total

RNA from was extracted. The OD260/280 of the

RNA was ranged between 1.5-1.8 and analyzed

through formaldehyde denaturing gel

electrophoresis indicating reasonably good purity for RNA-based work.

3. RT-PCR and in situ hybridization analysis of sFRP2 in normal MG and primary MGT cells RT-PCR and in situ hybridization showed sFRP2 was abundantly expressed in primary MGT cells, but not in normal MG cells. Following northern blotting hybridization, three mRNA species of approximately 1.5 kb, 2.2 kb, and 4.2 kb hybridized to the sFRP2 cDNA probe of which the 2.2 kb mRNA species was the major abundant transcript. sFRP2 gene in the dog was abundantly expressed only in the MGT cells, not in normal MG cells nor human breast cancer cell MCF7. RT-PCR and in situ hybridization results are shown in the Figure 3.

4. Protein analysis of sFRP2 in primary MGT cell lines and other cell lines by Western blotting and immunohistochemistry

The primary culture of the MGT cell lines and normal MG cells was analyzed by Western blotting and immunohistochemical staining of sFRP2. Again, the sFRP2 protein was abundantly accumulated in the MGT cells, not in normal MG cells and other commercial non-MGT cell lines (Figure 4).

5. Transfection of sFRP2 into primary MGT cells and normal MG cells

Cells was transfected with the pcDNA4

mammalian expression vector (Invitrogen)

containing no insert (mock), and canine sFRP2 cDNA, by using Lipofectamine reagent (Gibco)

according to manufacturer’s protocol. The

transfected vector are useful tool for further analysis of sFRP2 functions in tumor cells from the next stage.

參考文獻

1. Abu-Jawdeh, G., Comella, N., Tomita, Y., Brown, L.F., Toognazzi, K., Sokol, S.Y. and Kocher, O. (1999) Differential expression of

frpHE: A novel human stromal protein of the secreted frizzled gene

family, during the endometrial cycle and malignancy. Laboratory

Investigation 79, 439-447.

2. Melkonyan, H.S., Chang, W.C., Shapiro, J.P., Mahadevappa, M., Fitzpatrick, P.A., Kiefer, M.C., Tomei, L.D. and Umansky, S.R. (1997) sARPs: A family of secreted apoptosis-related proteins.

13636-13641.

3. Morrison, W.B. (1998) Canine and feline mammary tumors. In: Morrison W.B. Cancer in dogs and cats. Philadelphia, Williams & Wilkins, 591-598.

4. Rattner, A., Hsien, J.C., Smallwood, P.M., Gilber, D.J., Copeland, N.G., Jenkins, N.A., and Nathans, J. (1997) A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. Proceedings of the National Academy

of Sciences of the USA 94, 2859-2863.

5. Wolf, V., Ke, G., Dharmarajan, A.M., Birlke, W., Artuso, L., Saurer, S. and Friis, R. (1997) DDC-4, an apoptosis-associated gene, is a secreted frizzled relative. FEBS Letters 417, 385-389.

6. Zhou, Z., Wang, J., Han, X., Zhou, J. and Linder S. (1998) Up-regulation of human secreted frizzled homolog in apoptosis and its down-regulation in breast tumors. International Journal of

Cancer 78, 95-99.

II. Year 2 (1 Aug. 2003 - 31 July 2004) 三、結果

1. Overexpression of SFRP2 results in decrease of sensitivity to UV-induced apoptosis

Previously, SFRP2 has been reported to possess anti-apoptosis activity. To explore the molecular mechanisms underlying the anti-apoptosis effect of SFRP2 in mammary tissues, vector encoding C-terminally Flag-tagged SFRP2 was constructed and delivered into canine and human mammary cancer cell lines. The canine cell line MPG was isolated and purified in our lab. After being

selected by G418, the stable clones of

MPG/pCMV-cSFRP2-Flag and MCF7/pCMV-cSFRP2-Flag were collected and the expression of SFRP2 was analyzed by Western blotting. Fig.

1a showed that MPG cells have basal expression

of SFRP2, and increasing amount of SFRP2-Flag protein expression was observed in both MPG

and MCF7 cells. To further examine the

functional activity of SFRP2, the stable cell lines described in Fig. 1a were exposed to UV, and subjected to DNA fragmentation analysis. Fig. 1b showed that overexpression of SFRP2 in MPG

and MCF7 cells could inhibit the DNA

fragmentation caused by UV (lanes 3 and 6). The caspase 3 activity in MPG cells was also decreased in the presence of SFRP2 (Fig. 1c). Taken together, these results demonstrated that canine SFRP2 has anti-apoptosis activity in mammary cancer cells.

