肺腺癌中新抗凋亡因子的表現與臨床預後

Expression of Novel Anti-Apoptotic Factors Associated with Clinical Outcome in Lung Adenocarcinoma

方信元(Hsin-Yuan Fang)

指導教授 ： 柯文哲 ； 楊偉勛

共同指導教授 ： 林清淵(Ching-Yuang Lin)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.02416

摘要

肺癌(lung cancer)是一個全球與台灣最常見的惡性疾病，台灣的肺癌死亡比率在四十年來增加近12倍。2009年台灣因肺癌而死亡的病患中，男性與女性皆居第一位，肺癌的盛行率在台灣迅速地增加，增長率大概是全世界最高，男性與女性的比率大致保持在2:1左右。肺癌的組織學型態可分為非小細胞肺癌和小細胞肺癌，非小細胞肺癌包括鱗狀上皮癌、腺癌、大細胞癌等。根據衛生署統計，台灣肺腺癌(lung adenocarcinoma)的數目與比例一直持續在增加中，已經超越鱗狀上皮癌，尤其在女性病患當中。台灣肺腺癌(lung adenocarcinoma)也與國外有所不同，根據國衛院研究發現，台灣肺腺癌病患有達55%具有表皮生長因子受體的基因突變，較國外高出許多，同時台灣肺腺癌病患接受化學治療或標靶治療的臨床反應，預期效果也比國外好。這顯示台灣的肺腺癌的致病機轉與疾病行為，可能與國外有明顯不同，進一步了解肺腺癌細胞的存活、增殖、凋亡、局部侵犯及遠端轉移的機轉，在臨床上就顯得相當重要，藉由這方面的研究，以期進而影響與改善肺腺癌病患的存活率。
細胞凋亡(apoptosis)是細胞死亡方法的一種，對於癌細胞的生長、分化、死亡扮演重要關鍵角色，癌細胞為了增加其存活率，適應生存環境，避免被身體免疫細胞殺死，發展出一套對抗細胞凋亡的方法，避免細胞死亡。在真核細胞生物中，粒線體(mitochondria)是細胞內供應能量及調控細胞生死的重要胞器，細胞凋亡發生時，粒線體藉由釋放凋亡誘導因子(apoptosis induced factor; AIF)與細胞色素C (cytochrome C; cyt C)進入細胞質與細胞核，分解細胞核內去氧核糖核酸 (deoxyribonucleic acid; DNA)，使細胞走向凋亡一途。因此癌細胞發展出一套穩定粒線體的方法，在環境不良的情況下，預防細胞凋亡的產生，使癌細胞能存活下來。根據我們之前的研究與文獻回顧顯示，凋亡誘導因子蛋白在肺腺癌細胞凋亡中，扮演相當重要的關鍵角色。
腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATPase family, AAA domain containing 3A; ATAD3A)屬於AAA家族，具有腺嘌呤核苷三磷酸酶(adenosine triphosphatase; ATP)活性，分布在內質網(endoplasmic reticulum; ER)與粒線體(mitochondria)膜狀部上，與促使粒線體分裂相關蛋白1 (dynamin-related protein 1; DRP1)、粒線體融合蛋白2 (mitofusin-2)、第一型視神經萎縮蛋白(optic atrophy type 1; OPA1)一起將凋亡誘導因子(AIF)從內質網，形成運輸小泡，再將凋亡誘導因子(AIF)運輸到粒線體中，這些蛋白與粒線體的分裂，融合與運輸有關，腺嘌呤核苷三磷酸酶家族AAA區域包含3A同時可穩定粒線體，避免凋亡因誘導子與細胞色素C釋放到細胞質中，進而避免細胞凋亡。第一型視神經萎縮蛋白為Dynamin家族成員之一，是參與粒線體融合的重要蛋白；也與粒線體依賴的細胞凋亡機制有關。