田 垚 陳 璐 張 斌 曹旭晨
Runx2轉(zhuǎn)錄因子參與骨骼的形成,在骨骼發(fā)育過程中起重要作用。在乳腺組織中,Runx2也參與某些基因的表達(dá)調(diào)控,在乳腺上皮細(xì)胞分化過程中發(fā)揮重要作用,并與乳腺腫瘤細(xì)胞株的侵襲性密切相關(guān)[1]。Runx2的過表達(dá)與乳腺癌某些特征有關(guān),如在三陰性乳腺癌,Runx2與雌激素受體信號(hào)轉(zhuǎn)導(dǎo)通路之間存在著交互串話,其與乳腺癌細(xì)胞株發(fā)生轉(zhuǎn)移,及其引發(fā)的溶骨性病變有相關(guān)性[2]。本文就Runx2在乳腺癌中的作用綜述如下。
在哺乳動(dòng)物中有3種基因編碼α亞基,因與果蠅Runt基因有同源性,故稱為Runx基因,這3個(gè)基因分別編碼不同的蛋白R(shí)unxl、Runx2、Runx3,它們具有共同的DNA結(jié)合域——runt域。Runx家族基因與腫瘤發(fā)展密切相關(guān)[3],Runx2在乳腺上皮細(xì)胞中發(fā)揮生理性調(diào)節(jié)作用[4],并在乳腺癌細(xì)胞株中表達(dá)上調(diào)[5]。尤其是在侵襲性較高的乳腺癌細(xì)胞株中,Runx2的表達(dá)明顯上調(diào),提示Runx2與乳腺癌細(xì)胞的侵襲性有關(guān)。Runx2還與乳腺癌的轉(zhuǎn)移有關(guān),其可調(diào)節(jié)血管生成相關(guān)基因的表達(dá),如骨涎蛋白(BSP)、骨橋蛋白(OPN)、基質(zhì)金屬蛋白酶(MMPs)和血管內(nèi)皮生長因子(VEGF)[6]。Javed等[7]認(rèn)為這與腫瘤細(xì)胞行為直接相關(guān),抑制Runx2的表達(dá),可使其靶基因的表達(dá)下調(diào),從而降低溶骨細(xì)胞的活性。
Runx2與溶骨性病變關(guān)系密切,乳腺癌細(xì)胞中Runx2的表達(dá)缺失將下調(diào)其抑制成骨細(xì)胞分化,增強(qiáng)破骨細(xì)胞分化的功能[8]。Runx2通過增強(qiáng)核因子-κB(NF-κB)受體活化因子配體(RANKL)的表達(dá)和降低護(hù)骨素的表達(dá),促進(jìn)破骨細(xì)胞的分化,當(dāng)乳腺癌細(xì)胞產(chǎn)生過多的甲狀旁腺激素相關(guān)蛋白(PTHrP)時(shí),則導(dǎo)致骨溶解作用增強(qiáng),Runx2可通過Hedgehog信號(hào)通路調(diào)節(jié)PTHrP的表達(dá)水平。Runx2結(jié)合到印刺因子(IHH)并在乳腺癌細(xì)胞中表達(dá),使PTHrP進(jìn)一步增加,從而增強(qiáng)骨溶解作用。在乳腺癌MDA-MB-231細(xì)胞株中敲除Runx2,可抑制IHH和PTHrP的表達(dá),從而降低溶骨性疾病的發(fā)生率[9]。因此,Runx2在轉(zhuǎn)移瘤細(xì)胞中過表達(dá),通過IHHPTHrP信號(hào)通路作用,使得骨溶解加劇,釋放促生長因子,進(jìn)而影響腫瘤進(jìn)展。此外,Runx2還可調(diào)節(jié)乳腺癌細(xì)胞分泌細(xì)胞因子,如白細(xì)胞介素-11(IL-11)和粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF),增強(qiáng)破骨細(xì)胞的活性[10]。IL-11可誘導(dǎo)MDA-MB-231細(xì)胞中的Runx2過表達(dá)[11],而GM-CSF是刺激破骨細(xì)胞發(fā)育的重要因子,同時(shí)也是NF-κB信號(hào)通路的作用靶點(diǎn)。研究還證實(shí),Runx2在MDA-MB-231細(xì)胞內(nèi)表達(dá),具有抑制成骨細(xì)胞分化的作用[10]。
Runx2的表達(dá)與HER2/ErbB2陽性腫瘤的形成密切相關(guān),并影響雌激素受體陰性腫瘤患者的臨床預(yù)后情況[12]。Khalid等[13]通過對(duì)779例乳腺癌活檢分析,發(fā)現(xiàn)Runx2的表達(dá)與雌激素受體α(ERα)的表達(dá)呈負(fù)相關(guān);同時(shí)刺激MCF7細(xì)胞中的Runx2和雌激素受體,兩者靶基因表達(dá)譜表現(xiàn)為相互拮抗[14]。雌二醇(E2)結(jié)合的ERα直接通過其DNA結(jié)合域與Runx2產(chǎn)生強(qiáng)烈作用,而間接方式由其N末端和配體結(jié)合域完成,受ERα調(diào)節(jié)的Runx2或許在成骨細(xì)胞和乳腺上皮細(xì)胞分化過程中發(fā)揮重要作用。此外,通過軟瓊脂集落形成實(shí)驗(yàn)證明,E2促進(jìn)軟瓊脂集落形成,而Runx2阻斷該過程,Runx2可抑制雌激素受體對(duì)乳腺癌細(xì)胞的作用,而雌激素受體同時(shí)也抑制Runx2的表達(dá)[13]。
