探討C型血管內皮生長因子在人類肺腺癌之角色及調控機轉

The Role of Vascular Endothelial Growth Factor-C (VEGF-C) in Human Lymphangiogenesis and Metastasis of Lung Adenocarcinoma

蘇振良(Jen-Liang Su)

指導教授 ： 郭明良

國立臺灣大學/醫學院/毒理學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.00775

摘要

根據世界衛生組織統計，現今世界每隔六秒就有一個生命被癌症所剝奪，形成國人健康之一大隱憂。癌轉移不但是癌症治療失敗的主因更是癌症致死的第一殺手，癌細胞可經由釵h途徑進行轉移，令人防不勝防。癌轉移之途徑可分為：1.直接侵襲周邊組織 2.藉由血管達到轉移 3.藉由淋巴系統達到轉移。理論上而言,對於腫瘤轉移過程中任一步驟進行抑制,皆可達到治療之目的。對於腫瘤細胞進行轉移之機轉了解越清楚，我們對於癌症治療就可以增加更多的可能性。淋巴管新生（Lymphangiogenesis）是腫瘤藉由淋巴系統轉移的一項重要過程，也是目前在生物醫學研究上較具潛力的治療癌症的新方向。
C型血管內皮生長因子（VEGF-C）是促進淋巴管新生之重要因子，就細胞分子層面而言，VEGF-C能和血管內皮生長因子受體3號（VEGFR3/Flt-4）結合，活化下游之基因導致淋巴管內皮細胞增生，進而促使淋巴管生成，但時至今日，關於這些淋巴管新生因子的調控仍是不甚明瞭。Flt-4是位於細胞膜上之receptor tyrosine kinase，一但與結合子（ligand）結合，則會產生自體磷酸化（auto-phosphorylation）。近來有釵h研究指出，VEGF-C/Flt-4此一結合子/受體系統亦表現在非淋巴內皮細胞上，例如一些腫瘤細胞，包括肺癌、大腸直腸癌、頭頸部癌等等。更有臨床報告指出腫瘤細胞所表現的VEGF-C/Flt-4與其預後有相關性存在，因此VEGF-C/Flt-4對腫瘤細胞應有直接之生理弁遄A但此仍屬未知之數。在本篇論文中，將針對VEGF-C之調控機制加以釐清並探討VEGF-C/Flt-4除了淋巴管新生外之生理弁遄C
發炎反應與癌症之間的關聯性已發現酗[，釵h科學實驗證據顯示第二型環氧化酵素(Cyclooxygenase-2, COX-2)此一參與發炎反應之主要酵素和腫瘤的形成有相當密切的關聯。早期與COX-2基因有關的癌症研究大多集中在腸胃道腫瘤，但近來發現 COX-2 在腸胃道之外的器官腫瘤也扮演了相當重要的角色，例如肺癌。在腫瘤形成的過程中，COX-2基因的表現與細胞增生及侵襲轉移的能力密不可分，並且可以調控細胞抗凋亡的能力。而在近來的報導中除了指出 COX-2 可作為預後不良的指標外，更顯示了 COX-2 表現較高的腫瘤細胞較具有淋巴轉移能力，這些結果在在都說明了COX-2在腫瘤形成過程中的多弁鄔吨峟垠n性。在第一章中，我們發現COX-2和VEGF-C不論是在肺腺癌組織，或是肺腺癌組織細胞株中皆具有正相關性之表現。在利用外加COX-2之生理產物或是以過度表現COX-2基因之方式，可以發現VEGF-C之表現確實受到COX-2所誘發；反之，處理COX-2抑制劑或是以antisense COX-2減少COX-2表現，皆會降低VEGF-C在肺腺癌細胞株之表現量。文章中更進一步釐清COX-2誘發VEGF-C之表現是透過EP1-Src-HER2/Neu此一訊息傳遞路徑。除此之外，對肺腺癌組織進行免疫組織染色結果發現COX-2之表現與淋巴管之密度呈現正相關性。此項研究是首次證實COX-2可以促進VEGF-C之表現，並推測因此而產生較多的淋巴管以利腫瘤細胞進行轉移，因此在針對以抑制淋巴管新生為治療癌症的策略中，COX-2可當作一個具有潛力之標的分子。
腫瘤細胞之移動性及侵襲能力是決定癌轉移之重要因素，而在肺癌細胞本身之VEGF-C與Flt-4表現情形及弁酮假臚G章之研究重點。在第二部份中，我們發現不論是在肺腺癌組織之腫瘤細胞或是肺腺癌細胞株皆有VEGF-C與Flt-4之表現，且琪表現之情形與肺腺癌患者之淋巴轉移程度及細胞株之侵襲能力呈現高度之正相關。由in vitro及in vivo的實驗中，我們更證實了VEGF-C會藉由活化Flt-4而增加其侵襲能力並增加其肺轉移之機率。我們更發現contactin-1此一細胞黏著分子(Cell Adhesion Molecule,CAM)會受到VEGF-C所誘發且為VEGF-C促進腫瘤細胞進行轉移所必需，此一論述在動物實驗中亦得到相同結果支持。就機制層面探討，我們發現VEGF-C誘發contactin-1需經由活化Flt-4，接著透過Src-p38 MAPK此一訊息傳遞路徑。這些結果說明了VEGF-C除了會促進淋巴管新生外，更可藉由活化肺癌細胞之Flt-4而有利於癌細胞轉移。除了給予VEGF-C心的生理弁鄍~，我們更是首次報導contactin-1與肺癌轉移之關係。因此，藉由對VEGF-C/Flt-4在癌轉移之角色的探討，將可對癌轉移之治療增加可能之作用目標。
綜觀前述，我們的研究對VEGF-C在腫瘤發展過程中的多弁鄔吨峟垠n性提供了嶄新且直接的證據，並將發炎反應與癌症藉由VEGF-C作為連結。隨著生物醫學的日新月異及對VEGF-C、Flt-4與其相關之訊息傳遞路徑的了解，以VEGF-C做為臨床治療之標的分子是相當具有潛力的。

