宋新貌(綜述),王勝資(審校)

(復(fù)旦大學(xué)附屬眼耳鼻喉科醫(yī)院放療科，上海 200031)

頭頸腫瘤已成為第6位高發(fā)的腫瘤，全球每年約有65萬(wàn)例新增病例，約35萬(wàn)患者死亡[1]。目前針對(duì)頭頸腫瘤的治療方式主要有手術(shù)、放療、化療，但是頭頸腫瘤的預(yù)后仍然不容樂(lè)觀，復(fù)發(fā)和轉(zhuǎn)移是治療失敗的主要原因。較多研究證實(shí)頭頸腫瘤中表皮生長(zhǎng)因子受體(epithelial growth factor receptor,EGFR)的表達(dá)率高達(dá)90%以上，其突變體表皮生長(zhǎng)因子受體三型突變(EGFR varient Ⅲ,EGFRvⅢ)在頭頸腫瘤也有表達(dá)。EGFR/EGFRvⅢ通過(guò)多種信號(hào)途徑在促進(jìn)腫瘤細(xì)胞增殖、誘導(dǎo)血管新生、提高細(xì)胞侵襲和轉(zhuǎn)移能力、拮抗腫瘤對(duì)化療和放療的敏感方面起著重要作用，因而其過(guò)表達(dá)影響腫瘤的預(yù)后和治療。深入研究發(fā)現(xiàn),這些惡性生物性行為的呈現(xiàn)與腫瘤細(xì)胞發(fā)生的上皮-間質(zhì)化轉(zhuǎn)移(epithelial-mesenchymal transition,EMT)變化有密切的關(guān)系[2]。對(duì)EGFR和EGFRvⅢ的結(jié)構(gòu)及其配體、兩者參與的信號(hào)通路、在腫瘤中的表達(dá)及對(duì)預(yù)后的影響、促進(jìn)EMT變化的機(jī)制和對(duì)治療的影響作如下綜述。

1 EGFR/EGFRvⅢ的結(jié)構(gòu)及其配體

EGFR(ErbB1和Her1)屬于ErbB家族一個(gè)成員，有富含半胱氨酸的細(xì)胞外結(jié)構(gòu)與相應(yīng)的配體相結(jié)合，一個(gè)跨膜結(jié)構(gòu)能將受體錨定在胞膜上，膜內(nèi)區(qū)具有典型的ATP結(jié)合位點(diǎn)及酪氨酸激酶結(jié)構(gòu)。EGFR常見的配體有表皮生長(zhǎng)因子、腫瘤生長(zhǎng)因子α、雙調(diào)蛋白、肝素結(jié)合的表皮生長(zhǎng)因子樣生長(zhǎng)因子、B細(xì)胞生長(zhǎng)因子、表皮調(diào)節(jié)素。

EGFR及其下游信號(hào)系統(tǒng)：Ras/Raf/絲裂原活化蛋白激酶的激酶/細(xì)胞外信號(hào)調(diào)節(jié)激酶/絲裂原活化蛋白激酶通路；磷脂酰肌醇3-激酶/蛋白激酶B/哺乳動(dòng)物雷帕霉素靶蛋白通路；Janus激酶/信號(hào)轉(zhuǎn)導(dǎo)子和轉(zhuǎn)錄激活子3通路；磷酸激酶C-γ1/蛋白激酶C通路；蛋白激酶C-δ通路觸發(fā)效應(yīng)細(xì)胞在增殖、遷移、黏附等正常生理功能中發(fā)揮重要作用。EGFR在腦、肺、乳腺、卵巢、前列腺、胰腺、頭頸等多種腫瘤中過(guò)表達(dá)，參與腫瘤細(xì)胞的增殖、生存、轉(zhuǎn)移及新生血管的形成，改變轉(zhuǎn)錄因子的活性，調(diào)節(jié)細(xì)胞凋亡，調(diào)整細(xì)胞周期，促進(jìn)轉(zhuǎn)移的惡性生物學(xué)行為[3]。