2. Interaction of SFRP2 with FN

The immunohistochemical staining results of canine MGT tissues showed that SFRP2 existed

in connective tissues (Fig 2a). Furthermore, the presence of SFRP2 protein can be detected in the media from the stably expressed SFRP2 cells (Fig

2b). Thus, consistent with previous reports,

SFRP2 is indeed a secreted protein. To assess the possibility of an interaction between SFRP2 and ECM, the immunoprecipitated SFRP2 complexes from culture media were subjected to Western blotting using anti-FN antibody. Fig 2c showed

that the SFRP2 antibody could

co-immunoprecipitate FN from MPG and MCF7 cell culture media. FN has been reported to interact with integrin receptor (Pytela et al., 1985; Akiyama and Yamada, 1985). To dissect the signaling pathways of SFRP2, we further examine whether integrin receptor is present in the SFRP2 protein complexes. Expression of integrin 51 in both the MPG and MCF7 cells was confirmed (Fig 2d), and co-immunoprecipitation experiment using the anti-SFRP2 antibody revealed that FN as well as integrin 51 receptor associate with SFRP2 in MPG and MCF7 cells (Fig 2e). Taken together, theses results demonstrated that SFRP2 is a secreted protein and an interacting component of the FN-integrin receptor protein complexes. 3. The expression of SFRP2 could promote cell adhesion

To confirm the interaction between SFRP2 and FN, a reciprocal co-immunoprecipitation assay

was performed using anti-FN antibody.

Interestingly, although the amounts of

precipitated FN were nearly identical in the two cell clones, we observed that the amounts of precipitated integrin receptors were in direct proportion to those of precipitated SFRP2 (Fig

3a). It appeared that the presence of SFRP2 could

enhance the formation of FN-integrin receptor complexes. Based on the knowledge that the interaction of FN and integrin receptor promotes the cell adhesion, we set out to compare the level of cell adhesion in the presence or absence of SFRP2. As shown in Fig 3b, overexpression of SFRP2 in MPG cells could promote cell adhesion. SFRP2 also could further promote cell adhesion

in the presence of Mn2+, an integrin 51

receptor stimulator (Mould et al., 1995). It is possible that, in addition to the integrin 51-FN pathway, the SFRP2 may promote cell adhesion

through additional pathways. Fig 3c showed that the stimulatory effect of SFRP2 was specific to FN. The similar results were obtained in MCF7 cells (Fig 3d). Thus, we can conclude that SFRP2

promotes cell adhesion through enhancing

integrin51-FN binding.

4. SFRP2-FN interaction is required to reduce cell susceptibility to UV-induced apoptosis We have provided evidence that expression of SFRP2 could protect cell from UV-induced apoptosis and that SFRP2 interacts with FN. To examine whether SFRP2’sinteraction with FN is essential for its anti-apoptosis activity, we used inhibitors to interrupt the interaction between SFRP2 and FN. Heparin has been reported to be able to bind to either SFRP2 or FN and release FRP from the cell surface (Finch et al., 1997).

Immunoprecipitation assay using anti-SFRP2

antibody was done on lysates prepared from cells previously treated with heparin. As shown in Fig

4a, the level of co-immunoprecipitated FN was

decreased in the presence of heparin. In addition, anti-integrin51 antibody appeared to block the association of integrin with the SFRP2-FN

complexes, as revealed by the

co-immunoprecipitation experiment (Fig 4b). Moreover, result of the DNA fragmentation analysis indicated that the anti-apoptosis effect of SFRP2 was abrogated in the presence of heparin or anti-integrin51 antibody (Fig 4c).

四、討論

Despite the well-established role of Wnt signaling in oncogenesis (Peifer and Polakis, 2000), the involvement of the SFRP gene family in cancer is still under investigation. In the past few years, several reports have described the different expression patterns of SFRPs in various cancers. For example, SFRP1 is down-regulated in breast cancer (Zhou et al., 1998; Ugolini et al., 1999; Wong et al., 2002), but is overexpressed in uterine leimyoma (Fukuhara et al., 2002). frpHE

(SFRP4) is up-regulated in the stroma of

endometrial and breast carcinomas (Abu-Jawdeh

et al., 1999) and SFRP1 and SFRP2 are produced

by the majority of long-term and ex vivo malignant glioma cell lines (Roth et al., 2000). Our previous study demonstrated that canine

SFRP2 was expressed in MGTs, but absent in normal mammary glands. In this study, we showed that overexpression of canine SFRP2 in MCF7 and our cloned canine MGT cell line could decrease cell sensitivity to UV-induced apoptosis. This result is consistent with the observation that mouse homologue of SFRP2 could also enhance the cell viability of MCF7 cells in response to the presence of TNF or ceramide (Melkonyan et al., 1997). Apoptosis is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. When apoptosis malfunctions, the results may be: cancer and autoimmune diseases when there is too little apoptosis occurs; or possibly stroke damage or the neurodegeneration of Alzheimer’s disease when there is too much apoptosis. The anti-apoptotic activity of SFRP2 offers a clue that it may be involved in either the formation or progression of mammary tumors. Also consistent with this idea, investigating new

drugs that potentially restore the apoptotic

defense mechanism (such as heparin or

anti-integrin 51 antibody mentioned in this study) will possess substantial therapeutic benefit for mammary tumors.

