| 摘要: | Main Project: Cell Signaling Pathway in Cancer Progression 人?細胞訊息傳遞途徑的研究提供?許多資訊，進而讓我們對於多種疾病(包括癌症)有??進一步的?解。生 長因子受體像是EGFR 和ErbB2 所活化的訊息傳遞在癌細胞生長、存活、細胞週期調控、腫瘤起始與腫瘤發 展/轉移中扮演必要的角色，???針對這些訊息傳遞途徑(包括ER、EGFR/ErbB2 和PI3K/mTOR)新穎的治 ?性標靶化合物已被成功地研發出?，此外，許多的研究證實，小分子RNA 可與mRNA 結合以調控基因表現， 因而在腫瘤發展中亦扮演重要的角色。在本整合型計畫中，我們將會進一步研究EGFR 在細胞核內的非典型 功能、與其他細胞訊息傳遞分子如ADAM9、CDK1、EZH2 和L1CAM 間可能的相互作用、其在?胺酸激?抑 制物如lapatinib 或抗癌藥物如E1A 處?下對於小分子RNA 調控的影響。本計畫的?即目標是?解核內EGFR 相關調控以及與其他分子間在多種癌症(包括但?限於乳癌和口腔癌)的起始與進展中的交互作用。本整合型計 畫包括7 個子計畫，以及1 個支援的?政核心與動物核心設施，各個計畫皆具有獨?的目標，並與整合型計 畫中的其他計畫相互依存。計畫1 和2 將界定核內EGFR 在細胞增殖與腫瘤發展中以及抗?射特性中的角色、 計畫3 和4 將研究與小分子RNA 有關的抗癌藥物抗性、計畫5、6 和7 將檢視EZH2、CDK1、ADAM9 和L1CAM 在癌症轉移中扮演的角色。本計畫的達成將使我們進一步?解細胞表面受體在核內對於癌症進展的影響，並且 提供抗癌藥物發展的新方向。 Project 1: Nuclear EGFR as a Chromatin Remodeling Enzyme 表皮生長因子受體(EGFR)在傳統上被認為是細胞表面的膜蛋白，其具有?氨酸蛋白激?活性，且在細胞增殖 與腫瘤形成上扮演重要的角色。然而，EGFR 卻可在細胞核中被偵測到，而且，愈?愈多的研究顯示，核內 EGFR 在癌症發展中扮演極為重要的角色。我們發現RHA、SWI/SNF 和MCM 複合體可在細胞核內與EGFR 交互作用，這些蛋白已有研究顯示會?與腫瘤生成，因此，我們假設核內EGFR 藉由調控腫瘤啟動基因的轉 ?以及與染色體重塑關鍵蛋白複合物的交互作用，進一步促成腫瘤生成。本計畫的長程目標是?解核內蛋白的 新穎功能以及其在腫瘤發展中所扮演的角色。三大具體目標如下，目標1:?解核內EGFR 的轉?調控與腫瘤 發展之關?；目標2: 確定核內EGFR 與染色體重塑複合物SWI/SNF 在乳癌發展中所扮演的角色；目標3:釐 清MCM7 在EGFR 依賴性與細胞增殖中的作用。長期以?EGFR 一直被認為是細胞表面的受體，因而忽?? 其在細胞核中的功能，隨著對EGFR 核內功能的逐漸?解，以及核內EGFR 在?同癌症癒後價值的認知，本 計畫適時地針對極為重要但幾乎被忽視的問題提供?研究目標。Project 2: Roles of Nuclear EGFR in Radioresistance of Cancer 致癌蛋白 EGFR 被發現存在細胞核內已經超過十??。在過去的研究中發現，當快速增生的肝臟其肝細胞? 斷地增生時常緊密伴隨著核內EGFR 表現的增加。同時在許多癌症檢體中包括乳癌檢體常常也發現有核內 EGFR 過?表現，且最近已經被證實核內EGFR 可以被用?當作癌症病患的癒後分子標記。經由我們最近的 研究發現，核內EGFR 能透過磷酸化增生細胞核抗原(PCNA)?強化其蛋白的穩定性並調控細胞對基因毒性逆 境(genotoxic stress)的耐受性。而抑制EGFR 激?活性能?低PCNA 蛋白的穩定性進而減弱DNA 損傷修補的 功能。然而我們對於EGFR 在癌細胞核內?與?射抗性反應中的角色並?完全清楚。因此，在這次所提的計 畫中，我們首先將持續地尋找核內EGFR 能和哪些蛋白作用而?與癌細胞的?射抗性反應。除?PCNA 之外， 我們也將進一步研究核內EGFR 與 RNA 水解酵素(PNPase)之間的調控與其?與在癌細胞?射抗性反應的角 色。最後，根據我們的研究發現，磷酸化的PNPase 蛋白能破壞其蛋白的RNA 水解活性且可能在癌症發展過 程中扮演重要角色。因此，我們將試著運用干擾性的PNPase 胜?藉由影響PNPase 的磷酸化進而抑制具有 ?射抗性的癌細胞生長?發展新穎且有效的癌症治?方法。因此本計畫的三個目標如下: 第一，鑑定與分析癌細胞內?與在?射抗性反應中新穎的核內EGFR 目標蛋白與其複合體。第二，探討核內EGFR 與PNPase 如 何調控以及在癌細胞?射抗性反應中的角色。第三，運用干擾性的PNPase 胜??發展對於具有?射抗性癌 症的新穎治?方法。這個計畫可以讓我們??解EGFR 在癌細胞核內?與?射抗性反應的訊息網?。因此完 成此計畫?僅可以提供?一個很好的機會?進一步?解核內EGFR 在癌細胞?射抗性反應中所扮演的角色也 有助於未?成功發展出新穎且針對具有?射抗性反應的癌症治?方法。Project 3: MicroRNA Regulation and Epigenetic Molecular Mechanisms in E1A-mediated Anti-cancer Activity 腺病毒蛋白 type 5 E1A (E1A)與多種抗癌應用有關，並且目前已被運用於多種癌症如乳癌、大腸癌及頭頸癌 的?床基因治?試驗上。因此，?解E1A如何調控抗癌特性是十分重要。最近的研究證據顯示多種微型核?核 酸具有調控訊號分子的能?，並且?與到細胞凋亡、細胞增生、癌化以及癌轉移能?進而造成腫瘤的新生。本 計劃的目的在於探?微核醣核酸的調控及其表分子機轉在E1A抑癌作用的影響。為?達成此計劃的目的，我們 將把重點擺放在E1A如何抑制腫瘤生長及轉移以及研究其微核醣核酸的調控及其表分子機轉。實驗的設計將劃 分為以下四個主要目標?完成： 目標一：研究E1A所調控之微核醣核酸表現 目標二：驗證E1A是否藉由調控微核醣核酸抑制腫瘤的作用 目標三：E1A其表分子機轉如何調控微核?體核酸 目標四：藉由動物模式探討E1A調控的微核?核酸所扮演的角色 ?成功執?此計畫，將會使我們建??周全訊息傳遞網，且?加?解微核?核酸是如何被調控以及其表分子機 轉在E1A抑癌作用的影響。以上將包括微核?核體如何在E1A誘導下造成細胞凋?，及抑制上皮細胞轉形成間 ?細胞，發炎所造成的癌化以及轉移的過程 Project 4: The Role of microRNAs in Conferring Acquired Resistance to EGFR TKIs HER2 及EGFR 的過?表現常?於乳癌及肺癌等各種癌症細胞，並與癌細胞轉移及?化有極大之相關 性。近??，針對HER2 及EGFR 所發展出?的標的治?方式，包括單株抗體及小分子抑制劑均可有效 抑制癌細胞生長。其中Lapatinib (Tykerb?, GW-572016), 為可口服之EGFR and HER2 tyrosine kinases 抑制劑，已被核准使用於對化學治?及賀癌平(Herceptin, Trastuzumab)失效的乳癌病患，並具有延長病 人生命的?效。然而，此藥物在?床上的使用效果及發展卻常在服用一?