在頭頸腫瘤中常見EGFR基因外顯子2～7區(qū)的缺失突變，這種突變引起801個(gè)堿基對(duì)的缺失和胞膜外區(qū)域結(jié)構(gòu)變短，而產(chǎn)生一個(gè)150×103的EGFRvⅢ蛋白，它不需要配體的激活就能夠自發(fā)磷酸化觸發(fā)胞內(nèi)效應(yīng)[4]。盡管EGFRvⅢ缺乏細(xì)胞外配體結(jié)合區(qū)域，但能刺激下游的信號(hào)途徑有磷脂酰肌醇3-激酶/蛋白激酶B-1而不能激活Ras/Raf/絲裂原活化蛋白激酶[5]。其實(shí)EGFRvⅢ最先在人膠質(zhì)母細(xì)胞瘤中被發(fā)現(xiàn)，后證實(shí)在頭頸、肺、卵巢等多種腫瘤中表達(dá)，但在正常組織中不表達(dá)[6]。EGFRvⅢ在腫瘤的發(fā)生、進(jìn)展中的主要作用包括促進(jìn)細(xì)胞增殖、誘導(dǎo)血管新生、提高腫瘤侵襲和轉(zhuǎn)移能力、拮抗腫瘤細(xì)胞對(duì)化療和放療的敏感性[7]。另外發(fā)現(xiàn)表達(dá)EGFRvⅢ的腫瘤細(xì)胞甚至具有腫瘤干細(xì)胞的特征和自我更新的能力[8]。

2 EGFR/EGFRvⅢ在頭頸腫瘤中的表達(dá)及預(yù)后

EGFR在哺乳動(dòng)物組織中，尤其是在其上皮、間質(zhì)、神經(jīng)源性器官中廣泛表達(dá)，EGFR在上皮性腫瘤(如乳腺癌、前列腺癌、肺癌、神經(jīng)膠質(zhì)瘤)中均廣泛表達(dá)[9]。在頭頸腫瘤中表達(dá)較為常見：87.5%(35/40)的口腔鱗癌[10]，89%(48/54)的鼻咽癌[11]，93.3%(42/45)的喉癌[12]組織均有EGFR的過(guò)表達(dá)，其中68%(44/65)頭頸腫瘤的患者呈高表達(dá)(+2,+3)[13]。不僅蛋白水平有表達(dá)異常，通過(guò)原位雜交技術(shù)發(fā)現(xiàn)80%(82/102)的患者EGFR mRNA上調(diào)，17.2%(11/64)的患者發(fā)生EGFR基因擴(kuò)增[14]。EGFR的高表達(dá)與腫瘤的體積、T分期有密切相關(guān)性，且與腫瘤進(jìn)展有關(guān)[15]；高表達(dá)的EGFR使腫瘤細(xì)胞具有更強(qiáng)的侵襲性表型，而且下調(diào)對(duì)放射和化療的敏感性，顯著縮短疾病進(jìn)展時(shí)間[16]。

EGFRvⅢ不僅存在于神經(jīng)膠質(zhì)瘤，后來(lái)證實(shí)在其他惡性腫瘤，如非小細(xì)胞性肺癌、乳腺癌、卵巢癌中也有表達(dá)、尤其在頭頸部腫瘤中EGFRvⅢ表達(dá)較常見。Szabó等[17]回顧性分析71例頭頸腫瘤患者病理資料，其中15例(21%)有EGFRvⅢ的缺失突變。Sok等[18]認(rèn)為EGFRvⅢ在42%(14/33)的頭頸腫瘤中表達(dá)。EGFRvⅢ的表達(dá)誘發(fā)腫瘤細(xì)胞提高遷移能力和抗凋亡能力也表現(xiàn)對(duì)放療、化療藥物和靶向治療的拮抗性[19-20]。

3 EGFR/EGFRvⅢ促進(jìn)EMT或轉(zhuǎn)移的機(jī)制

EMT使上皮細(xì)胞失去了細(xì)胞極性等上皮表型，獲得了較高的遷移與侵襲等間質(zhì)細(xì)胞表型，其特征性變化：上皮細(xì)胞失去正常立方形態(tài)而變成紡錘性結(jié)構(gòu)；細(xì)胞連接蛋白上皮鈣黏素減少，而間質(zhì)細(xì)胞的標(biāo)志物波形蛋白增加[21]。EMT涉及細(xì)胞間的黏附破壞、細(xì)胞與基質(zhì)間黏附增強(qiáng)、細(xì)胞極性變化、細(xì)胞骨架重組、細(xì)胞外基質(zhì)成分分子合成與聚集變化、細(xì)胞外蛋白酶的上調(diào)與激活等細(xì)胞形態(tài)和功能的變化。

目前，大量證據(jù)已經(jīng)證實(shí)EGFR和EGFRvⅢ能夠誘導(dǎo)腫瘤細(xì)胞發(fā)生EMT樣變化，那么EGFR和EGFRvⅢ是如何誘導(dǎo)腫瘤細(xì)胞EMT變化的呢？主要表現(xiàn)在以下幾個(gè)方面。

