陳洪巖，劉芝華

·基礎(chǔ)研究·

乳腺癌細(xì)胞雌激素受體非配體依賴性激活對(duì)來(lái)曲唑耐藥的機(jī)制研究

陳洪巖，劉芝華

目的研究雌激素受體(ER)轉(zhuǎn)錄調(diào)節(jié)變化在乳腺癌細(xì)胞對(duì)來(lái)曲唑耐藥中的作用及其機(jī)制。方法以來(lái)曲唑耐藥細(xì)胞(MCF-7-LR-1、MCF-7-LR-2)和對(duì)照細(xì)胞(MCF-7-Aro、MCF-7-T)為研究對(duì)象。通過(guò)Western blotting檢測(cè)ER和磷酸化ER[pER(Ser118)]在兩組細(xì)胞中的表達(dá)水平。通過(guò)染色質(zhì)免疫共沉淀(ChIP)方法檢測(cè)雌二醇(E2)處理前后細(xì)胞中ER與X盒結(jié)合蛋白1(XBP-1)增強(qiáng)子和三葉因子1(TFF-1)啟動(dòng)子區(qū)的結(jié)合情況。通過(guò)Real-time qRT-PCR比較XBP-1和TFF-1 mRNA的表達(dá)，以及雌激素反應(yīng)性基因CA2、GJA1、PGR、CTSD mRNA的表達(dá)。結(jié)果與對(duì)照細(xì)胞相比，來(lái)曲唑耐藥細(xì)胞中pER(Ser118)水平顯著增加，但ER表達(dá)水平并無(wú)明顯變化。ChIP結(jié)果表明，與對(duì)照細(xì)胞相比，MCF-7-LR-2細(xì)胞中ER在非配體激活條件下(未加E2處理)及配體激活條件下(10nmol/L E2處理45min)與XBP-1增強(qiáng)子區(qū)及TFF-1啟動(dòng)子區(qū)的結(jié)合均顯著增加。qRT-PCR結(jié)果表明，與未經(jīng)E2處理的MCF-7-Aro細(xì)胞相比，耐藥細(xì)胞中XBP-1和TFF-1表達(dá)均顯著增加；與MCF-7-T細(xì)胞相比，耐藥細(xì)胞中CA2和GJA1表達(dá)增高，PGR和CTSD的表達(dá)降低。結(jié)論在來(lái)曲唑耐藥的乳腺癌細(xì)胞中，ER以配體非依賴方式激活，pER(Ser118)水平增加，從而促進(jìn)相關(guān)靶基因的轉(zhuǎn)錄，該機(jī)制在乳腺癌細(xì)胞對(duì)來(lái)曲唑耐藥的過(guò)程中發(fā)揮重要作用。

乳腺腫瘤；受體，雌激素；抗藥性，腫瘤；X盒結(jié)合蛋白質(zhì)1；三葉因子1

乳腺癌是一種伴隨特定的形態(tài)學(xué)/組織學(xué)改變、遺傳學(xué)/表觀遺傳學(xué)改變和基因表達(dá)產(chǎn)物障礙的疾病[1]，嚴(yán)重危害女性的身心健康。雌激素受體(ER)信號(hào)通路在調(diào)控乳腺細(xì)胞增殖和凋亡等生理功能中發(fā)揮著重要作用。ER信號(hào)通路失調(diào)可導(dǎo)致相關(guān)基因表達(dá)失衡或改變胞質(zhì)內(nèi)的蛋白功能，促使乳腺細(xì)胞過(guò)度增殖或凋亡受阻，從而導(dǎo)致乳腺癌發(fā)生，另一方面還可促使乳腺癌由雌激素依賴型向雌激素非依賴型轉(zhuǎn)化，造成治療抵抗[2]。選擇性雌激素受體調(diào)節(jié)劑(他莫昔芬，tamoxifen)和芳香化酶抑制劑是ER陽(yáng)性乳腺癌患者的主要治療藥物。他莫昔芬在乳腺癌臨床治療中取得了令人矚目的療效，但會(huì)增加?jì)D女發(fā)生血栓栓塞和子宮內(nèi)膜癌的概率，且患者對(duì)藥物耐藥會(huì)導(dǎo)致癌癥復(fù)發(fā)[3-5]。與他莫昔芬相比，抑制雄激素轉(zhuǎn)換成雌激素的芳香化酶抑制劑具有更好的療效和耐受性，能有效延長(zhǎng)患者生存期[6-8]。非甾體類第三代芳香化酶抑制劑來(lái)曲唑(letrozole)作為絕經(jīng)后乳腺癌患者的一線臨床用藥，具有很好的臨床療效和耐受性，但長(zhǎng)期應(yīng)用后仍會(huì)發(fā)生藥物抵抗[9-10]。