內因產生嚴重抗藥性而受到相 當大的限制，造成lapatinib 抗藥性的機制目前仍尚未清楚。微小核醣核酸(microRNA) 是由長?只有17-24 鹼基對所組成的單股、非轉譯成蛋白的RNA，可經由結合於特定基因mRNA 上的3 端非轉譯區 (3?UTR)，而具有造成mRNA ?解或是抑制基因轉譯的作用，因而?與細胞生長、分化及凋亡等多項功 能。最近研究指出，microRNA 亦?與癌症形成、進展、轉移及抗藥性的過程。然而，microRNA 是否影 響乳癌細胞對lapatinib 的敏感?則尚未有研究報導。因此，本計劃的研究目的將探討在長期給予lapatinib 治?之後，是否藉由改變microRNA 的表現?，因而使細胞對lapatinib 產生抗藥性。第一?將?用 microRNA array 及RT-qPCR 找出在lapatinib 抗藥細胞株中，何種microRNA 的表現產生變化，並證明 其對lapatinib 產生抗藥性的因果關係；第二?將找出與lapatinib 抗藥性相關的microRNA 是透過影響那 些基因之表現，因而?與lapatinib 抗藥性之形成；第三?將進一步發展microRNA 的抑制劑antagomir 或 microRNA sponges ，測試其對?低癌細胞產生lapatinib 抗藥性的效果。經由本研究的成果，除?可以 解開對lapatinib 產生抗藥性的分子機制，?有助於發展新一代EGFR/HER2 抑制劑或針對所發現的相關 基因進?合併?法。Project 5: The interplay between CDK1 and EZH2 in Modulation of Cancer Progression and Metastasis polycomb group 蛋白質EZH2 是PRC2 複合蛋白中的催化亞基，具有組蛋白?氨酸甲基轉移?的活性，可甲 基化組蛋白H3 的第27 個?氨酸（H3K27），導致基因默化。在許多癌症中EZH2 的大?表現和腫瘤發展及癌 轉移有關。此外，抑制EZH2 表現可減少癌細胞的增生，並且造成細胞週期G2/M 期進?的延遲，顯示EZH2 ?與 G2/M 期的進?。?常細胞週期調控被認為是造成腫瘤發展的一個關鍵因素。由於 EZH2 在細胞週期 G2/M 扮演重要角色，而 CDK1 是調控G2/M 期進?的主要激?，因此，我們推測，EZH2 可能受CDK1 調控。 有趣的是，我們的初步結果顯示，CDK1 會和EZH2 交互作用，抑制 CDK1 可增加H3K27 的trimethylation， 造成EZH2 靶基因的表現減少。因此，根據這些初步結果，本計畫擬從下?四點深入探討CDK1 和EZH2 間 的相互作用及在腫瘤發展和癌轉移之角色：（1）分析CDK1 磷酸化EZH2 之胺基酸位點;（2）探討CDK 經由 調控EZH2 活性進而影響PRC 功能之角色;（3）?用系統性的研究方法找尋受CDK1/EZH2 調控之基因;（4） 探討CDK1 磷酸化EZH2 對於腫瘤發展形成和癌轉移之影響。本計劃的研究成果?僅可增進我們對於 EZH2/CDK1 調控腫瘤發展和癌轉移之分子機轉?進一步的?解， 並有助於找尋新的腫瘤診斷標記及抗癌藥 物新標的。Project 6: Face-off of ADAM9: The Regulation of Integrin-dependent and -independent Cancer Metastasis 癌症細胞已被證實具有多種轉移和入侵其他器官的機制，這其中包含?integrin-dependent 及 integrin-independent。?用調控黏合分子受器的結合?做為標靶治?癌症時，在?床上證實其效果是?好的。 因此我們必須?解到腫瘤細胞面對?同環境時，會去對外在的壓?做出反應，並修改其轉移機制。最近研究顯 示腫瘤生長時EGF sheddase 的表現?會增加(?如：ADAM9)。?用體外分子研究及動物體內之?低ADAM9 的表現時，同時也顯著的?低腫瘤細胞的轉移、侵入和生長。然而、這樣的一個結果在含有基質分子的遷移實 驗上是相反的，當?低ADAM9 的表現時，反而促進細胞的遷移能?。這暗示ADAM9 可能會阻礙integrin 和 基質分子的結合，?用integrin-independent 機制下促進腫瘤細胞的轉移能?。?解並認?細胞層次及分子層 次上ADAM9 促進細胞的轉移和入侵機制，可以幫助我們發展出新的治?腫瘤之方法。Project 7: Role of L1CAM in Epithelial-to-Mesenchymal Transition in Oral Cancer 口 腔癌是頭頸部癌症中最普遍且具高侵襲和轉移的癌症。病患5 ?存活?也沒有因醫?技術進步而明顯改善。 反映出對口腔癌細胞生長及轉移的分子機制?解甚少。 L1 細胞吸附分子 (L1CAM) 已被證明在?經發育及一 些腫瘤?化過程中扮演重要角色，但其功能尚未在口腔癌中被探討。我們在細胞株實驗發現L1CAM 表現?? 僅與口腔癌細胞株的侵?能?成正相關性，並能直接促癌?化與造成癌上皮細胞間質性轉形(EMT)。?重要 的，我們發現L1CAM 與表皮生長因子受器 (EGFR)在細胞內有直接的結合。因此我們假設L1CAM 可藉由與 EGFR 的共同作用導致癌細胞間質性轉形進而造成口腔腫瘤?化與轉移。本計畫之目標一：進一步在活體中驗 證L1CAM 與口腔癌腫瘤進展及EMT 的關?性，評估以L1CAM 當作EMT 標記?早期診斷與預測口腔癌轉移 的可?性。目標二：探討L1CAM 調控口腔癌腫瘤進展機制是否透過與EGFR 訊息傳導?徑的?結。目標三： 研發一個以EMT 為標靶的口腔癌基因治?試藥，在動物模型上驗證其?效及安全性，並評估其與?床使用的 EGFR 標靶治?藥物作為混合治?的優勢。這個研究?僅能增進我們對口腔癌細胞EMT 分子機制的?解， 並對標靶治?試藥的研發有極大助?。 Project 9: Establishment of Animal Core for cancer Research 分子醫學中心動物核心成?的目的，是將提供研究員在實?研究計畫時，對於動物模型使用的知?與能? 有?好的資源管道。動物核心也應用最低的花費達到?合研究員在?用動物模型的研究計畫上共享研究設備、 專門技能和動物資源。這將是最經濟的動物模型研究方式。因此為?配合其它七項研究計畫的進?，本動物核 心成?將有三項目標。目標一：確保有效?的動物實驗設計。目標二：提供必要的設備、人員和專業知?。目 標三: 提供最新?鼠癌症病?學的專門知?。目標四: 協助進?七項研究計畫中的動物實驗。
