摘要:肺癌是台灣及其他工業化國家最常見之癌症死亡原因之一(第二位)。肺癌之預後極差,在目前之治療模式下,其五年存活率低於15%。因此找尋其他有效之輔助治療方法極為重要。最近研究提供了一個重要之觀念,那就是發炎是腫瘤形成與發展之重要步驟。越來越多支證據顯示,許多惡性腫瘤之形成是被慢性感染誘發,或是與慢性發炎有關。在腫瘤微環境中之發炎細胞被證實可促進癌細胞之細胞增生、細胞存活、細胞移行侵略以及血管新生,因此可促進腫瘤之發展。我們之前的研究也顯示發炎細胞素如COX-2及IL-8 在非小細胞肺癌中過度表現,以及巨噬細胞與肺癌細胞交互作用可調控肺癌細胞中之基因表現 (Yuan, A et al. Am J Respir Crit Care Med 2000; 162: 1957、Int.J. Cancer 2005, 115, 545、Clin. Cancer Res. 2003: 9, 729, J. Clin. Oncol, 2005,23:953)。巨噬細胞為腫瘤微環境(tumor microenvironment)中發炎細胞之多數成員。癌細胞可藉由分泌化學吸引物(chemoattractants)來吸引血液中巨噬細胞聚集至腫瘤組織中,而形成腫瘤巨噬細胞。雖然巨噬細胞之前被認為具有抑制腫瘤作用之免疫細胞,最近之證據顯示巨噬細胞可被癌細胞或腫瘤微環境更改,轉而變為具有刺激腫瘤生長及擴散等促腫瘤作用之細胞。關於腫瘤巨噬細胞在人類癌症之進展轉移上扮演之功能,以及對癌症預測病人預後之重要性最近被熱烈的研究。高腫瘤巨噬細胞密度被報告在乳癌、子宮頸癌、黑色素瘤、膀胱癌、攝護腺癌及肺癌中,與癌細胞增生指數、腫瘤大小、高度血管新生、高度淋巴結轉移以及病患不良預後成正相關。然而在攝護腺癌、胃癌及肺癌中,也有報告指出高腫瘤巨噬細胞密度與早期臨床分期、較少淋巴結轉移、分化較好組織型以及較佳病患預後有關。在我們先前之研究中發現,腫瘤巨噬細胞密度及COX-2表現與肺癌血管新生及病患不良預後成正相關,而巨噬細胞與肺癌細胞株交互作用後,會刺激肺癌細胞IL-8 及其他五十個基因之表現,其中包括與血管新生、發炎及代謝等相關基因 (Yuan A et al. Clin Cancer Res 2003, Am J Respir Cell Mol Biol 2005,J ClinOncol 2005, Int J. Cancer 2005)。然而決定腫瘤巨噬細胞扮演抑制腫瘤作用,或是促腫瘤作用之確切機轉仍未清楚,但可能受巨噬細胞與癌細胞、間質細胞以及腫瘤微環境間交互作用之影響。證據顯示巨噬細胞在不同刺激下,可分化成不同表現型之M1(第一型,典型活化巨噬細胞)及M2 (第二型,另類活化巨噬細胞,又包括M2a, M2b 及M2c) 巨噬細胞,並可能擁有不同之功能。我們之前針對M1 或M2 (a, c) 型巨噬細胞對於肺癌細胞之腫瘤形成、侵略轉移等行為之影響,以及對肺癌細胞基因表現之調節功能,結果發現不同表現型之腫瘤巨噬細胞(M1 vs M2a/M2c)在實驗室內及活體內對調節肺腺癌細胞之增生、移行、侵略、血管新生、腫瘤生成及藥物抗藥性上具有不同及反相之功能。不同表現型之腫瘤巨噬細胞亦能活化或抑制肺腺癌細胞內不同之訊息傳遞路徑,而這些活化或抑制之基因產物可做為預測肺癌病患之臨床存活預後之有意義指標。這些結果顯示不2同表現亞型之巨噬細胞對肺癌之生物行為及基因調控有不同之影響 (圖一~圖四)。微小核醣核酸(microRNA)是體內大量內生性的非蛋白質製造(non-proteincoding)之小核醣核酸,最近被發現是重要之基因調節因子。微小核醣核酸是非蛋白製造、由約22 核苷酸組成之單股核醣核酸,是植物及動物體內新發現之基因調節者。它們可負向調控訊息核醣核酸(mRNA)之表現,其機轉為分解訊息核醣核酸(cleavage),或是抑制訊息核醣核酸之轉譯(translation)。至於經由何種機轉,則決定於微小核醣核酸與訊息核醣核酸間序列之互補性。最近之證據顯示,微小核醣核酸之異常表現與癌症之若干特徵,如腫瘤形成、腫瘤分化、病患預後及化療反應間呈相關性。微小核醣核酸表現與癌症之相關性,最早在血癌中被發現;微小核醣核酸-15 (miR-15) 及微小核醣核酸-16 (miR-16)在68%之B細胞慢性淋巴球性白血病中,被發現有降低表現之情況。而微小核醣核酸-let-7 也在肺癌組織中有降低表現之情形,let-7 之降低表現並與肺癌病患之較短術後存活相關。miR-15、miR-16 及let-7 微小核醣核酸作用之標靶,最近亦被證實為致癌蛋白BCL-2及RAS 之訊息核醣核酸。這些新發現顯示微小核醣核酸,可經由抑制抑癌基因蛋白或致癌基因蛋白之表現,而成為作用像致癌基因或抑癌基因之微小核醣核酸(oncogenicmicroRNAs or tumor suppressor microRNAs)。然而關於調節發炎過程之微小核醣核酸,以及腫瘤間質中之發炎過程對癌症微小核醣核酸表現之影響,目前並不清楚。有限之研究發現,在人類巨噬細胞中可找出與發炎有關之微小核醣核酸;例如lipopolysacharide 可誘發單核細胞中miR-146a/b,miR-132 and miR-155 之表現,而INF-β 及 Toll-like receptor ligands 可引發巨噬細胞中miR-155 之表現。但是關於巨噬細胞對肺癌細胞中微小核醣核酸表現之影響,目前仍不清楚。對於不同表現型巨噬細胞如何透過調控肺癌細胞中之微小核醣核酸之表現,進而影響肺癌細胞之基因表現以及生物行為如腫瘤生成、侵略轉移及血管新生等之改變,目前仍未知。肺癌細胞中可被不同表現型巨噬細胞誘發或抑制之可能的”致癌微小核醣核酸”及”抑癌微小核醣核酸",目前仍未被確認。而這些在肺癌細胞中受不同表現型巨噬細胞影響而表現之微小核醣核酸,其可能作用標的,亦仍未被證實。而在肺癌細胞中過度表現或降低表現這些相關受巨噬細胞調控之微小核醣核酸,對癌症擴展之影響目前仍然未知。另外抗發炎藥物對肺癌細胞中受巨噬細胞調控之微小核醣核酸表現之影響,也全然未知。此計畫是一個三年計畫。在第一年計畫中,我們將研究不同表現型巨噬細胞對肺癌細胞中微小核醣核酸表現之影響,以及在肺癌細胞與巨噬細胞交互作用中調控發炎相關訊息上所扮演之角色;並嘗試找出肺癌細胞中可受不同表現型巨噬細胞調控之”致癌微小核醣核酸”及”抑癌微小核醣核酸”。我們將非小細胞肺癌細胞與偏極化成M1、M2a及M2c 之不同表現型之巨噬細胞共同培養,並以微小核醣核酸基因微陣列晶片及即時性定量反轉錄聚合脢連鎖反應,來評估肺癌細胞中微小核醣核酸表現之改變。然後我們將利用生物資訊預測演算電腦程式,來預測這些受巨噬細胞調控之相關微小核醣核酸之推定之作用標靶。