已有研究表明，ER磷酸化水平增加能促進(jìn)相關(guān)靶基因的轉(zhuǎn)錄，從而在他莫昔芬耐藥中發(fā)揮重要作用[11]。其中，ER經(jīng)典的靶基因X盒結(jié)合蛋白1 (XBP-1)和三葉因子1(TFF-1)備受關(guān)注[12]。體外研究表明，XBP-1與他莫昔芬耐藥密切相關(guān)，臨床研究進(jìn)一步顯示XBP-1高表達(dá)與接受他莫昔芬治療的乳腺癌患者的不良預(yù)后相關(guān)[2]。同時(shí)，ER非配體依賴性的激活可增強(qiáng)TFF-1的轉(zhuǎn)錄，從而促進(jìn)乳腺癌細(xì)胞非激素依賴性增殖，在乳腺癌內(nèi)分泌治療抵抗中發(fā)揮著重要作用[13]。鑒于ER在乳腺癌發(fā)生發(fā)展和他莫昔芬抵抗中的重要作用，我們推測(cè)其在來(lái)曲唑耐藥中可能具有同等作用。本實(shí)驗(yàn)采用人乳腺癌細(xì)胞株MCF-7[14]研究來(lái)曲唑耐藥細(xì)胞中ER非配體依賴性激活對(duì)靶基因XBP-1和TFF-1轉(zhuǎn)錄調(diào)節(jié)的影響，探討ER在乳腺癌細(xì)胞來(lái)曲唑耐藥過(guò)程中的作用機(jī)制。

1 材料與方法

1.1主要試劑 睪酮、β-雌二醇(β-estradiol)、來(lái)曲唑購(gòu)自美國(guó)Sigma公司；RNA提取試劑盒(RNeasy Plus Mini Kit)購(gòu)自美國(guó)Qiagen公司；反轉(zhuǎn)錄試劑盒(high-capacity cDNA reverse transcription kits)和實(shí)時(shí)定量PCR試劑(SYBR Green PCR Master Mix)購(gòu)自美國(guó)ABI公司；BCA蛋白定量試劑盒、化學(xué)發(fā)光試劑(ECL)購(gòu)自美國(guó)Pierce公司；蛋白酶抑制劑(PI)購(gòu)自德國(guó)Roche公司；G418購(gòu)自美國(guó)Invitrogen公司；pER (Ser118) 和Calnexin抗體購(gòu)自美國(guó)Cell Signaling公司；ER抗體購(gòu)自美國(guó)Santa Cruz公司；過(guò)氧化物酶標(biāo)記的二抗購(gòu)自美國(guó)Sigma公司。

1.2耐藥細(xì)胞和對(duì)照細(xì)胞的建立 來(lái)曲唑耐藥的乳腺癌細(xì)胞(MCF-7-LR-1、MCF-7-LR-2)及其對(duì)照細(xì)胞(MCF-7-Aro、MCF-7-T)由本實(shí)驗(yàn)室建立，細(xì)胞株的建立方式如下。MCF-7-Aro細(xì)胞：課題組前期研究建立的過(guò)表達(dá)芳香化酶的細(xì)胞株[15]。MCF-7-T細(xì)胞：為對(duì)照細(xì)胞，將MCF-7-Aro細(xì)胞培養(yǎng)在加入睪酮和來(lái)曲唑溶劑對(duì)照(DMSO)的去激素培養(yǎng)基中。MCF-7-LR-1和MCF-7-LR-2細(xì)胞：在100nmol/L和200nmol/L來(lái)曲唑作用下篩選獲得的耐藥細(xì)胞。耐藥細(xì)胞篩選步驟：將MCF-7-Aro培養(yǎng)于含有睪酮和芳香化酶抑制劑來(lái)曲唑的去激素培養(yǎng)基，盡管有睪酮存在，但來(lái)曲唑可抑制芳香化酶將睪酮轉(zhuǎn)換為雌激素，所以在篩選過(guò)程中大部分細(xì)胞死亡，經(jīng)過(guò)3個(gè)月的篩選，最終獲得耐藥細(xì)胞。