Main Project: Cell Signaling Pathway in Cancer Progression Studies of the cell signaling pathways, which are central to human development, have uncovered important information for understanding a variety of diseases including cancer. Signaling pathways activated by growth factor receptors such as EGFR and ErbB-2 as well as others have been shown to play an essential role in cancer cell growth, survival, cell cycle deregulation, tumor initiation, and tumor progression/metastasis. Over the years, novel therapeutics targeting components within these pathways have been successfully developed. Specific pathways that have been targeted for drug development include ER, EGFR/HER2 and PI3K/mTOR among many others. More recently, numerous studies have demonstrated that microRNAs, which are small non-coding RNAs that bind to mRNAs to regulate gene expression, also play an important role in tumorigenesis. In our proposed program project, we are working together to further study the non-canonical function of EGFR in the nucleus, its potential interactions with other cell signaling components such as ADAM9, CDK1, EZH2 and L1CAM, its affect on regulations of micro-RNAs under the treatment of tyrosine kinase inhibitors like lapatinib or novel anti-cancer therapeutics such as E1A. The immediate goals of this Program Project Grant (PPG) are to understand the role of nuclear EGFR-mediated regulation and to decipher its interactions with other molecules that lead to cancer initiation and progression of various cancer types including but not limited to breast and oral cancer. This PPG consists of 7 interactive projects that are supported by one administrative and one animal core. Each of the 7 projects has its own unique set of specific aims and is interdependent on components of other projects in the PPG. Project 1 and 2 will delineate the role of nuclear EGFR in cell proliferation and progression as well as in radioresistance. Project 3 and 4 will focus on identification and characterization of microRNA-mediated resistance to anti-cancer drugs. Project 5, 6 and 7 will examine the role of EZH2, CDK1, ADAM9 and L1CAM in cancer metastasis. The outcome of this PPG will advance our understanding of the effect of cell surface receptor in the nucleus on regulation and progression of cancer and may shed light on new directions for development of effective cancer therapies. Project 1: Nuclear EGFR as a Chromatin Remodeling Enzyme EGF Receptor (EGFR) traditionally regarded as a cell surface membrane-bound protein harboring a tyrosine kinase activity plays crucial roles in cell proliferation and tumor progression. Unorthodoxly, EGFR could be detected in the nucleus. However, due to its ambiguous functionality, the role of nuclear EGFR is still a puzzle. Emerging data indicated that the nuclear EGFR may be an integral part of cell proliferation and tumor progression. For instance, nuclear EGFR expression could serve as a poor prognosis tumor marker for multiple cancer types including breast cancer. We identified several interesting proteins interacting with EGFR in the nucleus including RHA, the SWI/SNF complex and MCM complex. RHA is a DNA-binding protein that recognizes the DNA sequence identical to the ATRS sequence and may be a potential candidate to be a DNA-binding partner of nuclear EGFR, which does not have a DNA binding domain. SWI/SNF is a protein complex involved in chromatin remodeling. A nuclear MCM hexameric