Runx2是在MPA小鼠非依賴型乳腺腫瘤上皮細(xì)胞的DNA微陣列研究中最具上調(diào)性的基因,且Runx2在“基底細(xì)胞樣”即雌激素受體陰性腫瘤細(xì)胞株(如MDA-MB-231、MDA-MB-157、HCC38)中過表達(dá)[5]。一項(xiàng)針對(duì)原發(fā)性乳腺癌的全部乳腺組織提取物微陣矩分析顯示,Runx2在乳腺癌細(xì)胞中過表達(dá),提示Runx2與ER/PR/HER2-陰性疾病有關(guān)[2]。
3.1 Runx2的表達(dá)與乳腺癌細(xì)胞侵襲性的關(guān)系 侵襲性和非侵襲性乳腺癌細(xì)胞的對(duì)比研究表明,Runx2是最具上調(diào)性的基因之一[1]。MDA-MB-231細(xì)胞株及MCF7細(xì)胞株內(nèi)Runx2過表達(dá)時(shí),細(xì)胞株的侵襲性增強(qiáng)[15]。Runx2通過Wnt和轉(zhuǎn)化生長因子(TGF)-β信號(hào)轉(zhuǎn)導(dǎo)通路表達(dá),可誘導(dǎo)MCF7細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化(EMT)[16]。TGF-β主要通過經(jīng)典的Smad和旁路絲裂原激活的蛋白激酶(mitogen-activated pro?tein kinase,MAPK)通路傳導(dǎo)信號(hào),而 Runx2正是 Smad和MAPK通路下游的共同作用靶點(diǎn),并且作為轉(zhuǎn)錄調(diào)節(jié)因子在Smad信號(hào)通路中發(fā)揮重要作用。在MCF10A細(xì)胞株的3D培養(yǎng)實(shí)驗(yàn)中發(fā)現(xiàn),Runx2過表達(dá)抑制乳腺小泡正常結(jié)構(gòu)形成,培養(yǎng)結(jié)果顯示細(xì)胞增殖增加,細(xì)胞凋亡減少及基底膜形成障礙,而在MDA-MB-231細(xì)胞株培養(yǎng)實(shí)驗(yàn)中,抑制Runx2表達(dá),可促使乳腺小泡結(jié)構(gòu)恢復(fù)正常[17]。此外,Runx2還可通過磷脂酰肌醇-3羥基激酶/絲氨酸激酶(phosphatidylinositol3-ki?nase,PI3K/Akt)信號(hào)通路激活,Akt磷酸化使Runx2過表達(dá),導(dǎo)致乳腺癌細(xì)胞株侵襲性增強(qiáng)[18]。
E2可拮抗Runx2的促轉(zhuǎn)移作用,并阻礙SNAI2上調(diào),SNAI2是Runx2賦予細(xì)胞轉(zhuǎn)移行為的基因靶點(diǎn)[14],E2通過抑制SNAI2的表達(dá),拮抗Runx2誘導(dǎo)EMT的發(fā)生,最終降低乳腺癌細(xì)胞的侵襲性[16]。Runx2是miR-203的直接靶基因,沉默miR-203可使SNAI2表達(dá)上調(diào),導(dǎo)致乳腺癌細(xì)胞的侵襲性增強(qiáng)[19]。因此,可通過監(jiān)測(cè)原發(fā)性乳腺癌樣本中Runx2/SNAI2的表達(dá)水平,進(jìn)而預(yù)測(cè)癌發(fā)生轉(zhuǎn)移的可能性[20]。
3.2 Runx2與乳腺癌骨轉(zhuǎn)移的關(guān)系 Runx2不僅調(diào)節(jié)成骨細(xì)胞分化,而且與乳腺癌發(fā)生骨轉(zhuǎn)移相關(guān)[3]。Runx2通過調(diào)節(jié)血管生成相關(guān)基因的表達(dá)促發(fā)乳腺癌細(xì)胞發(fā)生骨轉(zhuǎn)移。BSP與導(dǎo)管內(nèi)轉(zhuǎn)移性乳腺癌相關(guān),可能在誘導(dǎo)乳腺癌細(xì)胞定向骨轉(zhuǎn)移過程中發(fā)揮作用,OPN可以介導(dǎo)乳腺癌骨轉(zhuǎn)移中腫瘤細(xì)胞與骨組織表面的連接,并且與骨轉(zhuǎn)移中破骨細(xì)胞引發(fā)骨吸收活性增加相關(guān)。