在第二年計畫中,我們將用luciferase 檢測法及蛋白檢測法,來3分析並確認肺癌細胞中之受巨噬細胞調控之相關微小核醣核酸的作用標靶蛋白。另外我們將在非小細胞肺癌細胞中,轉染過度表現或降低表現這些受巨噬細胞調控之相關微小核醣核酸,並利用實驗室內/活體外模式評估肺癌細胞生物特性之改變,包括癌細之細胞增生、移行侵略以及血管新生能力之改變。在第三年計畫中,我們將以免疫不全鼠腫瘤移植之活體內模式,來評估這些受巨噬細胞調控之相關微小核醣核酸對調控癌症擴展之影響。最後我們將篩檢現有之抗發炎藥物,並找出可影響肺癌細胞中之受巨噬細胞調控之微小核醣核酸表現的有效藥物;並以活體外或活體內動物模式來評估這些藥物對抑制非小細胞肺癌擴展之效果。在此計畫之第一部份,我們將發展一個包括約600 個微小核醣核酸之微陣列基因晶片,並用此晶片來評估肺癌細胞之微小核醣核酸表現。然後我們將選擇各種肺癌細胞株如A549、CL1-0 及CL1-5 與各種不同表現型之巨噬細胞如M1、M2a 及M2c 共同培養。然後以微小核醣核酸之微陣列基因晶片來評估肺癌細胞中受巨噬細胞調控之相關微小核醣核酸之表現。然後以即時性定量反轉錄聚合脢連鎖反應,來確認在肺癌細胞株中受巨噬細胞調控之相關微小核醣核酸表現之變化。我們將利用生物資訊預測演算電腦程式,來預測肺癌細胞中受巨噬細胞調控之相關微小核醣核酸的推測之作用標靶。如果微小核醣核酸推測之作用標靶與癌擴展(包括腫瘤生成、轉移與血管新生等)之訊息路徑有關,我們則稱此微小核醣核酸為可能參與調控非小細胞肺癌擴展過程之” 受巨噬細胞調控之相關微小核醣核酸”。在此計畫之第二部份,我們將利用3’UTR luciferase 檢測法及蛋白檢測法,來分析並確認肺癌細胞中受巨噬細胞調控之相關微小核醣核酸的作用標靶蛋白。另外我們將在肺癌細胞株中轉染過度表現或降低表現” 受巨噬細胞調控之相關微小核醣核酸",並在活體外模式中評估這些” 受巨噬細胞調控之相關微小核醣核酸”對肺癌細胞株生物特性之影響(包括細胞增生、細胞移行侵略及血管新生等),以確認” 受巨噬細胞調控之相關微小核醣核酸"之作用及功能。此舉將有助於找出肺癌細胞中受不同表現型巨噬細胞調控之相關微小核醣核酸,並有助於在肺癌細胞中發現新的受巨噬細胞調控與發炎發炎相關之致癌性及抑癌性微小核醣核酸。而對於” 受巨噬細胞調控之相關微小核醣核酸"作用標靶之確定,亦有助於建立一個與巨噬細胞調控非小細胞肺癌擴展有關之新的微小核醣核酸訊息路徑(microRNAs mediated signal pathways)以及相對應之基因訊息路徑。在此計畫之第三部份,我們將在免疫不全鼠腫瘤移植模式中,評估並確認” 受巨噬細胞調控之相關微小核醣核酸"對肺癌細胞之腫瘤生成、轉移及血管新生之影響。最後我們將篩選抗發炎藥物,找出可調控非小細胞肺癌中” 受巨噬細胞調控之相關微小核醣核酸"之有效藥物,並在活體內及活體外之模式中檢測其抑制肺癌擴展之能力。此計畫將有助於確認肺癌細胞中受巨噬細胞調控之相關微小核醣核酸,並釐清4這些” 受巨噬細胞調控之相關微小核醣核酸”在肺癌中,調控癌症擴展之可能作用。此計畫亦有助於找出非小細胞肺癌中受不同表現型巨噬細胞調控之”致癌微小核醣核酸”及”抑癌微小核醣核酸”,此舉對未來我們利用調控腫瘤微環境中發炎細胞作用之基因治療來治療非小細胞肺癌,提供了有價值之資訊。此計畫之結果亦對非小細胞肺癌之分類,提供了一個以”發炎巨噬細胞調控之相關微小核醣核酸”表現特徵為基礎的新的分類方法;此分類法將有助於預測病患預後,以及選擇適當病患接受抗發炎輔助治療。關於” 受巨噬細胞調控之發炎相關微小核醣核酸”以及其作用標靶之資訊,亦可作為將來評估抗發炎藥物對非小細胞肺癌之抑制效果之新工具;另外我們亦可以此新評估工具來研發可作為非小細胞肺癌輔助治療之新的抗發炎藥物。
Abstract: Lung cancer is the most common cause of cancer-related death in Taiwan(top 2) and other industrialized nations, and it has poor prognosis with the five-yearsurvival rate was less than 15% current treatment modalities. Therefore, the searchfor other effective adjuvant therapy is important for treatment of lung cancer.Recent data have established the concept that inflammation is a criticalcomponent of tumor initiation and progression. The inflammatory cells in tumor microenvironmenthad been shown to be able to enhance cancer cell proliferation, cellsurvival, cell migration, and angiogenesis, thereby promoting tumor development. Ourprevious studies also showed that the Inflammatory cytokines such as COX-2 andInterleukin-8 were over-expressed in NSCLC, and interaction between macrophageand lung cancer cell can regulate gene expression in lung cancer (Yuan, A et al. AmJ Respir Crit Care Med 2000; 162: 1957、Int. J. Cancer 2005, 115, 545、Clin.Cancer Res. 2003: 9, 729, J. Clin. Oncol, 2005, 23:953)Macrophages constitute a large proportion of the inflammatory cell infiltrate intumor microenvironments. Tumor-associated macrophages (TAM) were previouslyregarded as potent immune cells that have anti-tumor activity. However, recentevidences showed that