SFRP2 is a secreted glycoprotein. Functional characterization of this protein is our main research focus in this project. Interactions of cells with the extracellular environment regulate many basic cellular functions, including differentiation, migration, cell growth, and apoptosis (Aplin et al., 1999; Ilic et al., 1997; Sonoda et al., 1999; Lee and Juliano, 2000). Most normal cells require attachment to ECM for survival. Cell adhesion to ECM is mainly mediated by integrins, a family of cell surface receptors widely expressed on all

tissues. The  integrin is heterodimeric

transmembrane receptors with overlapping

specificity toward ECM components (Damsky and Werb, 1992; Meredith et al., 1993; Howlett et

al., 1995; Giancotti and Ruoslanhti, 1999). One

of ECM components, FN, is particularly effective in providing survival signals for several cell types; and these survival signals were found to be transduced by FAK (Frisch et al., 1996; Ilic et al., 1998). The physical interaction of SFRP2 with FN-integrin protein complex was demonstrated by co-immunoprecipitation, and it was found that cell adhesion could be promoted in the presence

of SFRP2. Although we cannot rule out the existence of other signaling pathways by which SFRP2 mediates its anti-apoptotic effect, our results clearly showed that when the interaction of SFRP2 with FN-integrin complex was blocked, the protective effect of SFRP2 on UV-induced apoptosis was dramatically diminished. In an attempt to examine the possible underlying mechanisms of such anti-apoptotic function, we

discovered that, under UV-stimulation, the

overexpression of SFRP2 causes the activation of

FAK, elevation of -catanin protein, and

inactivation JNK. According to the published

observations, these cellular features are an

indication that cells is in a survival mode (Sonoda

et al., 1999; Chen et al., 2001; Harding et al.,

2001). Moreover, our studies highlight the importance that SFRP2, in addition to the Wnt/Frizzled pathway, can potentially modulate

other signaling pathway(s). The detailed

mechanism underlying the SFRP2-mediated

signaling will be a subject of future research. SFRPs, Wnt and FN possess a highly basic region that confers strong affinity for heparin (Finch et al., 1997; Üren et al., 2000). If tissue proteoglycan content is decreased, either by genetic or biochemical manipulation, the Wnt signaling is impaired (Hacker et al., 1997; Binari

et al., 1997). For instance, heparin or endogenous

HSPG might promote SFRP1/Wg binding by stabilizing conformation of either SFRP1 or Wg that would increase their mutual affinity or by enhancing ligand or receptor oligomerization (Üren et al., 2000). Heparinase treatment of

endothelial cells inhibited endothelial cell

proliferation and in vivo neovascularization

(Sasisekharan et al., 1994), emphasizing the role of heparin-like molecules in tumorigenesis.

Estrogens are known to stimulate the

proliferation of hormone-dependent breast and ovarian cancers through their interaction with estrogen receptors (ERs) and the subsequent expression of a number of genes, some of them involved in the control of cell proliferation (Dickson et al., 1986; Vignon et al., 1986). In addition, several recent in vitro and in vivo studies have demonstrated that ECM proteins, including FN, may be important paracrine factors in mammary gland growth, morphogenesis, and lactation (Xie and Haslam, 1997; Woodward et

al., 2000). The progestin, R5020, significantly

stimulates proliferation of primary mouse

mammary epithelial cells cultured on FN but not on collagen I (Col I), laminin (LM), or tenascin (Xie and Haslam., 1997). In this study, our results link FN to mammary tumorigenesis as part of the SFRP2-regulated integrin-mediated anti-apoptotic signaling pathway. Taken together, the available data suggest that estrogen/ER and FN/SFRP2

both play critical roles in breast cancer

development. However, some studies have

demonstrated that the ER-positive breast cancer cells are in general poorly invasive in vivo (better prognosis) and in vitro as compared with ER-negative cancer cells (Price et al., 1990). One hypothesis for this contrasting observation is that FN/SFRP2 is a key factor. In related studies, estradiol and fibulin-1 can inhibit human ovarian-and breast-cancer cell motility induced by FN ovarian-and therefore have the potential to inhibit tumor invasion (Hayashido et al., 1998). The expression

levels of the ECM protein FN and the 51

integrin are enhanced by estrogen in normal mouse mammary gland (Woodward et al., 2001) and by the Wnt/Frizzled-mediated -catenin/Tcf transcription (Gradl et al., 1999). In contrast to FN, the concentrations of Col I, Col IV, and LM do not exhibit major changes during mammary development (Woodward et al., 2001). Therefore, ER-positive breast cancer patients have better

prognosis due to the enhanced

FN/SFRP2-mediated signal. Additionally, further studies can be undertaken to determine whether SFRP2 and FN might be ideal prognostic markers in breast cancers.