細(xì)胞培養(yǎng)：MCF-Aro細(xì)胞培養(yǎng)于含10%胎牛血清、丙酮酸鈉、100U/ml青霉素、100U/ml 鏈霉素、400μg/ml G418的DMEM培養(yǎng)基中，置于37℃、5% CO2培養(yǎng)箱中培養(yǎng)。將MCF-7-LR細(xì)胞培養(yǎng)于含有睪酮和來(lái)曲唑的去激素培養(yǎng)基[去除酚紅，含10%活性炭吸附的胎牛血清(CFBS)、丙酮酸鈉、100U/ml青霉素、100U/ml鏈霉素的DMEM培養(yǎng)基]。MCF-7-T細(xì)胞培養(yǎng)在加入睪酮和DMSO的去激素培養(yǎng)基中。

1.3Western blotting檢測(cè) 取來(lái)曲唑耐藥細(xì)胞(MCF-7-LR-1、MCF-7-LR-2)及對(duì)照細(xì)胞(MCF-7-Aro、MCF-7-T)，在去激素培養(yǎng)基中培養(yǎng)3d，收集細(xì)胞，離心獲得細(xì)胞沉淀，加入含1×PI的RIPA裂解液(1×DPBS，1% Nonidet P-40，0.5%脫氧膽酸鈉，0.1%SDS)進(jìn)行裂解，冰上放置20min，13 000×g離心收集上清。采用BCA法行蛋白定量，取20μg蛋白進(jìn)行聚丙烯酰胺凝膠電泳，將蛋白轉(zhuǎn)至PVDF膜，封閉、一抗孵育、洗膜，用辣根過(guò)氧化物酶標(biāo)記的二抗孵育、洗膜，采用化學(xué)發(fā)光ECL系統(tǒng)檢測(cè)ER的表達(dá)和磷酸化(pER，Ser118)水平，以鈣聯(lián)蛋白(calnexin)作為上樣對(duì)照。