半胱氨酸蛋白酶14(caspase-14; casp-14)屬於半胱氨酸蛋白酶家族成員之ㄧ，分布在細胞質與細胞核中，並不在粒線體內，在蛋白質交互作用分析中顯示，半胱氨酸蛋白酶14會與凋亡誘導因子(AIF)產生交互作用，一但凋亡誘導因子(AIF)從粒線體中被釋放出來，半胱氨酸蛋白酶14會與凋亡誘導因子(AIF)結合，抑制凋亡誘導因子的(AIF)活性，進而避免細胞凋亡，使細胞存活下來。由以上現象，我們推測腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATAD3A)，第一型視神經萎縮蛋白(OPA1)與半胱氨酸蛋白酶14(casp-14)都會影響凋亡誘導因子在粒線體與細胞質的活性與表現，腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATAD3A)與第一型視神經萎縮蛋白(OPA1)並在粒線體生理功能調控上也佔有重要的地位，這三種蛋白皆與肺腺癌細胞的凋亡，存在明顯相關。
本研究主要探討項目包括：腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATAD3A)，第一型視神經萎縮蛋白(OPA1)與半胱氨酸蛋白酶14(casp-14)如何參與肺腺癌細胞的凋亡、其所在細胞內分布位置、與粒線體及凋亡誘導因子(AIF)的關係，如何磷酸化與作用、改變細胞內包器形態、運輸方式，影響細胞對化學治療藥物以及與肺腺癌病患臨床預後之間的相關。本研究以肺腺癌細胞株與肺腺癌病患手術後肺部組織檢體為材料，我們利用逆轉錄聚合酶連鎖反應方法偵測訊息核糖核酸(messenger RNA; mRNA)在肺腺癌細胞與組織檢體的表現，進一步利用蛋白表現系統，生產重組蛋白來製備抗體，再利用西方墨點法偵測蛋白在肺腺癌細胞株及病患檢體中的表現，以細胞免疫螢光染色與組織免疫染色觀察，腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATAD3A)在細胞分布的主要位置為粒線體與內質網，第一型視神經萎縮蛋白(OPA1)為粒線體，半胱氨酸蛋白酶14(casp-14)在細胞分布的位置為細胞質與細胞核，並用電子顯微鏡觀察粒線體與內質網型態，與腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATAD3A)表現量有關的變化，同時證明腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATAD3A)與半胱氨酸蛋白酶14(casp-14)表現量與病患臨床上的預後有明顯相關。再利用短干擾核糖核酸(small interfering RNA; siRNA)將腺嘌呤核苷三磷酸酶家族AAA區域包含3A(ATAD3A)與半胱氨酸蛋白酶14(casp-14)的表現降低，會影響肺腺癌細胞在順鉑(cisplatin)中的耐受度，進而推估與臨床化學治療的抗藥性有關，本實驗更證明腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATAD3A)、第一型視神經萎縮蛋白(OPA1)與半胱氨酸蛋白酶14(casp-14)有磷酸化的情形，與其蛋白穩定度及功能有關。同時發現，腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATAD3A)會與粒腺體相關蛋白粒線體分裂相關蛋白1(mitofusin-1)，粒線體融合蛋白2(mitofusin-2)、第一型視神經萎縮蛋白(OPA1)與產生交互作用。
綜合以上結果顯示，本研究證明腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白(ATAD3A)、第一型視神經萎縮蛋白(OPA1)與半胱氨酸蛋白酶14(casp-14)為肺腺癌中新的抗凋亡因子，並與臨床預後有高度相關，希望經由此發現，進一步能影響肺腺癌的臨床判斷，改進治療效果，增進肺腺癌病患預後。