macrophages can be modified by cancer cells andmicroenvironment, and are directed towards stimulating tumor growth and progression,and thus have pro-tumorigenesis activity. The possible function of TAM on progressionand invasion of human cancer, and the significance of TAM in patients’ prognosis arethe topics under intensive investigation recently. A high TAM density was reported tocorrelate with a high proliferation index, large tumor size, high tumor angiogenesis,high regional lymph node metastasis, and a poor patients’ prognosis in several humancancers including breast, cervix, melanoma, bladder, prostate cancer and lung cancer.However, several studies showed the opposite results as that high TAMs areassociated with less advanced clinical stage and decreased lymph node metastasis,well differentiated histological type, and favorable patient prognosis in prostate, gastricand lung cancers. In our previous studies, we also showed that TAM density andCOX-2 expression correlates with angiogenesis and adverse prognosis in patients withnon-small cell lung cancer, and macrophage and cancer cell interaction can stimulateIL-8 and about 50 genes expression (involved in angiogenesis, inflammation,metabolism etc) in lung cancer cell (Yuan A et al. Clin Cancer Res 2003, Am JRespir Cell Mol Biol 2005, J Clin Oncol 2005, Int J. Cancer 2005). Whether TAMsshow pro- tumorigenesis or anti-tumor activity depends on the interactions betweenTAMs and the cancer cells, other stromal cells, and the tumor microenvironment, andthe exact mechanism is still under investigation.2Recent evidence showed that depending on the activating stimuli, TAMscan differentiate into different subsets macrophage: classically (M1) or alternatively(M2) activated macrophages (including M2a, M2b and M2c), which has differentfunctions. We have previously studied the effects and possible functions of M1 or M2 (a,b or c) macrophage subsets on the lung cancer cells’ biologic behaviors and on thegene expression regulation in lung cancer cell. The results showed that the differentphenotype macrophages (M1vs M2a/M2c) have different and opposite in vitro and invivo effects on regulation of lung cancer cell behaviors, such as proliferation, migration,invasion, angiogenesis, tumorigenesis and drug resistance. In addition, M1 andM2a/M2c macrophages can activate or inhibit different gene expression signaltransduction pathways in lung cancer cells, and these activated or inhibited geneexpression signals can be used as significant indicators for patients’ survival in lungcancers. These results implied that different phenotype TAMs have different impacts onregulation of cancer cell progression and gene expression in lung cancer (Fig 1~Fig 4).MicroRNAs (miRNAs) are an abundant class of endogenous smallnon-protein- coding RNAs, which were found recently to be important