梅麗麗，　史志周

(昆明理工大學(xué)醫(yī)學(xué)院，云南 昆明 650500)

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鈣離子激活的氯離子通道蛋白ANO1在腫瘤發(fā)病機(jī)制中的作用*

梅麗麗，史志周△

(昆明理工大學(xué)醫(yī)學(xué)院，云南 昆明 650500)

惡性腫瘤具有高發(fā)病率和高死亡率的特點(diǎn)，嚴(yán)重威脅人類的健康。盡管人們不斷探索新的腫瘤治療方法，但現(xiàn)階段惡性腫瘤的預(yù)后仍然較差。因此深入研究腫瘤的發(fā)病機(jī)制，鑒定致癌基因，無(wú)論對(duì)于分子標(biāo)志物的開發(fā)還是治療靶點(diǎn)的確定都具有重要意義。近年的研究表明，惡性腫瘤常發(fā)生鈣離子激活氯離子通道蛋白1(anoctamin 1，ANO1)的擴(kuò)增和過(guò)表達(dá)，并且其功能改變與腫瘤的發(fā)生發(fā)展密切相關(guān)，因此本文將綜述ANO1在惡性腫瘤細(xì)胞增殖、轉(zhuǎn)移中的作用機(jī)制，及在分子標(biāo)志物和抗腫瘤治療方面的最新研究進(jìn)展。

1　ANO1的結(jié)構(gòu)與功能

ANO1又稱為跨膜蛋白16A(transmembrane protein 16A，TMEM16A)、DOG1(discovered on gastrointestinal stromal tumor 1)、ORAOV1(oral cancer overexpressed 1)和腫瘤擴(kuò)增及過(guò)表達(dá)序列2(tumor-amplified and overexpressed sequence 2，TAOS2)[1-4]，是鈣離子激活氯離子通道蛋白家族TMEM16A～K或anoctamin 1～10的成員之一[5]。ANO1基因定位于11q13.3，包含26個(gè)外顯子，編碼含8個(gè)跨膜結(jié)構(gòu)域的膜蛋白，其蛋白的N端和C端均面向胞質(zhì)一側(cè)。ANO1蛋白包含多個(gè)鈣離子結(jié)合位點(diǎn)，但準(zhǔn)確的數(shù)目尚待進(jìn)一步研究[14]。ANO1在感覺(jué)傳導(dǎo)、上皮分泌和平滑肌收縮等生理過(guò)程中發(fā)揮重要作用[6-9]，在小鼠心肌細(xì)胞中具有鈣離子激活氯離子通道典型的陰離子轉(zhuǎn)運(yùn)功能特性[10]，同時(shí)ANO1在乳腺癌、頭頸鱗癌和食管鱗癌等多種惡性腫瘤中高頻擴(kuò)增并過(guò)表達(dá)，且與患者的淋巴結(jié)轉(zhuǎn)移和不良預(yù)后相關(guān)[11-13]。功能研究揭示過(guò)表達(dá)ANO1具有促進(jìn)腫瘤細(xì)胞增殖和遷移的作用，而降低ANO1的表達(dá)水平或活性能夠顯著抑制癌細(xì)胞的增殖和轉(zhuǎn)移。由此可見(jiàn)ANO1有可能成為潛在的分子標(biāo)志物和抗腫瘤治療的一個(gè)重要靶點(diǎn)。

2　ANO1在腫瘤中的表達(dá)和調(diào)控機(jī)制

迄今為止，已經(jīng)在胃腸道間質(zhì)瘤、乳腺癌、膀胱癌、頭頸部鱗狀細(xì)胞癌、食管鱗癌、前列腺癌、結(jié)直腸癌和胰腺癌等多種惡性腫瘤中發(fā)現(xiàn)ANO1的過(guò)表達(dá)，并且其過(guò)表達(dá)與多種腫瘤的預(yù)后相關(guān)，在功能上具有促進(jìn)腫瘤細(xì)胞的增殖和轉(zhuǎn)移的作用[14-15]。

在細(xì)胞正常生長(zhǎng)和癌變過(guò)程中，組蛋白去乙酰化酶(histone deacetylase, HDAC)是調(diào)控細(xì)胞增殖及分化的關(guān)鍵酶。最新研究發(fā)現(xiàn)，在腫瘤細(xì)胞中，ANO1受組蛋白去乙?；傅恼{(diào)控[16]。在ANO1高頻擴(kuò)增的惡性腫瘤中，HDAC3抑制劑可以下調(diào)乳腺癌細(xì)胞系YMB-1和前列腺癌細(xì)胞系PC-3中ANO1的表達(dá)，進(jìn)而抑制了鈣離子激活氯離子通道活性和癌細(xì)胞的增殖和遷移[17-18]。鑒于目前panobinostat、vorinostat和romidepsin等組蛋白去乙?；敢种苿┮呀?jīng)用于淋巴瘤等的臨床治療，而ANO1是HDAC下游重要調(diào)控基因，因此ANO1也有可能成為腫瘤治療的新靶點(diǎn)。