ring complex is involved in the process of DNA replication licensing by recruiting DNA polymerases to initiate DNA synthesis. EGFR expression in tumor tissues, especially in the more advanced stages, is positively correlated with certain members of the MCM complex. These newly identified EGFR interacting proteins have been shown to play a role in tumor progression. Thus, we hypothesize that nuclear EGFR contributes to tumor progression through transcriptional upregulation of tumor promoting genes and interacting with critical protein complexes involved in chromatin remodeling. The long-term goal of this proposal is to understand the novel functions of nuclear EGFR and their roles in the tumor progression. Three specific Aims are proposed: Specific Aim 1: Transcriptional regulation and tumor progression of nuclear EGFR; Specific Aim 2: Nuclear EGFR and chromatin remodeling complex SWI/SNF in breast tumor development; Specific Aim 3: The role of MCM7 in EGFR dependent DNA synthesis and cell proliferation. As EGFR has long been considered a cell surface receptor, its nuclear functions have been overlooked for decades. With increasing evidence of receptor tyrosine kinases in the nucleus and gradual discovery of their nuclear functions and prognostic value of nuclear EGFR in multiple human cancers, the current proposal represents a timely project that addresses the critical but “almost neglected” issues. Project 2: Roles of Nuclear EGFR in Radioresistance of Cancer Nuclear existence of epidermal growth factor receptor (EGFR) has been documented for more than a decade. In regenerating liver, increased nuclear EGFR expression is closely associated with active hepatocyte proliferation. In cancer, nuclear expression of EGFR is frequently associated with multiple tumor types, including breast cancer, and has been shown to be a poor prognosis tumor marker for cancer patients. We recently demonstrated that nuclear EGFR can also regulate cell sensitivity to genotoxic stress through PCNA. Inhibition of EGFR kinase activity rendered PCNA destabilized from the chromatin so that its DNA damage repair function was also impaired. However, the role of nuclear EGFR in radioresistance of cancer remains elusive. In this current proposal, we continue to identify the nuclear EGFR and its associated proteins that may be involved in radioresistance of cancer. In addition to PCNA, we will extend our study to investigate the regulation between nuclear EGFR and hPNPase, a 3 to 5 exoribonuclease, and their role in radioresistance during cancer progression. From our studies, the serine phosphorylated PNPase abolishs its RNase activity and may contribute to inhibit cancer progression, we will plan to develop an anti-radioresistance cancer therapy by engineering PNPase-derived interference peptides to protect PNPase functions may further enhance tumor-cell killing. Three Specific Aims are proposed: 1. Identification of novel nuclear EGFR associated proteins involved in radioresistance of cancer; 2. Characterization of nuclear EGFR/hPNPase and their role in radioresistance during cancer progression; and 