gene regulators.MicroRNAs are non-coding, single-stranded RNAs of ~22 nucleotides and constitute anovel class of gene regulators that are found in both plants and animals. They cannegatively regulate their targets (mRNA) in one of two ways (cleavage of mRNA andinhibition of mRNA translation) depending on the degree of complementarity betweenthe microRNA and the target mRNA.Recent evidence has shown that deregulation of microRNAs correlates withcertain features of diverse cancers such as tumorigenesis, differentiation status,outcome of tumor patients and response to chemotherapy. The connection betweenmicroRNA expression and cancer was first reported in hematological malignancy inwhich miR-15 and miR-16 are deleted or down-regulated in 68% of B cell chroniclymphocytic leukemias (B-CLL) patients. Decreased expression of let-7 was shown inlung cancer specimens, and reduced let-7 expression was shown to be correlatedwith shorter post-operative survival of lung cancer patients. The targets for miR-15/16,let-7 suppression action were proved to be BCL-2, and RAS mRNAs (oncoprotein).These novel findings confirmed that microRNAs can function as oncogenic or tumorsuppressor microRNAs, by down-regulated the tumor-suppression protein oroncogenic proteins’ translation.The relation between inflammation signals and the microRNAs expressionregulation is an emerging avenue of investigation, and the effect of inflammation instroma on the microRNAs expression in cancers is still not clear. Only limited studiesidentified the possible inflammation related microRNAs in human cells, which3including that lipopolysacharide (LPS) can induce miR-146a/b, miR-132 and miR-155expression, and up-regulation of miR146 a/b expression may be through NF-kBactivation in monocyte. Moschos SA et al showed that the LPS can induce timedependent increases in 46 microRNAs expression in different cell components in lungtissue including in alveolar and bronchial epithelial cell. However, the relationshipbetween inflammatory cells TAMs in tumor strom and microRNAs expression in lungcancer is still unclear. The effects of different phenotype TAMs on regulation ofmicroRNAs expression in non-small cell lung cancer cells are also unknown. Thepotential TAMs regulatory “oncogenic microRNAs” and “tumor suppressormicroRNAs” in lung cancer cells are still undefined. The putative targets of theseTAMs regulatory miRNAs in lung cancer cell are un-determined. The effects ofoverexpression or knockdown of the TAMs regulatory microRNAs in lung cancer cellson regulation of the gene expression and the biologic behavior of the lung cancer cells(such as invasion, metastasis and angiogenesis) still remained unknown. In addition,whether the anti-inflammatory agents (drugs) can modulate these TAMs regulatorymicroRNAs in lung cancer cells is also still unknown.This project