3　ANO1對(duì)腫瘤細(xì)胞增殖的影響

ANO1在腫瘤細(xì)胞中發(fā)揮促進(jìn)細(xì)胞增殖的作用。最新的研究表明過(guò)表達(dá)ANO1通過(guò)激活表皮生長(zhǎng)因子受體(epidermal growth factor receptor，EGFR)和鈣調(diào)素依賴蛋白激酶II(calmodulin-dependent protein kinase II，CAMK II)誘導(dǎo)AKT激活和絲裂原活化蛋白激酶(mitogen-activated protein kinases，MAPK)信號(hào)，從而促進(jìn)乳腺癌、頭頸鱗癌和食管鱗癌的進(jìn)展。Duvvuri 等[19]發(fā)現(xiàn)ANO1過(guò)表達(dá)是通過(guò)增加細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2(extracellular signal-regulated kinase 1/2，ERK1/2)激活誘導(dǎo)細(xì)胞周期蛋白D1(cyclin D1，CCND1)表達(dá)促進(jìn)腫瘤細(xì)胞的增殖和腫瘤的生長(zhǎng)。同時(shí)，研究表明ANO1過(guò)表達(dá)誘導(dǎo)RAS-RAF-MEK-ERK通路激活促進(jìn)腫瘤細(xì)胞的增殖。此外，在ANO1過(guò)表達(dá)的乳腺癌細(xì)胞系ZR75-1、HCC1954和MDA-MB-415中，頭頸鱗癌細(xì)胞系(Te11)和食管鱗癌細(xì)胞系(FaDu)中抑制ANO1的表達(dá)，可顯著抑制腫瘤細(xì)胞的增殖[20]。同時(shí)Mazzone等[21]用ANO1的特異性抑制劑T16Ainh-A01(10 μmol/L)作用于ANO1過(guò)表達(dá)的乳腺癌細(xì)胞系YMB-1和前列腺癌細(xì)胞系PC-3，結(jié)果表明癌細(xì)胞的增殖能力被顯著抑制[22-23]。綜上所述，ANO1通過(guò)其氯離子通道蛋白的活性發(fā)揮促進(jìn)腫瘤細(xì)胞增殖的的作用。

目前研究報(bào)道提出2個(gè)可能機(jī)制解釋ANO1通過(guò)氯離子通道活性參與對(duì)細(xì)胞增殖的調(diào)節(jié)。一是氯離子通道蛋白本身可能影響細(xì)胞內(nèi)信號(hào)通路[24]，或是增強(qiáng)細(xì)胞內(nèi)激酶活性而促進(jìn)腫瘤細(xì)胞增殖，例如ANO1通過(guò)激活MAPK直接促進(jìn)腫瘤細(xì)胞的增殖[19]；二是細(xì)胞內(nèi)氯離子濃度的改變會(huì)對(duì)氯離子通道活性產(chǎn)生影響，間接影響腫瘤細(xì)胞增殖，最新研究發(fā)現(xiàn)氯離子濃度有助于腫瘤形成[25-26]。雖然較多的研究表明ANO1可以促進(jìn)多種腫瘤細(xì)胞的增殖，但其詳細(xì)的調(diào)控機(jī)制仍然未被闡明。

4　ANO1在腫瘤轉(zhuǎn)移中的作用

ANO1不但促進(jìn)細(xì)胞細(xì)胞增殖，而且與腫瘤轉(zhuǎn)移也密切相關(guān)。目前研究表明，在頭頸鱗癌、胰腺癌、口腔鱗狀細(xì)胞癌和肺癌中ANO1過(guò)表達(dá)有助于癌細(xì)胞轉(zhuǎn)移[27]。Ayoub等[21]發(fā)現(xiàn)在頭頸鱗癌細(xì)胞系HEP-2中ANO1過(guò)表達(dá)能刺激細(xì)胞運(yùn)動(dòng)、附著、擴(kuò)散、分離和侵襲。Schroeder等[28]和Yang等[29]分別利用膜片鉗技術(shù)證明ANO1具有需鈣離子激活的氯離子通道活性，這一發(fā)現(xiàn)為闡明ANO1在腫瘤轉(zhuǎn)移中的作用指出了新的方向，即ANO1離子通道功能是否參與調(diào)控癌細(xì)胞的轉(zhuǎn)移過(guò)程及其機(jī)制是什么。當(dāng)ANO1過(guò)表達(dá)時(shí)，細(xì)胞膜上的鈣離子和氯離子通道增多，鈣離子和氯離子跨膜轉(zhuǎn)運(yùn)會(huì)增多，從而導(dǎo)致胞內(nèi)滲透壓的節(jié)律變化加快，促進(jìn)了細(xì)胞的遷移。Ruiz等[1]研究發(fā)現(xiàn)在頭頸鱗癌細(xì)胞系BHY和CAL-33中ANO1過(guò)表達(dá)誘導(dǎo)細(xì)胞中鈣離子激活氯離子電流、細(xì)胞運(yùn)動(dòng)和遷移，采用ANO1特異性抑制劑T16Ainh-A01作用后，結(jié)果顯示細(xì)胞的遷移速率明顯下降。ANO6是ANO家族中另一個(gè)離子通道，有研究表明，在艾氏腹水癌細(xì)胞中用siRNA穩(wěn)定沉默ANO1或ANO6，遷移分析表明ANO1沉默改變他們的遷移方向，而ANO6沉默顯示細(xì)胞遷移率降低[30]。因此，該項(xiàng)研究表明ANO1確定了腫瘤細(xì)胞遷移方向，而ANO6則確定腫瘤細(xì)胞遷移的速度。最新研究發(fā)現(xiàn)在胰腺癌細(xì)胞系CFPAC-1和口腔鱗癌細(xì)胞系SCC-25中ANO1過(guò)表達(dá)也促進(jìn)了癌細(xì)胞的轉(zhuǎn)移[22, 31]。因此，ANO1過(guò)表達(dá)有助于腫瘤細(xì)胞轉(zhuǎn)移。