3. To develop the novel anti-radioresistance cancer therapy through PNPase-derived interference peptides. The proposed work will greatly extend our knowledge on the signaling network of nuclear EGFR that may contribute to radioresistance during cancer progression. Success of outcome will not only allow us to further understand underlying mechanisms of cancer cell radioresistance regulated by nuclear EGFR, but eventually to design an effective therapeutic intervention for radioresistant cancer treatment. Project 3: MicroRNA Regulation and Epigenetic Molecular Mechanisms in E1A-mediated Anti-cancer Activity The adenoviral type 5 E1A (E1A) associates with multiple anti-cancer activities and has been tested in multiple clinical trials in a gene therapy setting for breast, ovarian and head and neck cancer patients. Thus, it is critical and timely to understand the detailed molecular mechanisms that associate with E1A-mediated anti-cancer properties. Recent evidences reveal that various microRNAs modulate the signal molecules involve in apoptosis, proliferation, angiogenesis, or metastasis and may in turn contribute to the tumorigenesis. The goal of this application is to understand the microRNA regulation and epigenetic molecular mechanisms of the E1A-mediated anti-cancer activities. To reach the goal, we will focus on how E1A may interact with the novel signaling pathways and exert suppression effects on tumor progression and metastasis in breast cancer and four SPECIFIC AIMS are proposed. SPECIFIC AIM 1: Identify the differential expression of microRNAs between breast cancers expression of control vector or E1A-expressing vector. SPECIFIC AIM 2: Define the microRNAs involving in E1A-mediated anti-cancer activity. SPECIFIC AIM 3: Study the epigenetic regulations and molecular mechanism involving in the regulation of microRNA by E1A. SPECFIC AIM 4: Investigate the role of E1A-mediated microRNA in animal model. Success of these SPECIFIC AIMS will help us to establish signaling networks to understand better the microRNA regulation and epigenetic molecular mechanisms of E1A-mediated anti-cancer activities. These will include how microRNAs involves in E1A-induced sensitization to apoptosis, and how E1A-mediated microRNAs may inhibit epithelial-mesenchymal transition (EMT), inflammation-induced tumorigenesis and homing process of metastases. Project 4: The Role of microRNAs in Conferring Acquired Resistance to EGFR TKIs EGFR and/or HER2 receptor tyrosine kinases were frequently found to be amplified and overexpressed in various cancer types and associated with a poor clinical outcome. The dysregulated signaling pathways, including PI3K/Akt and MAPK, from the oncogenic EGFR/HER2 leads to the gene expressions associated with the neoplastic transformation, initiation, cellular immortalization, and tumor progression. Thus, targeting the kinase activities of EGFR/HER2 has emerged as an attractive approach to treat malignancies driven by EGFR/HER2. Lapatinib (Tykerb?, GW-572016), an oral dual inhibitor of EGFR and HER2 tyrosine kinases, has proven to be efficacious and approved in combination