is a three-year project. In the first year, we will investigate the roleof different phenotypes TAMs on regulation of microRNAs expression in lung cancer,and identify the candidate “TAMs regulatory oncogenic microRNAs” and “TAMsregulatory tumor suppressor microRNAs” in lung cancer cells. We will polarize theTAMs into M1, M2a or M2c macrophage, and coculture different phenotype TAMs withnon-small cell lung cancer cell lines. We will evaluate the microRNAs expressionchanges in lung cancer cell lines by microRNAs oligoarray gene chip and real-timequantitative RT-PCR. Then we will use computational prediction algorithm to predictthe putative targets of these TAMs regulatory microRNAs in lung cancer. In thesecond year, we will verify the target protein regulated by these TAMs regulatorymicroRNAs by luciferase assay or protein assay of target protein expression in lungcancer cells. We will also transfect and forced- overexpress these TAMs regulatorycandidate microRNAs in non-small cell lung cancer cell lines, and evaluate thebiologic character change including proliferation, migration, invasion andangiogenesis capacity of the lung cancer cell line in vitro. In the third year, we willalso verify the biologic effects of these candidate TAMs regulatory microRNAs inregulation cancer progression in vivo using SCID mice tumor xenograft models withtransplantation of candidate microRNAs overexpressed or knockdown-expressed lungcancer cells. Finally, we will screen the effect of potential anti-inflammatory drugs ofother medicines that can regulate these “TAMs regulatory microRNAs” in NSCLC, andto evaluate the effect of these drugs on inhibit cancer progression in NSCLC by in vitroand in vivo SCID mice models.In the first part of this project, we will use a microRNA oligoarray gene chip4including around 600 known human microRNAs to evaluate the microRNAs expressionprofile in lung cancer cells in this project. We will choose lung cancer cell lines (such asA549, CL1-0 and CL1-5) to coculture with different phenotype TAMs such as M1, M2aand M2c. We will then use the microRNAs microarray gene chip to evaluate theup-regulation and down-regulation of microRNAs in these lung cancer cell lines. Wewill also use real-time quantitative RT- PCR to confirm the microRNAs expressionchanges in lung cancer cells affected by different phenotype TAMs.We will then use the published prediction algorithms to predict the putativetargets of these TAMs regulatory microRNAs (upregulated or down-regulatedmicroRNAs in A549, CL1-0 and CL1-5 after coculture different type TAMs), and if theputative targets is associated with cancer progression signal pathway (includingtumorigenesis, metastasis and angiogenesis activity), then the correspondingmicroRNAs are defined as TAMs regulatory candidate oncogenic or tumor suppressormicroRNAs involving in