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Endocrinol., 110, 227–236.

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III. Year 3 (1 Aug. 2004 - 31 July 2005) 三、結果

1. Overexpression of SFRP2 results in a decreased sensitivity to UV-induced apoptosis

To explore the molecular mechanisms

underlying the anti-apoptosis effect of SFRP2 in mammary tissues, vectors encoding full-length SFRP2 were constructed and delivered into MPG cells. After being selected by G418, the stable

clones of MPG/pcDNA4-pGFP and

MPG/pcDNA4-pGFP -cSFRP2 were collected and the percentage of cells expressing GFP or GFP-cSFRP2 fusion proteins was determined by

fluorescence microscope. The MPG cells

expressing GFP are shown in the Fig. 1a, 1b. The

MPG cells expressing GFP-cSFRP2 fusion

protein are shown in the Fig. 1c, 1d. To further examine the functional activity of SFRP2, the stably transfected cells described in the Fig. 1 were exposed to UV and subjected to DNA

fragmentation analysis. Fig. 2 shows that

overexpression of SFRP2 in MPG cells could inhibit the DNA fragmentation caused by UV

(lanes 8 and 9). The percentage of

TUNEL-positive cells was also decreased in the presence of SFRP2 (Fig. 3). Taken together, these results prove again that canine SFRP2 has anti-apoptosis activity in the canine mammary cancer cells.

2. Comparison of expression between fibronectin and SFRP2 in MGTs

we performed immunofluorescence staining in comparison with fibronectin. Nucleus was stained with DAPI (blue; Fig. 4a, 4d). The expression of fibronectin was found in the extracellular matrix (red, Fig. 4b, 4e). Interestingly, the same regions were also stained with SFRP2 antibodies (green, Fig. 4c, 4f). The data suggest that SFRP2 and fibronectin could be co-localized in extracellular matrix of MGTs. In our recent studies, we have demonstrated that SFRP2 was associated with fibronectin by co-immunoprecipitation (Lee et al., paper in preparation). In previous studies, binding of the SH3 domain of p130cas to prolinerich region 1 in FAK was found to be required for prevention of apoptosis on fibronectin following serum withdrawal. Therefore, we tried to evaluate the tyrosine phosphorylation status of FAK. When excess fibronectin was added to MPG cells harboring the SFRP2 expression vector, the tyrosine phosphorylation of FAK was enhanced (Fig. 5), reaching peak at 10-20 min. Furthermore, we also investigated that the presence of SFRP2

could enhance the formation of

fibronectin-integrin receptor complexes by

immunoprecipitation and adhesion assay (data not shown).

3. The possible downstream signaling pathway of the SFRP2-fibronectin interaction

To delineate the anti-apoptotic signaling pathways mediated by SFRP2, we attempted to examine the expression of a few related signaling

factors or transcription factors. The JNK

suppression and NF-kB activation are implicated in the FAK-induced resistance to apoptosis (Sonoda et al., 2000). Therefore, these two additional apoptotic effectors, JNK and NF-kB, were also analyzed. Fig. 6 shows that JNK could be activated by UV-irradiation in the control cell line (lane 3, 5), whereas it was down-regulated in the SFRP2-overexpressed cells (lane 1). The role

of NF-kB in the transmission of SFRP2

anti-apoptotic signal was established by

determining the level of active NF-kB in

UV-treated mammary cancer cells by EMSA (Fig. 7). The active NF-kB in UV-treated cells (Fig. 7, lane 1, 3) was markedly enhanced in comparison to control cells. Although the basal level of NF-kB activation was similar, the UV-induced

level of active NF-kB in MPG cells expressing SFRP2 was significantly greater than that in

control cells after being characterized by

supershift assay with anti-p50 (lanes 2 and 4) antibodies. The DNA-binding activity of NF-kB was significantly affected by SFRP2, suggesting

that SFRP2-mediated anti-apoptotic function

indeed involved NF-kB activation.

四、討論

The process of apoptosis is integral to normal mammary gland development (Medina 1996). Thus the mammary gland is an excellent

model for studying normal morphologic

development and the early steps of tumor formation (Medina 1996; Nandi 1959).