1.4染色質(zhì)免疫共沉淀(ChIP)檢測(cè) 為驗(yàn)證ER磷酸化水平增加對(duì)靶基因調(diào)節(jié)區(qū)域結(jié)合能力的影響，采用ChIP法檢測(cè)耐藥細(xì)胞及對(duì)照細(xì)胞中ER與XBP-1和TFF-1調(diào)節(jié)區(qū)域的結(jié)合情況，其中E2溶于乙醇，同時(shí)以乙醇處理細(xì)胞作為溶劑對(duì)照。根據(jù)文獻(xiàn)[16]選擇XBP-1第2個(gè)增強(qiáng)子區(qū)(XBP-1 ENH2)和TFF-1啟動(dòng)子區(qū)(TFF-1 Pro)用于判斷ER對(duì)靶基因調(diào)節(jié)區(qū)域的結(jié)合情況，以XBP-1啟動(dòng)子區(qū)(XBP-1 Pro)為無(wú)ER結(jié)合的陰性對(duì)照區(qū)。取MCF-7-Aro、MCF-7-T、MCF-7-LR-1和MCF-7-LR-2細(xì)胞，接種至去激素培養(yǎng)基，培養(yǎng)3d，至細(xì)胞融合約達(dá)90%，用10nmol/L E2處理45min，以1%甲醛于37℃固定細(xì)胞10min，預(yù)冷PBS洗滌細(xì)胞2次，加入1ml預(yù)冷PBS，用細(xì)胞刮收集細(xì)胞，3 000×g離心5min，收集細(xì)胞沉淀，加入300μl含PI的細(xì)胞裂解液[1% SDS，5mmol/L EDTA，50mmol/L Tris-HCl (pH8.1)]在冰上裂解10min。經(jīng)Bioruptor超聲破碎儀破碎細(xì)胞(使DNA片段集中在300～500bp)，4℃、14 000×g離心10min收集上清。取20μl作為Input對(duì)照，用Dilution buffer[1% Triton X-100，2mmol/L EDTA，150mmol/L NaCl，20mmol/L Tris-HCl(pH8.1)1×PI]按照1:10稀釋。加入鮭精DNA、預(yù)清除免疫球蛋白(IgG)和45μl Protein A-sepharose 在4℃預(yù)清除2h。收集上清液加入ER抗體，4℃過(guò)夜孵育，第2天加入45μl protein A-sepharose 4℃孵育1h，收集Beads，依次用TSE Ⅰ、TSE Ⅱ、Buffer Ⅲ和TE緩沖液洗滌，每次10min。此后用100μl Elution buffer (1% SDS，0.1mol/L NaHCO3)室溫10min洗脫DNA。經(jīng)65℃孵育6h，解交聯(lián)后用PCR純化試劑盒(Qiagen公司)進(jìn)行DNA純化，然后用下述引物進(jìn)行Real-time PCR分析。結(jié)果以相對(duì)于Input的百分比進(jìn)行計(jì)算，公式為

ChIP引物序列(3'-5')如下。XBP-1：正義TCTG GAAAGCTCTCGGTTTG，反義AATCCCTGGCCAA AGGTACT；XBP-1 Enh2：正義TTGCTGTGCAAAC AATAGCC，反義GTCCAAGGGCACATTCTCAT；TFF-1：正義CACCCCGTGAGCCACTGT，反義CTGCAGAAGTGATTCATAGTGAGAGAT。

1.5Real-time qRT-PCR檢測(cè) 取MCF-7-Aro細(xì)胞，在去激素培養(yǎng)基中培養(yǎng)3d，加入E2處理12h后收集細(xì)胞(稱為MCF-7-Aro-E2)。取MCF-7-Aro、MCF-7-Aro-E2、MCF-7-T和MCF-7-LR-2細(xì)胞，提取RNA并反轉(zhuǎn)錄成cDNA，用XBP-1、TFF-1和β-actin特異性引物進(jìn)行Real-time PCR檢測(cè)，結(jié)果以相對(duì)于β-actin的表達(dá)量表示。

根據(jù)文獻(xiàn)[17]、[18]選擇芳香化酶抑制劑及他莫昔芬耐藥細(xì)胞中表達(dá)改變的雌激素反應(yīng)性基因碳酸酐酶2(CA2)、縫隙連接α-1蛋白(GJA1)、孕激素受體(PGR)和組織蛋白酶D(CTSD)，通過(guò)Real-time qRT-PCR檢測(cè)上述基因在 MCF-7-T和MCF-7-LR-2細(xì)胞中的表達(dá)情況。以MCF-7-T和MCF-7-LR-2細(xì)胞的cDNA作為模板，用CA2、GJA1、PGR和CTSD特異性引物行Real-time qRT-PCR檢測(cè)，結(jié)果以相對(duì)于β-actin的表達(dá)量表示。

采用RNeasy Plus Mini Kit提取試劑盒提取RNA，去除DNA，使用Nanodrop分光光度計(jì)行RNA定量，取1μg RNA，用High-Capacity cDNA Reverse Transcription Kits反轉(zhuǎn)錄試劑盒行cDNA合成。使用SYBR Green PCR Master Mix試劑，以XBP-1、TFF-1和β-actin 特異性引物行Real-time PCR。同時(shí)檢測(cè)MCF-7-T和MCF-7-LR-2細(xì)胞中CA2、GJA1、PGR和CTSD的mRNA表達(dá)水平。特異基因的mRNA相對(duì)表達(dá)量采用如下公式進(jìn)行計(jì)算：2(40－Ct-target)/2(40－Ct-β-actin)。