關鍵字

血管內皮生長因子受體3號 ； C型血管內皮生長因子 ； 第二型環氧化酵素 ； 細胞黏著分子 ； 癌轉移 ； 訊息傳遞 ； 淋巴管新生

並列摘要

Mortality in cancer patients principally result from metastatic spread of cancer cells to distant organs. Tumor metastasis involves a series of complicated steps that includes the detachment of tumor cells from the primary tumor mass, microinvasion of tumor cells into stromal tissue, enter into the body’s circulatory system — known as intravasation and might extravasate from the circulation into the surrounding tissue. Clinical and pathological observations suggest that for many carcinomas, transport of tumor cells via lymphatics is the most common pathway of initial dissemination, with patterns of spread via afferent lymphatics following routes of natural drainage.
Lymphangiogenesis is an important step in tumor cells metastatic spread through the lymphatic system, and potent to be the next cancer target for therapeutic intervention. Clinical findings have long suggested that by providing a pathway for tumor cell dissemination, tumor-associated lymphatics are a key component of metastatic spread. Recent publications have demonstrated that vascular endothelial growth factor (VEGF)-C, ligand for the VEGF receptor-3, is the lymphangiogenic factors that induce the formation of lymphatic vessels within and around tumors. In addition, these studies demonstrated that VEGF-C-overexpressing tumors increase intratumoral lymphangiogenesis, enhancing metastatic spread via the lymphatics. However, the regulation of these lymphangiogenic factors and the lymphangiogenesis process are not clear until now. Flt-4 has been proposed as a specific marker for lymphatic endothelial cells. Recent studies indicated that Flt-4 also expressed in a variety of human malignancies, including lung adenocarcinoma, colorectal adenocarcinoma, head and neck carcinoma, neuroblastoma, and Kaposi's sarcoma. However, the biological significance of VEGF-C/ Flt-4 axis in cancer cells is completely unknown. In this dissertation, we attempted to elucidate the role of VEGF-C and Flt-4 in human lung adenocarcinoma, regarding the mechanisms by which expression of VEGF-C in tumors and defined the non-lymphangiogenic function of VEGF-C.
The association between chronic inflammation and the development of cancer has been recognized. Cyclooxygenase-2 (COX-2), the inducible form of the COX enzymes, catalyzes synthesis of large amounts of prostaglandins with diversified biological activities, and its dysregulation plays a vital role in inflammation, tissue damage, and tumorigenesis. A great amount of evidence suggests the close association of up-regulation of COX-2 with tumor invasion and metastasis in human colorectal, breast, and lung tumors. In human lung tumors, COX-2 is predominantly expressed in adenocarcinomas, especially in patients with lymph node metastasis. It is tempting to speculate that certain un-identified downstream gene(s) of COX-2 may have a role in tumor metastasis. In the chapter 1, our work suggests a cause-effect link between COX-2 overexpression and tumor lymphangiogenesis through VEGF-C activity in lung adenocarcinoma. The up-regulation of VEGF-C by COX-2 through EP1/Src/HER-2/Neu signaling pathway was demonstrated in this study. Our findings also provide new therapeutic modalities for lymphangiogenesis as lymph node metastasis inhibitor by targeting COX-2.
Expression of Flt-4 has been also reported that significantly correlated with the different stages and poorer survival in cervical and colorectal cancer, suggesting a role of VEGF-C/Flt-4 signaling in tumor progression. The migration and invasion ability of cancer cells play a critical role in tumor metastasis. In the chapter 2, we clearly provide a novel function of VEGF-C/Flt-4 axis in cell invasion and metastasis in lung adenocarcinoma. The promotion of cell mobility in response to VEGF-C was required the involvement of adhesion molecule contactin-1. The up-regulation of contactin-1 by VEGF-C through Src and p38 MAPK signaling pathway was demonstrated. VEGF-C/Flt-4 and/or its downstream effector contactin-1 may become a possible therapeutic target for patients with lung adenocarcinoma.
In summary, our studies provide evidences for regarding the mechanisms by which expression of VEGF-C in tumors and the non-lymphangiogenic role of VEGF-C in lung adenocarcinoma metastasis. With progress in molecular biology of VEGF-C and Flt-4, its value as atherapeutic target is highly promising.

並列關鍵字

invasion ； VEGF receptor-3 ； vascular endothelial growth factor–C ； cyclooxygenase-2 ； metastasis ； contactin-1 ； lymphangiogenesis

參考文獻

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