cancer progression of NSCLC.In the second part of this project, we will verify the putative targets regulated bythese candidate TAMs regulatory microRNAs by using 3’UTR luciferase reporter assay,and by protein assay (Western blot or ELISA assay) of target protein after transfectionand forced overexpression of the candidate TAMs regulatory microRNAs in lung cancercell. We will further confirm the function of these candidate TAMs regulatorymicroRNAs by transfection and overexpression or knockdown expression of thesemicroRNAs in lung cancer cells lines, and then evaluate the change of cancer cell’sbiological behaviors such as proliferation, migration, invasion and angiogenesis by invitro models.This will help to identify the down-stream microRANs regulated by differentphenotype TAMs, and to discover novel TAMs regulatory oncomir or tumor suppressormiRNAs in lung cancer. The putative target verification will also help to establish thenew “microRNAs mediated signal pathway”, which might enhance or suppress tumorprogression in non-small cell lung cancers.In the 3rd part of this project, we will evaluate the effect of these candidateTAMs regulatory microRNAs on the in vivo tumorigenesis, metastasis andangiogenesis capacity of lung cancer cells in a murine tumor xenograft model. After wetransfect and overexpress candidate TAMs regulatory microRNAs or knockdownexpress of candidate microRNAs by antisense mirRNA in lung cancer cell lines, we willtransplant the lung cancer cell line subcutaneously on SCID mice back, and evaluatethe effect of candidate microRNAs on regulation of tumorigenesis, metastasis andangiogenesis activity of tumor in SCID mice models. We also use published data basefor NSCLC patients to investigate the TAMs regulatory miroRNAs signatures that can5predict survival for patients with NSCLC. Finally, we will screen anti-inflammatoryagents to evaluate their effects on regulation of the TAMs regulatory oncomir or tumorsuppressor miRNAs in non-small cell lung cancer cell lines, and select the potentialanti- inflammatory agents that can inhibit TAM mediated cancer progression of NSCLCin SCID mice models.The efforts of this project will identify the TAMs regulatory microRNAs in lungcancer, and elucidate the possible function of TAMs regulatory microRNAs inregulation of tumor progression in lung cancers. These results will also help to identifythe candidate” TAMs regulatory oncomir” and the “TAMs regulatory tumor-suppressormicroRNAs, and this can provide the basic information for the anti-inflammation genetherapy in lung cancer in the future. The information about TAMs regulatory microRNAand their target signal pathway also can provide a new tool to evaluate the effect ofpotential anti-inflammation drugs, and develop new anti-inflammation drug fornon-small cell lung cancer adjuvant therapy in the future.