引物由上海英駿工程公司合成，序列(3'-5')如下。XBP-1：正義GCGCCTCACGCACCTG，反義GCTGCTACTCTGTTTTTCAGTTTCC；TFF-1：正義GTGTCACGCCCTCCCAGT，反義GGACCCCACG AACGGTG；CTSD：正義AGCTCCTGGACCAGAAC ATCT，反義GGGTGACATTCAGGTAGGACA；PGR：正義AGGTCTACCCGCCCTATCTC，反義CAGCTCCCACAGGTAAGGAC；GJA1:正義ATAGACAGGTCTGAGTGCCTGAA，反義CAGTTGAGTAGGCTTGAACCTTG；CA2:正義CCTCCTCTTCTGGAATGTGTG，反義CCAGTTGTCCACCATCAGTTC；β-actin:正義GGCGGCACCACCATGTACCCT，反義AGGGGCCGGACTCGTCATACT。

1.6統(tǒng)計(jì)學(xué)處理 采用 SPSS 19.0軟件進(jìn)行統(tǒng)計(jì)分析。數(shù)據(jù)結(jié)果以±s表示，多組間比較采用單因素方差分析法，進(jìn)一步兩兩比較采用獨(dú)立樣本t檢驗(yàn)。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

圖1 來(lái)曲唑耐藥細(xì)胞及對(duì)照細(xì)胞中ER表達(dá)和磷酸化(pER，Ser118)水平檢測(cè)(Western blotting)Fig. 1 Expression of ER and phosphorylation of ER at Ser-118 in letrozole resistant cells and control cells (Western blotting)

2.1Western blotting 檢測(cè)結(jié)果 由圖1可見，與去激素條件下培養(yǎng)的MCF-7-Aro和MCF-7-T細(xì)胞相比，MCF-7-LR-1和MCF-7-LR-2細(xì)胞中ER磷酸化(pER，Ser118)水平顯著增加，但ER總體表達(dá)水平無(wú)顯著變化，表明來(lái)曲唑耐藥細(xì)胞中ER以配體非依賴的方式激活。

圖2 來(lái)曲唑耐藥細(xì)胞及對(duì)照細(xì)胞中ER在靶基因XBP-1和TFF-1調(diào)節(jié)區(qū)域的結(jié)合(ChIP檢測(cè))Fig.2 Conjugation of ER on the regulatory regions of target genes in Letrozole resistant and control cells(Chromatin Immunoprecipitation)

2.2ChIP檢測(cè)結(jié)果 由圖2A可見，除MCF-7-LR-1中ER在TFF-1 Pro處的結(jié)合未被顯著誘導(dǎo)外，E2處理可顯著誘導(dǎo)各組細(xì)胞中ER在XBP-1 ENH2處和TFF-1Pro處的結(jié)合，但在XBP-1 Pro處的結(jié)合無(wú)明顯改變。由圖2B可見：無(wú)E2處理?xiàng)l件下，與對(duì)照細(xì)胞(MCF-7-Aro和MCF-7-T)比較，來(lái)曲唑耐藥細(xì)胞(MCF-7-LR-1和MCF-7-LR-2)中ER在XBP-1 ENH2處和TFF-1 Pro處的結(jié)合顯著增強(qiáng)。在E2處理?xiàng)l件下，與對(duì)照細(xì)胞MCF-7-Aro比較，100nmol/L來(lái)曲唑篩選獲得的耐藥細(xì)胞MCF-7-LR-1中ER在XBP-1 ENH2處的結(jié)合顯著增加，而在TFF-1 Pro處的結(jié)合并無(wú)顯著變化；與對(duì)照細(xì)胞MCF-7-T比較，MCF-7-LR-1細(xì)胞中ER在XBP-1 ENH2處和TFF-1 Pro區(qū)的結(jié)合均無(wú)明顯變化；與對(duì)照細(xì)胞(MCF-7-Aro和MCF-7-T)比較，200nmol/L來(lái)曲唑篩選獲得的耐藥細(xì)胞MCF-7-LR-2中ER在XBP-1 ENH2和TFF-1 Pro處的結(jié)合均進(jìn)一步增強(qiáng)。該結(jié)果表明來(lái)曲唑耐藥細(xì)胞中ER以配體非依賴的方式激活，從而促進(jìn)其與靶基因XBP-1和TFF-1調(diào)節(jié)區(qū)域的結(jié)合。