Estrogen is essential in mammary

development and plays a central role in

carcinogenesis of the breast (Pike et al., 1993). The majority of human breast tumors are estrogen

receptor (ER) -positive and respond to

anti-hormone therapy. However, most malignant tumors are ER-negative (MaGuire and Clark, 1985). The current therapeutic approach with antihormones, targeted at hormone receptors, is effective only in a fraction of breast cancer patients. All estrogen receptor negative (ER-) and also a fraction of ER positive (ER+) tumors do not respond to antihormone treatment (Jordan 1995; Hedden 1995). Thus, alternative treatment protocols aimed at different targets for these classes of antihormone nonresponsive breast cancers need to be explored. Consistent with this finding, we also investigated that MPG cells derived from canine malignant MGT were ER-negative (data not shown). Additionally, the potent mitogenic effect of Wnt-1 on mammary epithelial cells may not depend upon other mammogenic hormones either (Edwards 1992; Lin 1992. In a similar manner, we suggest that SFRP2 contributed to canine MGT may not depend upon mammogenic hormones. Moreover, MPG cells can serve as a good model for research on antihormone nonresponsive breast cancers.

The level of NF-kB has been shown to be elevated in ER- human breast cancers, as compared with ER+ cells (Nakashatri 1997; Bhat-Nakashatri 1998; Sovak 1997, Rayet 1999;

Biswas 2000). The role of NF-kB in tumorigenesis is circumstantial, such as higher levels of activated NF-kB in ER- tumor cells. In

recent studies, it has demonstrated the

antitumorigenic activity of a compound that inhibits activation of NF-kB without causing significant detectable cellular damage of vital organs in a model of mouse tumor (Biswas 2001).

Furthermore, selective activation of NF-kB

induced loss of tumorigenic potential of the parent CSMLO cells, thus strongly suggesting a role of this transcription factor in ER- mammary epithelial cell carcinogenesis (Biswas 2001). NF-kB exits in an inactive state in most cell types, except B lymphocytes (SEn 1986). Interestingly, MPG cells isolated from our lab had basal expression level of NF-kB, suggesting that NF-kB may play a role in canine MGTs. Moreover, numerous tumor cells with elevated levels of NF-kB are resistant to apoptosis induced by chemotherapy, radiotherapy, and TNFtreatment (Wang 1996; Liu 1996; Van Antwerp 1996). Therefore, inhibition of NF-kB would be a likely approach to enhance anti-tumor therapy in tumors with constitutive NF-kB activity.

Understanding of mechanisms implicated in the apoptosis control by NF-kB may lead to the development of new strategies to improve cancer therapy. Although several genes that may play a role in blocking apoptosis and whose expression is regulated by NF-kB, the precise understanding of the role of NF-kB in controlling apoptosis is still lacking. There are two general models about

Wnt-frizzled signaling by which NF-kB

transcription factors may regulate apoptosis: (1)

The sFRP2



WntFrizzledDishevelledGSK

-3-catenin-Tcf complex pathways can result in activation of NF-kB. SFRP-2 is a modulator of Wnt signaling (Finch 1997). Wnt-1 acts as a survival factor in PC12 cells by activating the death-suppressing transcription factor NF-kB and by inhibiting the apoptosis induced by serum withdrawal through a pathway that is partially independent of PI-3 kinase, but is dependent on the inactivation of GSK-3 (Hoeflich 2000; Bournat 2000). Moreover, overexpression of transfected SFRP2 in breast adenocarcinoma cells increases their resistance to apoptotic signals,

associated with increased intracellular levels of -catenin (Melkonyan 1997). Consistent with this finding, we have demonstrated that MPG cells

expressing SFRP2 could inhibit apoptosis,

associated with enhanced expression of-catenin. (2) The fibronectinintegrin receptorFAK PI-3KAkt pathways appear to cooperate for activation of NF-kB by phosphorylating different activating residues on IKK. In our previous studies, it was found that the presence of SFRP2

could enhance the formation of

fibronectin-integrin receptor complexes. One of ECM components, fibronectin, is particularly effective in providing survival signals for several cell types, and that these survival signals are transduced by FAK (Frisch 1996; Hungerford

1996; Ilic 1998). Additionally, PI-3K-Akt

survival pathway and NF-kB activation are involved in the FAK-induced resistance to apoptosis (Sonoda 2000). In this study, we show that SFRP2 can inhibit apoptosis, associated with increased FAK and NF-kB activities.

Several lines of evidences suggest that JNK

plays an important role in tumor cells.

Ras-induced tumorigenicity is suppressed by mutation of the JNK phosphorylation sites on

c-Jun (Behrens 2000). These data strongly

support the hypothesis that JNK is relevant to cancer. The JNK pathway has been implicated in both apoptosis and survival signaling (Ip 1998). Although one function of JNK is implicated in cancer, the mechanism of JNK action is unclear. During tumor development, it is likely that JNK-dependent stress-induced apoptosis must be suppressed. This would imply that components of the JNK pathway are potential tumor suppressor

genes. Established tumor cells must adopt

mechanisms to inhibit JNK-dependent apoptosis. In this study, we also demonstrated that MPG cells expressing SFRP2 inhibited apoptosis via JNK suppression. Recent investigations using anti-sense JNK oligonucleotides support the

conclusion that JNK functions are distinct

between normal and tumor cells. These studies imply that JNK inhibition may be useful for tumor therapy. Furthermore, these studies indicate that genetic interactions with cancer-associated genes may be critical for determining the outcome of JNK signaling.