with capecitabine for treating advanced HER2-postive breast cancer patients who failed to chemotherapy or Trastuzumab therapies. Lapatinib inhibits EGFR/HER2 tyrosine kinase activities, in turn, attenuating downstream signaling pathways that regulate tumor cell growth, survival, and metastasis. However, the anti-tumoral effects and survival benefit from lapatinib treatment as a monotherapy or in combination with chemotherapy are limited by the development of therapeutic resistance that typically occurs within 12 months of starting therapy. The determinants that confer the acquired resistance to lapatinib (Lapr) are not systemically characterized although some studies were done only focusing on certain molecules. MicroRNAs, a class of endogenous 17-24 base-long single-stranded noncoding RNAs, regulate an array of gene expressions via targeting the 3’ untranslated region (UTR) in a sequence-specific manner. MicroRNAs participate in the regulation of many cellular processes including proliferation, differentiation, apoptosis, and chemoresistance. But it remains unknown whether microRNAs also contribute to the development of acquired resistance to lapatinib. Therefore, the goal of this 3-year proposal is to study whether the development of Lapr involves the dysregulation of microRNAs. To achieve this goal, the following specific aims will be addressed. The first aim is to identify the microRNA expression profile in response to Lapr by using a non-biased approach. The differential microRNA expression between Lapr breast cancer clones and their parental cell lines will be analyzed by microRNA microarray. After confirming the result of microarray analysis by RT-qPCR, the causal relationship between these Lapr-associated microRNAs and the desensitization to lapatinib will be further addressed by re-introducing the downregulated microRNAs or by neutralizing the downregulated microRNAs in Lapr cells. The second aim is to identify the dysregulated genes targeted by Lapr–associated microRNAs. Once these target genes have been explored, their roles in conferring the development of Lapr in vivo will be validated in xenograft mouse model. Finally, novel therapeutic strategies against the Lapr-associated microRNAs or their targeting genes will be developed to circumvent Lapr. Results of this study will not only provide the insight into molecular mechanisms of acquired resistance to lapatinib, and also be helpful for the development of new strategies overcoming the dilemma. Project 5: The interplay between CDK1 and EZH2 in Modulation of Cancer Progression and Metastasis The Polycomb group (PcG) protein EZH2, a catalytic subunit of Polycomb repressive complex 2 (PRC2), is a histone lysine methyltransferase which catalyses the methylation of histone H3 at lysine 27 (H3K27) in target gene promoters, resulting in epigenetic silencing. Overexpression of EZH2 has been detected in aggressive solid tumors and implicated in cancer progression and metastases in diverse cancer types. In addition, knockdown of EZH2 has been shown to be able to decrease proliferation of cancer cells and cause a