3.1調(diào)節(jié)細(xì)胞外基質(zhì)成分 細(xì)胞外基質(zhì)成分是由大分子構(gòu)成的錯(cuò)綜復(fù)雜的網(wǎng)絡(luò)。為細(xì)胞的生成及活動(dòng)提供適宜的場(chǎng)所，并通過(guò)信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)影響細(xì)胞的形狀、代謝、功能、遷移、增殖和分化。細(xì)胞外基質(zhì)成分與生長(zhǎng)因子結(jié)合能影響細(xì)胞的表型和生物學(xué)行為，EGFR和EGFRvⅢ可調(diào)節(jié)纖溶酶原激活物抑制1、尿激酶纖維蛋白溶酶原激活劑和尿激酶型纖溶酶原激活物受體發(fā)揮細(xì)胞的纖維蛋白溶解作用[22]。EGFR可以通過(guò)細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2和磷脂酰肌醇3-激酶途徑增加基質(zhì)金屬蛋白酶9的表達(dá)來(lái)降解細(xì)胞外基質(zhì)成分和上皮鈣黏素使細(xì)胞發(fā)生EMT[23]；表皮生長(zhǎng)因子結(jié)合EGFR后能夠刺激透明質(zhì)酸的生產(chǎn)，透明質(zhì)酸的增加可以導(dǎo)致透明質(zhì)酸依賴的細(xì)胞外基質(zhì)增加，促進(jìn)上皮細(xì)胞的遷移能力及發(fā)生間質(zhì)化形態(tài)學(xué)變化，失去上皮標(biāo)志上皮鈣黏素而獲得間質(zhì)細(xì)胞的特性波形蛋白，另外透明質(zhì)酸與其表面受體CD44結(jié)合使細(xì)胞發(fā)生EMT變化[24]。

3.2重塑細(xì)胞骨架結(jié)構(gòu) EGFR T790M突變的非小細(xì)胞肺癌的細(xì)胞transmembrane 4 L six family member 5(TM4SF5)表達(dá)增加，TM4SF可以和整合素α2、α5協(xié)同作用改變細(xì)胞骨架結(jié)構(gòu)，誘導(dǎo)細(xì)胞發(fā)生EMT變化[25]；轉(zhuǎn)化生長(zhǎng)因子β1和EGFR信號(hào)協(xié)同調(diào)節(jié)TM4SF5的表達(dá)，而TM4SF5能夠增加α平滑肌激動(dòng)蛋白的表達(dá)使細(xì)胞間的黏附下降，因此細(xì)胞獲得EMT樣表型[26]；在宮頸癌細(xì)胞中EGFR過(guò)表達(dá)與上皮鈣黏素下調(diào)，神經(jīng)鈣黏素的上調(diào)呈平行關(guān)系，進(jìn)一步分析發(fā)現(xiàn)，整合素α5β1和細(xì)胞外基質(zhì)纖連蛋白調(diào)整表皮生長(zhǎng)因子誘導(dǎo)的宮頸癌細(xì)胞EMT過(guò)程[27]； Staurosporine(ST)是一種非典型的蛋白激酶C抑制劑，能迅速分解F肌動(dòng)蛋白束，導(dǎo)致細(xì)胞間黏附的不穩(wěn)定，擴(kuò)大EGFR信號(hào)的EMT作用[28]。在頭頸腫瘤細(xì)胞中EGFR能夠激活Rac1交換因子Vav2，激活的Vav2誘導(dǎo)肌動(dòng)蛋白構(gòu)象變化而調(diào)整細(xì)胞骨架的重組，這一系列變化使腫瘤細(xì)胞獲得黏附性、極性、侵襲性[29]。EGFR與CD44相互作用活化磷脂酶ε-Ca2+和Ras/Raf/細(xì)胞外信號(hào)調(diào)節(jié)激酶上調(diào)后激活鈣-鈣調(diào)節(jié)蛋白依賴性激酶Ⅱ使細(xì)絲蛋白磷酸化而增加頭頸腫瘤細(xì)胞的遷移力[30]。

3.3直接上皮鈣黏素的內(nèi)吞和降解 高表達(dá)EGFR的腫瘤細(xì)胞以EGF刺激后，細(xì)胞通過(guò)細(xì)胞質(zhì)膜微囊依賴的機(jī)制內(nèi)吞上皮鈣黏素；EGF刺激后導(dǎo)致β聯(lián)蛋白與上皮鈣黏素解離，一方面直接降解上皮鈣黏素，另一方面β聯(lián)蛋白進(jìn)入核內(nèi)激活T細(xì)胞因子/淋巴增強(qiáng)因子1[31]；在頭頸鱗狀細(xì)胞癌10A細(xì)胞系EGFR激活后通過(guò)基質(zhì)金屬蛋白酶9作用使上皮鈣黏素降解為可溶性的鈣黏素促進(jìn)的遷移和侵襲[23]。Rho家族三磷酸鳥苷酶Cdc42，激活EGFR信號(hào)通路接到Src的激活，引起上皮鈣黏素蛋白泛素化和溶酶體的降解，使細(xì)胞發(fā)生間質(zhì)化表型[32]。在頭頸腫瘤細(xì)胞中發(fā)現(xiàn)上皮鈣黏素的降解反過(guò)來(lái)短暫上調(diào)EGFR的表達(dá)，再次觸發(fā)下游通路的激活[33]。