In mammals, aberrant regulation of the Wnt pathway is implicated in tumorigenesis (Polakis 2000). The precise mechanism by which Wnt signaling ‘switches’ between the canonical Wingless/Wnt pathway or activation of the JNK pathway is unclear; however, recent evidence implicates the proteins Dishevelled and PAR-1. (Boutros 1998; Sun 2001). Dishevelled under enhancing Wnt pathway can activate JNK, whereas PAR-1 decreases JNK activity. Wnt signaling increases endogenous PAR-1 kinase

activity and subsequently potentiates the

canonical Wnt pathway at a level upstream of Axin and β-catenin. In contrast, activation of

PAR-1 inhibits Dishevelled-mediated JNK

activation (Sun 2001). Thus, PAR-1 is a positive regulator of the Wnt/β-catenin pathway and an inhibitor of the JNK pathway. Consistent with this idea, we found that MPG cells expressing SFRP2 enhanced the expression of β-catenin and

inhibited apoptosis via JNK suppression.

Therefore, we suggest that PAR-1 might play an

important role in MPG cells, associated

anti-apoptotic function.

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19. Bhat-Nakshatri P, Newton TR, Goulet R Jr, Nakshatri H. NF-kappaB activation and interleukin 6 production in fibroblasts by estrogen receptor-negative breast cancer cell-derived interleukin 1alpha. Proc Natl Acad Sci USA 95:6971–6976; 1998.

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32. Ilic D, Almeida EA, Schlaepfer DD, Dazin P, Aizawa S, Damsky CH. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J Cell Biol 143:547–560; 1998.

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35. Boutros M, Paricio N, Strutt DI, Mlodzik M. Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94:109-118; 1998.

36. Sun TQ, Lu B, Feng JJ, Reinhard C, Jan YN, Fantl WJ, Williams LT. PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling. Nat Cell Biol 3:628-636; 2001. 五、計畫成果自評

During this 3-year project, we have obtained important research data and progress. Firstly, we have established and characterized the apoptosis regulatory functions of sFRP2 in mammary cancer cell lines.

Secondly, the complex signaling pathways of sFRP2-mediated apoptosis regulation were investigated by a number of complex experiments. It is an important and the first finding regarding signaling pathways of sFRP2-mediated apoptosis regulation in cancer cell lines.

Thirdly, the relation of sFRP2-mediated anti-apoptosis with transcription factors or other signaling factors have been studied.

The research results expect to be helpful to

elucidate the relation between apoptosis,

signaling networks in sFRP2-mediated

anti-apoptosis in mammary cancers, and possible related pathogenesis of mammary cancers.

行政院國家科學委員會專題研究計

畫

成果報告

實驗結果圖表部分

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 (1/3)

計畫編號：NSC 90-2313-B-002-404

執行期限：91 年 8 月 1 日至 92 年 7 月 31 日

主持人：林中天

國立台灣大學獸醫學系

計畫參與人員: 李佳霖、林念儀

等研究生

Figures of Experimental Data

Fig. 1

A

C

B

Fig. 3

A

B

Fig. 4

A

Figure Legends

Fig. 1 Primary culture of canine MGT. (A, upper) Mix cell types of primary culture from MGT. (A, bottom) Primary culture after purification by cloning. (B) All colonies of primary culture were detected for cytokeratin 18 by western blotting. MCF7: positive. The expected size of cytokeratin 18 protein is 45 kDa. (C) MPG1 were detected immunohistochemically for cytokeratin 18 to prove the epithelial origin of the cultured cells. Bottom: negative control. Original magnification: x200. MPG: primary culture from canine MGTs. NMG: primary culture from canine normal MGs.

Fig. 2 Characterization of MPG and NMG cells. (A) Colony-forming efficiency assay.

Colony-forming efficiency was expressed as the ratio of the number of colonies at 14 days to the original number of cells per dish at the time of suspending in soft agar. (B) Proliferation assay. Proliferation rate was detected by MTT assay. MCF7 was served as a positive control. Results are represent the means ± SE of three independent experiments.