關鍵字

肺癌 ； 肺腺癌 ； 細胞凋亡 ； 粒腺體 ； 腺嘌呤核苷三磷酸酶家族AAA區域包含3A蛋白 ； 第一型視神經萎縮蛋白 ； 半胱氨酸蛋白酶14

並列摘要

Lung cancer is one of the most frequent malignant disease in Taiwan as well as in the world. The mortality of lung cancer in Taiwan increases nearly 12 times within recent 40 years. In 2009, lung cancer occupied the first position of cancer death in both male and female in Taiwan. The incidence rate of lung cancer in Taiwan is increasing rapidly and probably is the highest in the world. The male and female ratio in lung cancer maintains approximately about 2:1.The histological type of lung cancer can be divided into the small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) which includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. According to the annual statistical report of Department of Health in Taiwan, the number of lung adenocarcinoma continuously increases and has already surmounted the number of squamous cell carcinoma, especially in female. The behavior of lung adenocarcinoma in Taiwan is different from that in other countries. According to the studies from National Health Research Institutes in Taiwan, the mutation rate in epidermis growth factor receptor gene in lung adenocarcinoma patients is 55%, much higher than the rate in western countries. The clinical response rates of chemotherapy and target therapy in lung adenocarcinoma are also different. These evidences demonstrate that the clinical behavior of lung adenocarcinoma in Taiwan is obviously different from that in western countries. To investigate the mechanism of survival, proliferation, apoptosis, local invasion, and distant metastases in lung adenocarcinoma cells becomes quite important. By doing the research, we hope we can help lung adenocarcinoma patients and improve their survival rate.
Apoptosis is one of the cell death methods. It plays an important part in cellular growth, differentiation, and the death in cancer cells. In order to increase the survival rate, adapt to the micro-environment, prevent being killed by immunological cells, cancer cells develop many anti-apoptosis methods to prevent apoptosis. Mitochondrion is an important organelle for energy supply and also determines the cells to die or live. When the cell apoptosis is induced by mitochondrial pathway, the apoptosis induced factor (AIF) and cytochrome C are released to cytoplasm and nucleus from the inner membrane of mitochondria. AIF and cytochrome C will dissect the deoxyribonucleic acid in the nuclease with other associated enzymes which cause the cell to die. Therefore the cancer cells develop a method for stabilizing the mitochondria to prevent the apoptosis exposing to worse environment. This makes the cancer cells to survive. According to our previous research and literature review, AIF acts quite an important key role in the apoptosis of lung adenocarcinoma cells.
The ATPase family, AAA domain containing 3A (ATAD3A) protein belongs to AAA family. It contains the enzyme activity of adenosine triphosphatase. ATAD3A in cells distributes over endoplasmic reticulum and membrane portion of mitochondria. It is transported from endoplasmic reticulum to mitochondria through small vesicles which associate with dynamin-related protein 1 (DRP1), mitofusin-2, and optic atrophy type 1(OPA1). These proteins associate with mitochondrial fusion and fission. We consider that ATAD3A prevents cellular apoptosis through stabling mitochondria and stopping the release of AIF and cytochrome C. OPA1 is one of dynamin families protein involved in mitochondrial fusion. It also associates with cellular apoptosis via mitochondria through caspase dependent pathway. Caspase-14 belongs to the cysteine protease family members. It correlates with the epithelial cell differentiation. However, it is still not so clear in apoptosis pathway. In cells, caspase-14 is distributed in cytoplasm and nucleus, not in mitochondria. From the analysis of protein interaction, we know that caspase-14 interacts with AIF in cytoplasm when AIF released from mitochondria. Caspase-14 combines with AIF and blocks its function. It might stop the process of apoptosis through caspase independent pathway. By veiwing above phenomenon, we consider that ATAD3A, OPA1 and caspase-14 affect the activity of AIF and cytochrome C in cytoplasm and mitochondria. Moreover, ATAD3A is the key factor in controlling mitochondrial morphology and stability with other associated proteins. In this research, we focus on several parts as follows: (1) ATAD3A, OPA1 and caspase-14 involving the cellular apoptosis; (2) the distribution and location of ATAD3A, OPA1 and caspase-14 in cells; (3) the function and phosphorylation of these proteins; (4) the change of mitochondria and organelle morphology by ATAD3A, OPA1 and caspase-14; (5) the suppression of these proteins activity in cells affecting the response to chemotherapy; and (6) the association in the expression of these proteins and clinical outcome in lung adenocarcinoma patients.
In this research, we used lung adenocarcinoma cell lines and lung tissues after surgical resection. The messenger RNA expression in lung adenocarcinoma cells and tissue were detected by reverse transcription polymerase chain reaction method. We also prepared the monoclonal antibodies of ATAD3A, OPA1 and caspase-14 from mice. Using Western blotting, the protein expression of ATAD3A, OPA1 and caspase-14 were detected in lung adenocarcinoma cells and tissue. Confocal microscope and immunohistochemistry were used to localizate the position of ATAD3A, OPA1 and caspase-14 inside lung adenocarcinoma cells. We found the expression of ATAD3A in cells were in endoplasmic reticulum and mitochondria. The OPA1 was in the inner membrane of mitochondria. The caspase-14 was in cytoplasm and nucleus. Using electric microscope, we observed the morphologic change in endoplasmic reticulum and mitochondria after knocking down the ATAD3A and OPA1 expression. Clinically, the high expression of ATAD3A, OPA1 and caspase-14 in tumor tissue is related to local invasion and poor outcome. We used the small interfering RNA to decrease the expression of these proteins in cancer cells and examined the growth rate of lung adenocarcinoma cells with cisplatin. Moreover, we found that phosphorylation of ATAD3A, OPA1 and caspase-14 improved stability and function. Finally, we found the interaction among ATAD3A, mitofusion 1, mitofusion 2, AIF, caspase-14 and OPA1.
In conclusion, this research evidences ATAD3A, OPA1 and caspase-14 as new anti-apoptosis factors in lung adenocarcinoma. They have high correlation with the clinical prognosis. By these discoveries, we believed that they can affect and improve the clinical outcome and prognosis in lung adenocarcinoma in the future.

並列關鍵字

Lung cancer ； Lung adenocarcinoma ； Apoptosis ； Mitochondria ； ATAD3A ； OPA1 ； Caspase-14

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