2.3Real-time qRT-PCR檢測(cè)結(jié)果 由圖3A可見，與去激素條件下培養(yǎng)的MCF-7-Aro細(xì)胞相比，經(jīng)E2處理獲得的MCF-7-Aro-E2細(xì)胞中XBP-1和TFF-1 mRNA水平顯著增加；MCF-7-T細(xì)胞中，因芳香化酶將雄激素轉(zhuǎn)換成雌激素，所以XBP-1和TFF-1的表達(dá)維持在較高水平；而MCF-7-LR-2細(xì)胞中XBP-1和TFF-1的表達(dá)均顯著增加。

由圖3B可見，與MCF-7-T細(xì)胞相比，MCF-7-LR-2細(xì)胞中CA2和GJA1表達(dá)上調(diào)，而PGR和CTSD表達(dá)下調(diào)，表明來(lái)曲唑耐藥細(xì)胞中激素反應(yīng)性基因CA2和GJA1選擇性表達(dá)增加，而激素反應(yīng)性基因PGR和CTSD依然保持對(duì)來(lái)曲唑的敏感性。

3 討 論

圖3 來(lái)曲唑耐藥細(xì)胞及對(duì)照細(xì)胞中雌激素反應(yīng)性基因(ERGs)表達(dá)(qRT-PCR檢測(cè))Fig. 3 Expression of estrogen responsive genes(ERGs) in letrozole resistant cells and the control cells (quantitative reverse transcription PCR)