Fig. 3 mRNA analysis of SFRP2 expression. (A) Transcripts of canine SFRP2 were analyzed by

RT-PCR. Lane 1-4: MPG1-MPG4; Lane 5-6: NMG1-NMG2. Lane 7-8: RNA integrity was confirmed

by monitoring for -actin mRNA of NMG1-NMG2; M, molecular weight markers; Lane 9, negative

control. The expected sizes of SFRP2 and-actin PCR products are 980 bp and 156 bp, respectively. (B)

In situ hybridization of SFRP2 in MPG1. The cells were hybridized with a digoxigenin-labelled SFRP2

antisense (upper) and sense probe (bottom). Original magnification: x200. MPG: primary culture from canine MGTs. NMG: primary culture from canine normal MGs.

Fig. 4 Protein analysis of SFRP2 expression. (A) Whole cell extracts were prepared and 50 μg oftotal

protein and separated by SDS-PAGE (10%). Following membrane transfer, a Western analysis was performed with antibody against SFRP2. For internal control experiment, a parallel Western analysis

was performed on the blots using antibody to -tubulin. Lane 1-4: MPG1-MPG4; Lane 5-6:

NMG1-NMG2; Lane 7: 293T cells; Lane 8: Hela cells; Lane 9: K562 cells; Lane 10: P388D1 cells. The expected size of SFRP2 protein is 35 kDa. (B) Immunohistochemical detection of SFRP2 in MPG1. Bottom: negative control. Original magnification: x100. MPG: primary culture from canine MGTs. NMG: primary culture from canine normal MGs.

行政院國家科學委員會專題研究計

畫

成果報告

實驗結果圖表部分

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)

計畫編號：NSC 92-2313-B-002-133

執行期限：92 年 8 月 1 日至 93 年 7 月 31 日

主持人：林中天

國立台灣大學獸醫學系

計畫參與人員: 李佳霖、林念儀、謝岱儒

等研究生

Figures of Experimental Data (計畫編號：NSC 92-2313-B-002-133)

Figure 1

a

b

Figure 2

a

b

c

Figure 3

a

b

Figure 4

a

b

FIGURE LEGENDS

Figure 1 Overexpression of SFRP2 reduces cell sensitivity to UV-induced apoptosis. (a) Expression

of the endogenous or exogenous SFRP2. MPG and MCF7 cells were transfected with expression vectors for SFRP2 proteins (MPG/pCMV-cSFRP2-Flag, MCF7/pCMV-cSFRP2-Flag cells) or the vector backbone (MPG/pCMV-Flag, MCF7/pCMV-Flag cells) and selected by G418. Expression of the SFRP2 proteins was determined by Western blot analysis. (b, c) Apoptotic analysis of the MPG and MCF7 transfectants. After selection by G418, the cells were irradiated with UV at 200 J/m2 in b or 100, 200 J/m2 in c. 24 hours after irradiation, the cells were analyzed for DNA fragmentation by DNA laddering in b and Cell-Death Detection ELISA in c. Results are represent the means ± SE of three independent experiments. *: P< 0.05.

Figure 2 SFRP2 is secreted from the cells and associated with FN. (a) Expression of SFRP2 in tissue

sections of canine MGTs (magnification x 400). Expression of the SFRP2 proteins were detected by immunohistochemical analysis; anti-SFRP2 antibody as indicated (left) and negative control (right). Note the positive signal around cells (arrow). (b) Expression of the SFRP2 and FN proteins in cultured medium were determined by Western blot analysis. Proteins were prepared from cultured medium of MPG/pCMV-cSFRP2-Flag, MPG/pCMV-Flag, MCF7/pCMV-cSFRP2-Flag, and MCF7/pCMV-Flag cells. (c) Immunoprecipitation of the SFRP2 proteins in cultured medium. Proteins were prepared as in b. SFRP2 was immunoprecipitated with anti-SFRP2 antibody as indicated. SFRP2 and FN were detected by Western blot analysis. (d) Expression of the SFRP2, integrin 51 and FN proteins of whole cell extracts were determined by Western blot analysis. Whole cell extracts were prepared from the same cells as in b. (e) Immunoprecipitation of the SFRP2 proteins of whole cell extracts was done as in c. Figure 3 SFRP2 promotes integrin-mediated cell adhesion by enhancing integrin 51-FN binding affinity. (a) Immunoprecipitation of the FN proteins. Whole cell extracts were prepared from MPG/pCMV-cSFRP2-Flag and MPG/pCMV-Flag cells. FN was immunoprecipitated with anti-FN antibody as indicated. SFRP2, integrin 51, and FN were detected by Western blot analysis. (b, c) Adhesion assay. The same cells as in a were incubated without (left) or with (right) 2 mM of MnCl2for

30 min. Cells were replated on FN and allowed to adhere for 10, 20, 30, or 40 min in b, or on non-coated, 1%BSA, FN for 30 min in c, respectively. The percentage of adhesion was calculated as described in Materials and Methods. Data from three separate experiments were shown as means ± SE. *, **: P< 0.05.

