EGFR/EGFRvⅢ促頭頸腫瘤上皮-間質(zhì)轉(zhuǎn)移的研究

宋新貌(綜述),王勝資(審校)

(復旦大學附屬眼耳鼻喉科醫(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65萬例新增病例，約35萬患者死亡[1]。目前針對頭頸腫瘤的治療方式主要有手術、放療、化療，但是頭頸腫瘤的預后仍然不容樂觀，復發(fā)和轉(zhuǎn)移是治療失敗的主要原因。較多研究證實頭頸腫瘤中表皮生長因子受體(epithelial growth factor receptor,EGFR)的表達率高達90%以上，其突變體表皮生長因子受體三型突變(EGFR varient Ⅲ,EGFRvⅢ)在頭頸腫瘤也有表達。EGFR/EGFRvⅢ通過多種信號途徑在促進腫瘤細胞增殖、誘導血管新生、提高細胞侵襲和轉(zhuǎn)移能力、拮抗腫瘤對化療和放療的敏感方面起著重要作用，因而其過表達影響腫瘤的預后和治療。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)變化有密切的關系[2]。對EGFR和EGFRvⅢ的結(jié)構(gòu)及其配體、兩者參與的信號通路、在腫瘤中的表達及對預后的影響、促進EMT變化的機制和對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家族一個成員，有富含半胱氨酸的細胞外結(jié)構(gòu)與相應的配體相結(jié)合，一個跨膜結(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)合位點及酪氨酸激酶結(jié)構(gòu)。EGFR常見的配體有表皮生長因子、腫瘤生長因子α、雙調(diào)蛋白、肝素結(jié)合的表皮生長因子樣生長因子、B細胞生長因子、表皮調(diào)節(jié)素。

EGFR及其下游信號系統(tǒng)：Ras/Raf/絲裂原活化蛋白激酶的激酶/細胞外信號調(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/哺乳動物雷帕霉素靶蛋白通路；Janus激酶/信號轉(zhuǎn)導子和轉(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)效應細胞在增殖、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表達，參與腫瘤細胞的增殖、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)細胞凋亡，調(diào)整細胞周期，促進轉(zhuǎn)移的惡性生物學行為[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個堿基對的缺失和胞膜外區(qū)域結(jié)構(gòu)變短，而產(chǎn)生一個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[4]。盡管EGFRvⅢ缺乏細胞外配體結(jié)合區(qū)域，但能刺激下游的信號途徑有磷脂酰肌醇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EGFRvⅢ最先在人膠質(zhì)母細胞瘤中被發(fā)現(xiàn)，后證實在頭頸、肺、卵巢等多種腫瘤中表達，但在正常組織中不表達[6]。EGFRvⅢ在腫瘤的發(fā)生、進展中的主要作用包括促進細胞增殖、誘導血管新生、提高腫瘤侵襲和轉(zhuǎn)移能力、拮抗腫瘤細胞對化療和放療的敏感性[7]。另外發(fā)現(xiàn)表達EGFRvⅢ的腫瘤細胞甚至具有腫瘤干細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及預后

EGFR在哺乳動物組織中，尤其是在其上皮、間質(zhì)、神經(jīng)源性器官中廣泛表達，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[9]。在頭頸腫瘤中表達較為常見：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的過表達，其中68%(44/65)頭頸腫瘤的患者呈高表達(+2,+3)[13]。不僅蛋白水平有表達異常，通過原位雜交技術發(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基因擴增[14]。EGFR的高表達與腫瘤的體積、T分期有密切相關性，且與腫瘤進展有關[15]；高表達的EGFR使腫瘤細胞具有更強的侵襲性表型，而且下調(diào)對放射和化療的敏感性，顯著縮短疾病進展時間[16]。

EGFRvⅢ不僅存在于神經(jīng)膠質(zhì)瘤，后來證實在其他惡性腫瘤，如非小細胞性肺癌、乳腺癌、卵巢癌中也有表達、尤其在頭頸部腫瘤中EGFRvⅢ表達較常見。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]認為EGFRvⅢ在42%(14/33)的頭頸腫瘤中表達。EGFRvⅢ的表達誘發(fā)腫瘤細胞提高遷移能力和抗凋亡能力也表現(xiàn)對放療、化療藥物和靶向治療的拮抗性[19-20]。