大約2/3的乳腺癌患者ER表達(dá)陽(yáng)性。ER既是乳腺癌內(nèi)分泌治療的重要靶點(diǎn)，也是判斷治療反應(yīng)的重要生物標(biāo)志分子[19-20]。ER與配體E2結(jié)合后，與熱休克蛋白HSP90解離，促進(jìn)ER結(jié)合在雌激素反應(yīng)元件(ERE)上，調(diào)控靶基因轉(zhuǎn)錄，從而調(diào)節(jié)細(xì)胞增殖[21]。在缺乏雌激素的情況下，生長(zhǎng)因子激活的細(xì)胞內(nèi)信號(hào)途徑誘導(dǎo)ER與ERE結(jié)合以調(diào)節(jié)基因的轉(zhuǎn)錄。表皮生長(zhǎng)因子(EGF)通過(guò)EGF受體→Ras→Raf→MAPKKK→MAPKK→MAPK磷酸級(jí)聯(lián)反應(yīng)，使ER的Ser118磷酸化，導(dǎo)致ER激活[22]。應(yīng)用ER選擇性調(diào)節(jié)劑(如他莫昔芬)和芳香化酶抑制劑是ER陽(yáng)性乳腺癌患者內(nèi)分泌治療的主要策略，而ER信號(hào)通路異常改變可促使乳腺癌細(xì)胞由激素依賴型向激素非依賴型轉(zhuǎn)變，導(dǎo)致耐藥或內(nèi)分泌治療失敗[2]。研究表明，他莫昔芬耐藥細(xì)胞中ER磷酸化水平增加，并且在激素非依賴的MCF-7-LETD細(xì)胞(長(zhǎng)期培養(yǎng)在去激素培養(yǎng)基中篩選獲得的不再依賴雌激素增殖的MCF-7細(xì)胞)中ER磷酸化(pER，Ser118)水平也顯著增加[22-23]。本研究檢測(cè)來(lái)曲唑耐藥細(xì)胞中ER的表達(dá)和磷酸化水平，結(jié)果也顯示ER磷酸化水平增加，表明在來(lái)曲唑耐藥細(xì)胞中ER以配體非依賴的方式激活。ER磷酸化水平增加可增強(qiáng)ER共激活因子SRC3的結(jié)合并提高其對(duì)雌激素的敏感性，由此改變對(duì)靶基因的轉(zhuǎn)錄調(diào)節(jié)[22,24]。研究表明，ER磷酸化水平增高導(dǎo)致靶基因轉(zhuǎn)錄調(diào)節(jié)改變與他莫昔芬耐藥密切相關(guān)，并且ER在全基因組結(jié)合位點(diǎn)的改變與乳腺癌的臨床預(yù)后密切相關(guān)[25-26]。為了解ER磷酸化水平增加是否可以改變ER與靶基因調(diào)節(jié)區(qū)域的結(jié)合，本研究應(yīng)用ChIP法檢測(cè)E2處理前后ER在靶基因調(diào)節(jié)區(qū)域結(jié)合能力的改變。結(jié)果發(fā)現(xiàn)，來(lái)曲唑耐藥細(xì)胞中ER以配體非依賴的方式結(jié)合在其靶基因的調(diào)節(jié)區(qū)域，并且在200nmol/L來(lái)曲唑篩選獲得的耐藥細(xì)胞(MCF-7-LR-2)中E2處理可進(jìn)一步增強(qiáng)ER的結(jié)合能力，而100nmol/L來(lái)曲唑篩選獲得的耐藥細(xì)胞(MCF-7-LR-1)中E2處理并不能進(jìn)一步增強(qiáng)ER在TFF-1 Pro的結(jié)合能力，該結(jié)果可能是由于兩個(gè)濃度篩選獲得的耐藥細(xì)胞對(duì)來(lái)曲唑抵抗程度不同造成的。因此，我們選擇MCF-7-LR-2繼續(xù)此后的研究。有研究顯示ER的異常磷酸化可導(dǎo)致其在全基因組結(jié)合位點(diǎn)的改變，影響靶基因的轉(zhuǎn)錄，從而在他莫昔芬耐藥中發(fā)揮重要作用[27-29]。為此，本研究進(jìn)一步檢測(cè)了來(lái)曲唑耐藥細(xì)胞中XBP-1和TFF-1轉(zhuǎn)錄水平的改變，結(jié)果發(fā)現(xiàn)來(lái)曲唑耐藥細(xì)胞中TFF-1和XBP-1的轉(zhuǎn)錄水平顯著高于MCF-7-Aro細(xì)胞，表明來(lái)曲唑耐藥細(xì)胞中ER激活促進(jìn)了其在靶基因調(diào)節(jié)區(qū)域的結(jié)合以及靶基因的轉(zhuǎn)錄。由于芳香化酶的功能是將雄激素轉(zhuǎn)換成雌激素，從而影響雌激素反應(yīng)性基因的表達(dá)，而芳香化酶抑制劑可通過(guò)抑制芳香化酶的功能從而拮抗雌激素反應(yīng)性基因表達(dá)[30]。為了進(jìn)一步研究耐藥細(xì)胞中雌激素反應(yīng)性基因的表達(dá)改變，我們根據(jù)文獻(xiàn)[17]、[18]選擇芳香化酶抑制劑耐藥細(xì)胞中表達(dá)上調(diào)的基因CA2和GJA1，以及他莫昔芬耐藥細(xì)胞中表達(dá)下調(diào)的激素反應(yīng)性基因PGR和CTSD進(jìn)行檢測(cè)，結(jié)果發(fā)現(xiàn)來(lái)曲唑耐藥細(xì)胞中CA2和GJA1表達(dá)顯著上調(diào)，而PGR和CTSD表達(dá)下調(diào)，表明來(lái)曲唑耐藥細(xì)胞中雌激素反應(yīng)性基因的改變與芳香化酶抑制劑耐藥及他莫昔芬耐藥的細(xì)胞有一定相似性，進(jìn)一步證實(shí)了來(lái)曲唑耐藥細(xì)胞中ER對(duì)靶基因的轉(zhuǎn)錄調(diào)節(jié)過(guò)程紊亂。后續(xù)研究需進(jìn)一步闡明來(lái)曲唑耐藥細(xì)胞中ER轉(zhuǎn)錄調(diào)節(jié)過(guò)程紊亂的特征，以鑒定介導(dǎo)來(lái)曲唑耐藥的關(guān)鍵性功能分子，為尋找有效的治療靶點(diǎn)提供依據(jù)。