(d) MCF7 cells were incubated with cultured medium of MCF7/pCMV-cSFRP2-Flag (SFRP2+) or

MCF7/pCMV-Flag (SFRP2-) cells. The cells were replated on FN and allowed to adhere for 30 min as indicated. Steps were the same as in b. *: P< 0.05.

Figure 4 SFRP2 requires ECM components to decrease susceptibility to UV-induced apoptosis. (a, b) Immunoprecipitation of the SFRP2 proteins. MPG/pCMV-cSFRP2-Flag and MPG/pCMV-Flag cells

were incubated with 0.1 mg/mL Heparin in a or 0.1 mg/mL integrin 51 antibody in b. Experimental steps were the same as Figure 2-e. (c) Apoptotic analysis. The same cells as in a and b were irradiated with UV at 200 J/m2. 24 hours after irradiation, the cells were analyzed for DNA fragmentation by DNA laddering experiment.

行政院國家科學委員會專題研究計

畫

成果報告

實驗結果圖表部分

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 (3/3)

計畫編號：NSC 92-2313-B-002-022

執行期限：93 年 8 月 1 日至 94 年 7 月 31 日

主持人：林中天

國立台灣大學獸醫學系

計畫參與人員: 李佳霖、林念儀、謝岱儒

等研究生

Figures

Fig. 1.

Fig. 2.

Fig. 4.

Fig. 6.

Figure Legends

Fig. 1. Cell culture and stably transfected cells lines. MPG cells were transfected with expression

vectors for SFRP2 proteins (pcDNA4-pGFP-cSFRP2) or the vector backbone (pcDNA4-pGFP) and selected by G418. The percentage of cells expressing GFP or GFP-cSFRP2 fusion proteins was determined by fluorescence microscope. (a, b) MPG/ pcDNA4-pGFP cells. (c, d) MPG/

pcDNA4-pGFP-cSFRP2 cells. Original magnification: top panel, X40; bottom panel, X200.

Fig. 2. DNA fragmentation assay. Apoptotic analysis of the MPG transfectants. After selection by

G418, the cells were irradiated with UV at 50, 100, 200 J/m2. 24 hours after irradiation, the cells were analyzed for DNA fragmentation by DNA laddering.

Fig. 3. TUNEL assay. Apoptotic analysis of the MPG transfectants. After selection by G418, the cells

were irradiated with UV at 200 J/m2. 24 hours after irradiation, the cells were analyzed for DNA fragmentation by in situ DNA degradation assay - terminal nucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining. The percentage of apoptotic cells was determined by counting 1000 cells. Results represent the means ± SE of three independent experiments. **: P< 0.005.

Fig. 4. Immunofluorescence analysis of SFRP2 expression. The section of canine MGT was

double-labeled with antibodies specific for fibronectin (red; b, e) and for SFRP2 (green; c, f). Nucleus was stained with DAPI (blue; a, d). (A) Combination with a, b, c. (B) Combination with e, f. Original magnification: left panel, X200; right panel, X400.

Fig. 5. SFRP2 modulated the activation of FAK. Both MPG/ pcDNA4-pGFP (bottom) and MPG/

pcDNA4-pGFP-cSFRP2 (top)cellsweretreated with fibronectin (10 ng/μl)fortheindicated times,and whole cell extracts were subsequently prepared. FAK and FAK tyrosine phophorylation were detected by immunoprecipitation with anti-FAK antibody as indicated, followed by western blot analysis using anti-FAK antibody and antibody to phosphotyrosine.

Fig. 6. Decreased activation of JNK in MPG cells expressing SFRP2. Both MPG/ pcDNA4-pGFP

and MPG/ pcDNA4-pGFP-cSFRP2 cells were irradiated with or without UV at 200 J/m2. After 24 h of incubation, the cells were lysed and JNK and JNK tyrosine phophorylation were detected by western blot analysis using anti-JNK and phospho-JNK antibodies. Expression of JNK in top and phospho-JNK in bottom, respectively.

Fig. 7. Active NF-kB complex in MPG cells expressing SFRP2. Both MPG/ pcDNA4-pGFP and

MPG/ pcDNA4-pGFP-cSFRP2 cells were irradiated with UV at 200 J/m2. After 30 min of incubation, nuclear extracts from control and UV-treated cells were incubated in a standard EMSA reaction mixture containing [32P]-labeled double-stranded oligonucleotide. The NF-kB–[32P]DNA complex was characterized by supershift assay with anti-p50 (lanes 2 and 4) antibodies. Nuclear extracts were incubated with anti-p50 antibodies for 15 min at room temperature, followed by incubation for an additional 30 min in the presence of [32P] double-stranded NF-kB oligonucleotide, and subjected to EMSA as described. The supershifted complexes are as indicated in the autoradiograph.