delay in the G2/M transition of the cell cycle, indicating EZH2 is involved in G2/M transition. Aberrant cell cycle regulation is considered as a key factor in the evolution of tumor. Since EZH2 has a role in G2/M transition and CDK1 is one of the major G2/M kinases and both play a pivotal role in regulation of cell cycle progression, we hypothesize that EZH2 might be regulated by CDK1. Interestingly, our preliminary results show that CDK1 interacts with EZH2 and inactivation of CDK1 enhances trimethylation of H3K27, resulting in downregulation of EZH2 target gene expression. Thus, based on these preliminary results, the goal of this Project 5 is to elucidate the interplay between CDK1 and EZH2 in modulation of cancer progression and metastasis. To achieve the goal, the following approaches are proposed: (1) To determine the biological relevance of CDK phosphorylation sites on EZH2 in vivo; (2) To investigate the role of CDK activity in PRC function through regulation of EZH2; (3) To identify target genes regulated by CDK1/EZH2 using quantitative real-time RT-PCR array or genome-wide cDNA microarray analysis; (4) To evaluate the contributions of EZH2 phosphorylation in tumor progression/metastasis mediated by CDK1. Success of this proposal will not only further advance our understanding of molecular mechanism by which EZH2/CDK1 mediate tumor progression and metastasis, but also help to identify new predictive/prognostic markers of tumor progression and anti-tumor therapeutic drug targets. Project 6: Face-off of ADAM9: The Regulation of Integrin-dependent and -independent Cancer Metastasis Cancer cells possess a broad range of migration and invasion mechanisms. These include both integrin-dependent and integrin-independent strategies. Cancer therapeutics designed to target adhesion receptors have shown ineffective in clinical trials. This might because of the fact that cancer cells can response to stress and modify their migration mechanisms in response to different conditions. Recent study of EGF sheddase, ADAM9, showed increased in response to cancer progression. Knockdown of ADAM9 significantly decreased cell migration, invasion and proliferation in both in vitro and in vivo studies. However, this observation was in opposite in which increased cell migration when culture cells on matrix in ADAM9 knockdown cancer cells, suggests ADAM9 might block integrin-matrix interaction and induced cell migration under integrin-independent manner. Understand and learning more about the cellular and molecular basis of ADAM9 induced cell migration/invasion programs will help us to develop a new therapeutic approach. Project 7: Role of L1CAM in Epithelial-to-Mesenchymal Transition in Oral Cancer Oral squamous cell carcinoma (OSCC) is one of the most prevalent highly invasive and metastatic head and neck cancers and does not have a good prognosis. Despite advances in surgery and radiation therapy, the 5-year survival rate for OSCC has not improved significantly and remains at 50–55%. Novel therapeutic alternatives to standard therapy need to be established to improve the prognosis for patients