MDA-MB-231細(xì)胞株具高轉(zhuǎn)移性并可引發(fā)溶骨性病變,Barnes等[21]通過小鼠移植模型實(shí)驗(yàn)證明,下調(diào)MDA-MB-231細(xì)胞內(nèi)Runx2的表達(dá),可抑制溶骨性疾病的發(fā)生;在小鼠的脛骨髓腔內(nèi)注射MDA-MB-231細(xì)胞后,有超過80%的小鼠出現(xiàn)了溶骨性病變,而下調(diào)Runx2的表達(dá)后,小鼠出現(xiàn)溶骨性病變的概率只有5%。Runx2?230即Runx2的靶基因表達(dá)下調(diào),在顯性失活蛋白R(shí)unx2?230所表達(dá)的MDAMB-231細(xì)胞株中,BSP的啟動(dòng)子活性降低50%。在MDAMB-231細(xì)胞株培養(yǎng)實(shí)驗(yàn)中發(fā)現(xiàn),與成骨細(xì)胞相關(guān)的礦化結(jié)節(jié)形成減少,同時(shí)成骨細(xì)胞標(biāo)志物,如堿性磷酸酶、BSP、骨鈣素、Ⅰ型膠原等的表達(dá)缺乏,提示了該細(xì)胞株可抑制來源于骨髓間充質(zhì)干細(xì)胞(MSCs)的成骨細(xì)胞分化,而破骨細(xì)胞標(biāo)志物抗酒石酸酸性磷酸酶的表達(dá)增加,提示該細(xì)胞株可誘導(dǎo)破骨細(xì)胞分化。但是,在Runx2?230所表達(dá)的MDA-MB-231細(xì)胞株中,以上骨細(xì)胞分化效應(yīng)并未出現(xiàn)。研究表明,在MDA-MB-231細(xì)胞株中,Runx2調(diào)節(jié)OPN基因的表達(dá),并可持續(xù)激活BSP使其表達(dá)異常,抑制Runx2的表達(dá)可導(dǎo)致OPN和BSP的表達(dá)明顯下調(diào),提示Runx2在乳腺癌轉(zhuǎn)移中發(fā)揮著重要作用[12]。
MMP-3是一種具有侵襲性的MMPs,它是Runx2的靶基因。染色質(zhì)免疫沉淀(ChIP)分析提示,在MDA-MB-231細(xì)胞中,位于內(nèi)源性MMP-3啟動(dòng)子內(nèi)的2個(gè)Runx位點(diǎn)被Runx2占用,任一Runx位點(diǎn)的突變,均可抑制MMP-3啟動(dòng)子的活性[8]。此外,Runx2亦可調(diào)節(jié)MMPs家族中的其他基因,在MCF-7細(xì)胞株中,Runx2的過表達(dá)可明顯增強(qiáng)MCF-7細(xì)胞株的侵襲能力,并可提高若干轉(zhuǎn)移標(biāo)志性分子,如MMP-2、MMP-9、MMP-13和VEGF的表達(dá)[16]。在MDA-MB-231中,利用小干擾RNA沉默Runx2表達(dá)可降低MMP-9的表達(dá),并減弱癌細(xì)胞的侵襲能力,提示Runx2與乳腺癌轉(zhuǎn)移密切相關(guān)[15]。
Runx2在細(xì)胞核內(nèi)子域的正確定位對(duì)轉(zhuǎn)移性乳腺癌細(xì)胞的溶骨活性所需靶基因的表達(dá)具有重要影響[7]。Runx2的C-端區(qū)域包含一個(gè)核基質(zhì)靶向信號(hào)(NMTS),Runx2通過該信號(hào)與核基質(zhì)聯(lián)系,若該序列發(fā)生點(diǎn)突變(R398A和Y428A),Runx2與核基質(zhì)間的聯(lián)系將被阻斷,提示乳腺癌細(xì)胞誘發(fā)骨溶解與Runx2改變細(xì)胞核內(nèi)轉(zhuǎn)運(yùn)相關(guān)。當(dāng)MDA-MB-231細(xì)胞中Runx2發(fā)生NMTS突變時(shí),其與核基質(zhì)中間纖維的聯(lián)系中斷。Runx2核基質(zhì)靶向信號(hào)對(duì)成骨細(xì)胞分化的作用影響,與顯性失活蛋白R(shí)unx2?230所產(chǎn)生的效應(yīng)類似[21]。Runx2的突變抑制了MDA-MB-231細(xì)胞的侵襲性和溶骨性,導(dǎo)致其基底膜遷移能力降低,且體內(nèi)溶骨性病變減少[9]。通過對(duì)Runx2在MDA-MB-231細(xì)胞株中的作用分析,Runx2在乳腺癌細(xì)胞影響成骨分化,提示Runx2過表達(dá)可促使乳腺癌發(fā)生骨轉(zhuǎn)移[9]。
Runx2在乳腺癌的進(jìn)展和轉(zhuǎn)移中的作用機(jī)制仍存爭(zhēng)議。但多數(shù)學(xué)者認(rèn)為,Runx2能夠調(diào)節(jié)一系列基因的轉(zhuǎn)錄進(jìn)而影響乳腺癌細(xì)胞的侵襲能力,在乳腺癌發(fā)生骨轉(zhuǎn)移的過程中起重要作用,且與其預(yù)后密切相關(guān)。
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