3 EGFR/EGFRvⅢ促進EMT或轉(zhuǎn)移的機制

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

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

3.1調(diào)節(jié)細胞外基質(zhì)成分 細胞外基質(zhì)成分是由大分子構(gòu)成的錯綜復雜的網(wǎng)絡。為細胞的生成及活動提供適宜的場所，并通過信號轉(zhuǎn)導系統(tǒng)影響細胞的形狀、代謝、功能、遷移、增殖和分化。細胞外基質(zhì)成分與生長因子結(jié)合能影響細胞的表型和生物學行為，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)揮細胞的纖維蛋白溶解作用[22]。EGFR可以通過細胞外信號調(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的表達來降解細胞外基質(zhì)成分和上皮鈣黏素使細胞發(fā)生EMT[23]；表皮生長因子結(jié)合EGFR后能夠刺激透明質(zhì)酸的生產(chǎn)，透明質(zhì)酸的增加可以導致透明質(zhì)酸依賴的細胞外基質(zhì)增加，促進上皮細胞的遷移能力及發(fā)生間質(zhì)化形態(tài)學變化，失去上皮標志上皮鈣黏素而獲得間質(zhì)細胞的特性波形蛋白，另外透明質(zhì)酸與其表面受體CD44結(jié)合使細胞發(fā)生EMT變化[24]。

3.2重塑細胞骨架結(jié)構(gòu) EGFR T790M突變的非小細胞肺癌的細胞transmembrane 4 L six family member 5(TM4SF5)表達增加，TM4SF可以和整合素α2、α5協(xié)同作用改變細胞骨架結(jié)構(gòu)，誘導細胞發(fā)生EMT變化[25]；轉(zhuǎn)化生長因子β1和EGFR信號協(xié)同調(diào)節(jié)TM4SF5的表達，而TM4SF5能夠增加α平滑肌激動蛋白的表達使細胞間的黏附下降，因此細胞獲得EMT樣表型[26]；在宮頸癌細胞中EGFR過表達與上皮鈣黏素下調(diào)，神經(jīng)鈣黏素的上調(diào)呈平行關系，進一步分析發(fā)現(xiàn)，整合素α5β1和細胞外基質(zhì)纖連蛋白調(diào)整表皮生長因子誘導的宮頸癌細胞EMT過程[27]； Staurosporine(ST)是一種非典型的蛋白激酶C抑制劑，能迅速分解F肌動蛋白束，導致細胞間黏附的不穩(wěn)定，擴大EGFR信號的EMT作用[28]。在頭頸腫瘤細胞中EGFR能夠激活Rac1交換因子Vav2，激活的Vav2誘導肌動蛋白構(gòu)象變化而調(diào)整細胞骨架的重組，這一系列變化使腫瘤細胞獲得黏附性、極性、侵襲性[29]。EGFR與CD44相互作用活化磷脂酶ε-Ca2+和Ras/Raf/細胞外信號調(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使細絲蛋白磷酸化而增加頭頸腫瘤細胞的遷移力[30]。

3.3直接上皮鈣黏素的內(nèi)吞和降解 高表達EGFR的腫瘤細胞以EGF刺激后，細胞通過細胞質(zhì)膜微囊依賴的機制內(nèi)吞上皮鈣黏素；EGF刺激后導致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)蛋白進入核內(nèi)激活T細胞因子/淋巴增強因子1[31]；在頭頸鱗狀細胞癌10A細胞系EGFR激活后通過基質(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的遷移和侵襲[23]。Rho家族三磷酸鳥苷酶Cdc42，激活EGFR信號通路接到Src的激活，引起上皮鈣黏素蛋白泛素化和溶酶體的降解，使細胞發(fā)生間質(zhì)化表型[32]。在頭頸腫瘤細胞中發(fā)現(xiàn)上皮鈣黏素的降解反過來短暫上調(diào)EGFR的表達，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 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Snail的激活，EGFR抑制劑能夠抑制snail mRNA的表達[34]；EGFR信號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并促進核內(nèi)移和蛋白分解，因此上調(diào)Snail，使細胞核內(nèi)的Snail增多[27]；以小干擾RNA誘導序列特異性的沉寂EGFR后能終止EGF誘導上皮鈣黏素向神經(jīng)鈣黏蛋白的轉(zhuǎn)換，同時抑制Snail,Slug和ZEB1表達[35]，同樣以小干擾RNA誘導序列特異性的沉寂EGFR后人胰腺癌細胞的遷移和侵襲力受到抑制，上皮標志物上皮鈣黏素增加，間質(zhì)標志物波形蛋白、神經(jīng)鈣黏蛋白、纖連蛋白減少，Sail和slug的表達下調(diào)[36]；表皮生長因子/EGFR信號通路通過信號轉(zhuǎn)導與轉(zhuǎn)錄激活因子3調(diào)節(jié)Twist基因的表達誘導腫瘤細胞發(fā)生EMT變化，Twist的表達可以被EGFR和Janus激酶/信號轉(zhuǎn)導子和轉(zhuǎn)錄激活子3通路抑制劑所阻斷[37]；EGFRvⅢ陽性細胞通過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)移到細胞核內(nèi)激活轉(zhuǎn)錄因子T細胞因子/淋巴增強因子1促進細胞發(fā)生EMT變化[8]；激活的EGFR進一步引起核因子κB 的激活預示腫瘤發(fā)生侵襲，因為核因子κB可與波形蛋白基因啟動子調(diào)節(jié)序列結(jié)合，促進Twist表達，誘導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胞的ZEB1/ZEB2表達增強，抑制p15INK4B和p16INK4A，從而克服EGFR介導的衰老[39]。