綜上，來(lái)曲唑耐藥細(xì)胞中ER對(duì)靶基因的轉(zhuǎn)錄調(diào)節(jié)發(fā)生了選擇性改變，在后續(xù)研究中，我們擬通過(guò)染色質(zhì)免疫共沉淀結(jié)合高通量測(cè)序方法(ChIP-Seq)鑒定來(lái)曲唑耐藥細(xì)胞中ER在全基因組中結(jié)合位點(diǎn)的改變，并通過(guò)基于測(cè)序技術(shù)的轉(zhuǎn)錄組學(xué)方法(RNASeq)檢測(cè)、篩選表達(dá)改變的基因，結(jié)合ChIP-Seq和RNA-Seq的結(jié)果分析ER轉(zhuǎn)錄調(diào)節(jié)過(guò)程改變的特征，并鑒定來(lái)曲唑耐藥細(xì)胞中ER的功能性靶基因。

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The mechanism of ligand-independent activation of estrogen receptor in letrozole-resistant breast cancer cells

CHEN Hong-yan, LIU Zhi-hua*
State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
*

, E-mail: liuzh@cicams.ac.cn
This work was supported by PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (2012D19)

ObjectiveTo investigate the functions and mechanisms of transcriptional regulation changes in estrogen receptor (ER) on letrozole resistance in breast cancer cells.MethodsLetrozole resistant cells (MCF-7-LR-1 and MCF-7-LR-2) and control cells (MCF-7-Aro and MCF-7-T) were used in present study. The levels of ER phosphorylation at Ser-118 and expressions of ER in these two groups were determined by Western blotting. The conjugation of ER with XBP-1 enhancer and TFF-1 promoter with or without β-Estradiol (E2) was detected by Chromatin Immuno-coprecipitation (ChIP) assay. The mRNA expression levels of XBP-1 and TFF-1 were determined by quantitative reverse transcription PCR (qRT-PCR) analysis. Furthermore, the mRNA expressions of estrogen responsive genes (ERGs) including CA2, GJA1, PGR, and CTSD were assayed also by qRTPCR analysis.ResultsThe level of ER phosphorylation at Ser-118 significantly increased in MCF-7-LR cells than in control cells. However, the ER expression showed no significant change. ChIP assay demonstrated that, under ligand-independent (without E2 treatment) or ligand-dependent (with E2 treatment 10nmol/L E2 treatment for 45min) condition, the conjugation of ER with XBP-1 enhancer and TFF-1 promoter significantly increased in MCF-7-LR-2 cells than in control cells. qRT-PCR analysis showed that the mRNA expressions of XBP-1 and TFF-1 increased in MCF-7-LR-2 cells compared with those in MCF-7-Aro cells without E2 treatment. Furthermore, the mRNA expressions of CA2 and GJA1 increased, whereas the mRNA expressions of PGR and CTSD decreased in MCF-7-LR-2 cells versus MCF-7-T cells.ConclusionThe level of ER phosphorylation at Ser-118 increased in aligand-independent manner, thereby promoting the transcription of target genes in letrozole resistant breast cancer cells, and it plays an important role in letrozole resistance.

breast neoplasms; receptors, estrogen; drug resistance, neoplasm; X-box-protein 1; trefoil factor 1

R736.8

A

0577-7402(2013)06-0461-06

2013-03-23；

2013-04-14)

(責(zé)任編輯：沈?qū)?

協(xié)和青年基金；中央高校基本科研業(yè)務(wù)費(fèi)專項(xiàng)資金(2012D19)

陳洪巖，醫(yī)學(xué)博士，助理研究員。主要從事腫瘤發(fā)生發(fā)展的分子機(jī)制研究

100021 北京 中國(guó)醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院腫瘤醫(yī)院腫瘤研究所分子腫瘤學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室(陳洪巖、劉芝華)

劉芝華，E-mail：liuzh@cicams.ac.cn