5　ANO1是癌癥治療的潛在靶點(diǎn)

最新的研究表明，在小鼠體內(nèi)移植腫瘤中抑制ANO1的表達(dá)可以降低癌細(xì)胞的轉(zhuǎn)移和侵襲[27]。同時(shí)抑制ANO1的表達(dá)可顯著降低肺癌細(xì)胞的侵襲和轉(zhuǎn)移[32]。通過(guò)對(duì)11萬(wàn)個(gè)化合物的高通量篩選，Namkung等[33]發(fā)現(xiàn)了一種新型的小分子，它作為ANO1的特異性抑制劑被命名為T16Ainh-A01。研究發(fā)現(xiàn)，在癌細(xì)胞增殖過(guò)程中ANO1的特異性抑制劑T16Ainh-A01可以降低癌細(xì)胞的增殖，尤其在頭頸部鱗狀細(xì)胞癌(SCC1)、胰腺癌(CFPAC-1)和前列腺癌(T24)細(xì)胞系中更顯著[20, 24, 27]。但是這個(gè)抑制劑的作用機(jī)理還不清楚。最新的研究表明，艾地苯醌、咪康唑和白花丹醌被確定為ANO1新的抑制劑。艾地苯醌可以抑制ANO1的活性，從而抑制癌細(xì)胞的增殖和誘導(dǎo)癌細(xì)胞凋亡[34]。以上研究結(jié)果表明對(duì)ANO1抑制劑的進(jìn)一步研究將有助于癌癥治療。

6　小結(jié)和展望

大量研究均表明抑制ANO1會(huì)對(duì)某些腫瘤進(jìn)展起到明顯的抑制作用，因此，ANO1有望成為治療腫瘤的新靶點(diǎn)。但目前對(duì)于ANO1如何通過(guò)促進(jìn)腫瘤細(xì)胞增殖和轉(zhuǎn)移進(jìn)而加速腫瘤進(jìn)展的分子機(jī)制了解較少，因此未來(lái)研究的重點(diǎn)是揭示ANO1調(diào)控的下游重要分子，并闡明ANO1調(diào)控腫瘤生長(zhǎng)和轉(zhuǎn)移的分子機(jī)制。相信隨著分子生物學(xué)技術(shù)及實(shí)驗(yàn)條件的不斷發(fā)展，人們對(duì)ANO1將會(huì)有更深入的了解。

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(責(zé)任編輯：林白霜， 羅森)

Effects of ANO1 on tumorigenesis

MEI Li-li, SHI Zhi-zhou

(MedicalFacultyofKunmingUniversityofScienceandTechnology,Kunming650500,China.E-mail:zhizhoushi@126.com)

Anoctamin 1 (ANO1) is a calcium-activated chloride channel and is amplified and over-expressed in gastrointestinal stromal tumor, breast cancer, bladder cancer, head and neck squamous cell cancer, esophageal squamous cell cancer, prostate cancer and pancreatic cancer. The amplification and over-expression of ANO1 are associated with lymph node metastasis and poor prognosis. ANO1 promotes tumor formation and metastasis, and the drugs that inhibit the activity or expression of ANO1 show antitumor effects. Therefore, ANO1 may promote the tumorigenesis, and may be a molecular biomarker and a new target for cancer therapy.

Anoctamin 1； 腫瘤； 增殖； 轉(zhuǎn)移

Anoctamin 1; tumors; Proliferation; Metastasis

1000- 4718(2016)04- 0759- 04

2015- 10- 22

2015- 11- 24

國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 81460425)；云南省應(yīng)用基礎(chǔ)研究計(jì)劃青年項(xiàng)目(No. 2013FD012)

Tel: 0871-65920761; E-mail: zhizhoushi@126.com

R363

A

10.3969/j.issn.1000- 4718.2016.04.029

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