4 展 望

目前針對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)廣泛應用于臨床并取得一定療效。也有專門針對特異性結(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)靶向治療后并沒有期待的效果，甚至表現(xiàn)對靶向治療的拮抗。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的機制的明確，能夠更加清楚地認識這一變化過程在腫瘤的發(fā)生、發(fā)展中的作用。針對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EGFRvⅢ的靶向治療藥物，因為其僅在腫瘤中表達，那么理論上對正常組織的不良反應更少。

[1] Ferlay J,Shin HR,Bray F,etal.Estimates of worldwide burden of cancer in 2008:GLOBOCAN 2008[J].Int J Cancer,2010,127(12):2893-2917.

[2] Misra A,Pandey C,Sze SK,etal.Hypoxia activated EGFR signaling induces epithelial to mesenchymal transition (EMT)[J].PLoS One,2012,7(11):e49766.

[3] Vecchione L,Jacobs B,Normanno N,etal.EGFR-targeted therapy[J].Exp Cell Res,2011,317(19):2765-2771.

[4] Gan HK,Burgess AW,Clayton AH,etal.Targeting of a conformationally exposed, tumor-specific epitope of EGFR as a strategy for cancer therapy[J].Cancer Res,2012,72(12):2924-2930.

[5] Mukherjee B,Mcellin B,Camacho CV,etal.EGFRvIII and DNA double-strand break repair: a molecular mechanism for radioresistance in glioblastoma[J].Cancer Res,2009,69(10):4252-4259.

[6] Nedergaard MK,Hedegaard CJ,Poulsen HS.Targeting the epidermal growth factor receptor in solid tumor malignancies[J].BioDrugs,2012,26(2):83-99.

[7] Wnorowski AM,De Souza A,Chachoua A,etal.The management of EGFR inhibitor adverse events: a case series and treatment paradigm[J].Int J Dermatol,2012,51(2):223-232.

[8] Del Vecchio CA,Jensen KC,Nitta RT,etal.Epidermal growth factor receptor variant III contributes to cancer stem cell phenotypes in invasive breast carcinoma[J].Cancer Res,2012,72(10):2657-2671.

[9] Ri N,Jm G,Me H.EGFR and cancer prognosis[J].Eur J Cancer,2001,37(Suppl 4):S9-S15.

[10] Sarkis SA,Abdullah BH,Majeed BA,etal.Immunohistochemical expression of epidermal growth factor receptor (EGFR) in oral squamous cell carcinoma in relation to proliferation,apoptosis,angiogenesis and lymphangiogenesis[J].Head Neck Oncol,2010,2(13):1-8.

[11] Chua DT,Nicholls JM,Sham JS,etal.Prognostic value of epidermal growth factor recrptor expression in patients with advanced stage nasopharyngeal carcinoma treated with induciton chemotherapy and radiotherapy[J].Int J Radiation Oncology Biol Phys,2004,59(1):11-20.

[12] Starska K,Glowacka E,Lewy-Trenda I,etal.EGFR immunoexpression and peripheral blood cytokine secretion as potential biomarkers of tumor behavior in laryngeal squamous cell carcinoma[J].Med Sci Monit,2009,15(10):CR518-CR527.

[13] Sheikh Ali MA,Gunduz M,Nagatsuka H,etal.Expression and mutation analysis of epidermal growth factor receptor in head and neck squamous cell carcinoma[J].Cancer Sci,2008,99(8):1589-1594.

[14] Pectasides E,Rampias T,Kountourakis P,etal.Comparative prognostic value of epidermal growth factor quantitative protein expression compared with FISH for head and neck squamous cell carcinoma[J].Clin Cancer Res,2011,17(9):2947-2954.

[15] Leong JL,Loh KS,Putti TC,etal.Epidermal growth factor receptor in undifferentiated carcinoma of the nasopharynx[J].Laryngoscope,2004,114(1):153-157.

[16] Tinhofer I,Klinghammer K,Weichert W,etal.Expression of amphiregulin and EGFRvIII affect outcome of patients with squamous cell carcinoma of the head and neck receiving cetuximab-docetaxel treatmen[J].Clin Cancer Res,2011,17(15):5197-5204.

[17] Szabó B,Nelhubel GA,Kárpáti A,etal.Clinical signi cance of genetic alterations and expression of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas[J].Oral Oncology,2011,47(46):487-496.