3.4抑制上皮鈣黏素轉(zhuǎn)錄因子的表達(dá) 調(diào)節(jié)EMT的重要轉(zhuǎn)錄因子包括：snail、slug、E盒結(jié)合鋅指蛋白(zinc finger E-box-binding protein,ZEB)1/2、Twist。EGFR能促進(jìn)Snail的激活，EGFR抑制劑能夠抑制snail mRNA的表達(dá)[34]；EGFR信號(hào)通路使得糖原合成酶激酶3β失活，糖原合成酶激酶3β能磷酸化Snail并促進(jìn)核內(nèi)移和蛋白分解，因此上調(diào)Snail，使細(xì)胞核內(nèi)的Snail增多[27]；以小干擾RNA誘導(dǎo)序列特異性的沉寂EGFR后能終止EGF誘導(dǎo)上皮鈣黏素向神經(jīng)鈣黏蛋白的轉(zhuǎn)換，同時(shí)抑制Snail,Slug和ZEB1表達(dá)[35]，同樣以小干擾RNA誘導(dǎo)序列特異性的沉寂EGFR后人胰腺癌細(xì)胞的遷移和侵襲力受到抑制，上皮標(biāo)志物上皮鈣黏素增加，間質(zhì)標(biāo)志物波形蛋白、神經(jīng)鈣黏蛋白、纖連蛋白減少，Sail和slug的表達(dá)下調(diào)[36]；表皮生長(zhǎng)因子/EGFR信號(hào)通路通過(guò)信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄激活因子3調(diào)節(jié)Twist基因的表達(dá)誘導(dǎo)腫瘤細(xì)胞發(fā)生EMT變化，Twist的表達(dá)可以被EGFR和Janus激酶/信號(hào)轉(zhuǎn)導(dǎo)子和轉(zhuǎn)錄激活子3通路抑制劑所阻斷[37]；EGFRvⅢ陽(yáng)性細(xì)胞通過(guò)wnt/β聯(lián)蛋白途徑激活β聯(lián)蛋白，激活的β聯(lián)蛋白變得穩(wěn)定，轉(zhuǎn)移到細(xì)胞核內(nèi)激活轉(zhuǎn)錄因子T細(xì)胞因子/淋巴增強(qiáng)因子1促進(jìn)細(xì)胞發(fā)生EMT變化[8]；激活的EGFR進(jìn)一步引起核因子κB 的激活預(yù)示腫瘤發(fā)生侵襲，因?yàn)楹艘蜃应蔅可與波形蛋白基因啟動(dòng)子調(diào)節(jié)序列結(jié)合，促進(jìn)Twist表達(dá)，誘導(dǎo)EMT[38]。發(fā)現(xiàn)EGFR和p53突變體協(xié)同作用使富含EMT變化的人類食管癌細(xì)胞的ZEB1/ZEB2表達(dá)增強(qiáng)，抑制p15INK4B和p16INK4A，從而克服EGFR介導(dǎo)的衰老[39]。

4 展 望

目前針對(duì)EGFR的分子靶向治療主要包括單克隆抗體(如西妥昔單抗、尼妥珠單抗)和酪氨酸激酶受體抑制劑(如吉非替尼、厄洛替尼)，已經(jīng)廣泛應(yīng)用于臨床并取得一定療效。也有專門針對(duì)特異性結(jié)合EGFRvⅢ的單克隆抗體的研發(fā)，這種抗體具有較好的抑制腫瘤生長(zhǎng)的作用且不易被正常組織吸收。但是，很多腫瘤患者經(jīng)靶向治療后并沒(méi)有期待的效果，甚至表現(xiàn)對(duì)靶向治療的拮抗。EGFR/EGFRvⅢ引起的EMT變化在加速頭頸腫瘤的侵襲和轉(zhuǎn)移中發(fā)揮重要作用。隨著對(duì)EGFR/EGFRvⅢ分子病理和基因?qū)W水平的深入研究，以及其誘發(fā)EMT的機(jī)制的明確，能夠更加清楚地認(rèn)識(shí)這一變化過(guò)程在腫瘤的發(fā)生、發(fā)展中的作用。針對(duì)EGFR/EGFRvⅢ的抗腫瘤藥物研究和開發(fā)具有廣闊的前景，尤其是針對(duì)EGFRvⅢ的靶向治療藥物，因?yàn)槠鋬H在腫瘤中表達(dá)，那么理論上對(duì)正常組織的不良反應(yīng)更少。

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