with advanced oral cancer. Molecular therapy that targets molecules selectively expressed by cancer cells could be an effective treatment of localized and disseminated oral cancer. The L1 cell adhesion molecule (L1CAM) was shown to have tumor-promoting activity in different types of cancer but has not been investigated in OSCC. Our preliminary data have shown that the overexpression of L1CAM was correlated with a more aggressive phenotype of human tongue squamous cancer cells, which was in accordance with downregulated E-cadherin and increased vimentin expression that favor cancer cell undergoing epithelial-to-mesenchymal transition (EMT). More important, we also found a physical association between the L1CAM and epithelial cell growth factor receptor (EGFR), an oncogenic molecular strongly related to EMT. Thus, we hypothesize that L1CAM functionally cooperates with EGFR to promote OSCC progression and metastasis though the EMT, and could serve as a promising molecular target for OSCC in combination with EGFR tyrosine kinase inhibitors. Specific Aim 1 will assess the functional property of L1CAM on OSCC local tumor growth and distant metastasis, and its potential use as an EMT marker in experimental animal models. Characterization of a novel biomarker would lead to improvement in the diagnosis and prognosis of OSCC. Specific Aim 2 will define the molecular pathway underlying the regulation of L1CAM-mediated cellular alternations during OSCC progression, in a primary focus on the signaling convergence of L1CAM and EGFR. An understanding of how the cell adhesion molecule influences EMT is the basis for creating strategies to prevent or diminish disease occurrence. Specific Aim 3 will develop an L1CAM-directed gene therapy approach to eliminate the most aggressive EMT-like OSCC cells with highest gene transduction efficacy and safety as well as clinical beneficial to EGFR tyrosine kinase inhibitors in orthotopic OSCC animal models. This study will not only improve our understanding in the molecular mechanism of EMT in oral cancer, but also accelerate the development of novel targeted therapies for the treatment of local and disseminated OSCC. Project 9: Establishment of Animal Core for cancer Research The principle of Animal Core in Animal Center of Center for Molecular Medicine is to provide the investigators with a better understanding of the possible knowledge and aptitude to utilize animal models in the execution of the research projects. The Animal Core will also minimize the cost of utilizing animal models by consolidating the equipment, expertise and animal resources in their research program. This will ensure an economical use of animal model research. Therefore, there are three aims in this Animal Core to support the other seven research projects. Aim 1: To ensure the efficient planning of experimental animals. Aim 2: To provide the necessary facilities, faculty and staff expertise. Aim 3: To provide expertise in mouse pathology with up-to-date proficiency in cancer pathobiology. Aim 4: To assistant the animal experiment proceed of the seven research projects. |