[18] Sok JC,Coppelli F,Thomas S,etal.Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targeting[J].Clin Cancer Res,2006,12(17):5064-5073.

[19] Wheeler SE,Suzuki S,Thomas SM,etal.Epidermal growth factor receptor variant Ⅲ mediates head and neck cancer cell invasion via STAT3 activation[J].Oncogene,2010,29(37):5135-5145.

[20] Wang H,Jiang H,Zhou M,etal. Epidermal growth factor receptor variant Ⅲ enhances tumorigenicity and resistance to 5-fluorouracil in human hepatocellar carcinoma[J].Cancer Lett,2009,279(1):30-38.

[21] Klymkowsky MW,Savagner P.Epithelial-mesenchymal transition:a cancer researcher′s conceptual friend and foe[J].Am J Pathol,2009,174(5):1588-1593.

[22] Garnier D,Magnus N,D′asti E,etal.PL-05 genetic pathways linking hemostasis and cancer[J].Thromb Res,2012,129(Suppl 1):s22-s29.

[23] Zuo JH,Zhu W,Li MY,etal.Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin[J].J Cell Biochem,2011,112(9):2508-2517.

[24] Chow G,Tauler J,Mulshine JL.Cytokines and growth factors stimulate hyaluronan production:role of hyaluronan in epithelial to mesenchymal-like transition in non-small cell lung cancer[J].J Biomed Biotechnol,2010,2010:485468

[25] Lee MS,Kim HP,Kim TY,etal.Gefitinib resistance of cancer cells correlated with TM4SF5-mediated epithelial-mesenchymal transition[J].Biochim Biophys Acta,2012,1823(2):514-523.

[26] Kang M,Choi S,Jeong SJ,etal.Cross-talk between TGFbeta1 and EGFR signalling pathways induces TM4SF5 expression and epithelial-mesenchymal transition[J].Biochem J,2012,443(3):691-700.

[27] Lee MY,Chou CY,Tang MJ,etal.Epithelial-mesenchymal transition in cervical cancer: correlation with tumor progression, epidermal growth factor receptor overexpression, and snail up-regulation[J].Clin Cancer Res,2008,14(15):4743-4750.

[28] Hugo HJ,Wafai R,Blick T,etal.Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction-a model for cross-modulation[J].BMC Cancer,2009,9:235.

[29] Patel V,Rosenfeldt HM,Lyons R,etal.Persistent activation of Rac1 in squamous carcinomas of the head and neck:evidence for an EGFR/Vav2 signaling axis involved in cell invasion[J].Carcinogenesis,2007,28(6):1145-1152.

[30] Bourguignon LY,Gilad E,Brightman A,etal.Hyaluronan-CD44interaction with leukemia-associated RhoGEF and epidermal growth factor receptor promotes Rho/Ras co-activation, phospholipase C epsilon-Ca2+signaling,and cytoskeleton modification in head and neck squamous cell carcinoma cells[J].J Biol Chem,2006,281(20):14026-14040.

[31] Lu Z,Ghosh S,Wang Z,etal.Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of β-catenin, and enhanced tumor cell invasion[J].Cancer Cell,2003,(6):499-515.

[32] Shen Y,Hirsch DS,Sasiela CA,etal.Cdc42 regulates E-cadherin ubiquitination and degradation through an epidermal growth factor receptor to src-mediated pathway[J].J Biol Chem,2008,283(8):5127-5137.

[33] Wang D,Su L,Huang D,etal.Downregulation of E-cadherin enhances proliferation of head and neck cancer through transcriptional regulation of EGFR[J].Mol Cancer,2011,10:116.

[34] Forsyth CB,Tang Y,Shaikh M,etal.Alcohol stimulates activation of Snail, epidermal growth factor receptor signaling,and biomarkers of epithelial-mesenchymal transition in colon and breast cancer cells[J].Alcohol Clin Exp Res,2010,34(1):19-31.

[35] Cheng JC,Auersperg N,Leung PC.EGF-induced EMT and invasiveness in serous borderline ovarian tumor cells: a possible step in the transition to low-grade serous carcinoma cells?[J].PLoS One,2012,7(3):e34071.

[36] Chang ZG,Wei JM,Qin CF,etal.Suppression of the epidermal growth factor receptor inhibits epithelial-mesenchymal transition in human pancreatic cancer PANC-1 cells[J].Dig Dis Sci,2012,57(5):1181-1189.

[37] Lo HW,Hsu SC,Xia W,etal.Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression[J].Cancer Res,2007,67(19):9066-9076.

[38] Gavert N,Ben-Ze′e A.Epithelial-mesenchymal transition and the invasive potential of tumors[J].Trends Mol Med,2008,14(5):199-209.

[39] Ohashi S,Natsuizaka M,Wong GS,etal.Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors[J].Cancer Res,2010,70(10):4174-4184.