結締組織生長因子於人類大腸直腸癌之角色研究

Abstract

大腸直腸癌在台灣是造成國內癌症死亡原因的第三位，每年新增的病例數約為七千例，僅次於肝癌和肺癌。就癌症分期而言，第一期的五年存活率約在90%，第二期的五年存活率約在60-80%，第三期的五年存活率則約在30-60%，若是有遠端轉移的患者﹝第四期﹞，則其五年存活率大概只有5%。而造成這些癌症病人的死亡主因是局部的復發﹝例如術後的腹膜侵犯﹞和遠端器官﹝常見的器官依序是肝臟、肺與骨骼﹞的轉移。因此，尋找這群病人的可能危險因子以及了解其相關的分子機轉，對癌症病患的治療就顯得相當的重要。在過去十年內，一群新的蛋白質所構成的CCN家族，已經被發現並加以分析。現有的結果顯示，這群蛋白似乎和很多重要生理功能的調節有關，包括細胞的分化、生長、凋亡以及和細胞外基質的附著、血管新生等都有很大的相關。因此，我們就針對CCN家族中的結締組織生長因子(CTGF)在大腸直腸癌所扮演的角色來作一系列的研究。由於學生本身是一位臨床醫師，因此在研究方向上，主要是針對結締組織生長因子和造成這些癌症病人死亡的主因中最主要的腹膜侵犯和遠端器官轉移之間的可能影響和臨床意義來著手。 首先，我們的病理組織切片免疫染色的結果顯示，結締組織生長因子(CTGF) 在第二、三期大腸直腸癌患者中，具有預測病人預後的能力。也就是說當病人的免疫染色結果為結締組織生長因子高度表現時，其五年存活率高於低表現者，且具有統計顯著性；同時其五年的疾病復發率也較低表現性者來的有義意的減少。另一方面在細胞株的研究上也獲得類似的結果：當在高表現結締組織生長因子、低侵犯性的大腸直腸癌細胞株(CT26)予以轉殖anti-sense CTGF 質體時，其侵犯能力會增加；同時這樣的細胞株(CT26/ anti-CTGF)在模擬大腸直腸癌肝臟轉移的小鼠脾臟內注射的動物實驗中，也顯示出其肝臟轉移的能力比母細胞株(CT26/neo)來的強。這些證據都明白地指出：結締組織生長因子的表現能影響大腸直腸癌細胞的侵襲和轉移能力。至於大腸直腸癌已知的致病序列機轉中，都會強調Wnt/β-catenint/Tcf-4的訊息傳遞；也就是在大腸直腸細胞癌化的過程中，Wnt/ β-catenin/Tcf-4訊息會被啟動而造成下游的作用因子如：MMP-7， cyclin D1，uPAR等被表現出來。在我們的研究中也指出結締組織生長因子(CTGF)的表現確實會在Wnt/β-catenin/Tcf-4的reporter assay 中減少其數值。 另一方面，大腸直腸癌的腹腔轉移是在治癒性大腸直腸切除後排名第二位的轉移形式 (25-35%)，僅次於肝臟轉移。在臨床上，一開始診斷出有大腸直腸癌時，大約有10﹪的病人就已經有癌細胞的腹腔轉移。這類的病人其預後相當的不好（平均存活時間只有六個月），因此一般皆被認為是癌末的情況，而只接受緩解性的治療而已。雖然近來有報告指出，若選擇性的使用減縮手術和腹腔內化學治療，可以增加病人的存活率，但其術後的高併發症發生率（22.9%）和致死率（4% ）無形中也限制了這項治療方法的使用空間。因為癌細胞腹腔轉移的步驟是自原病灶脫離，而能在腹腔液中存活，接著是沾附至腹膜而進一步的增殖生長。所以我們分析結締組織生長因子是否也在此步驟中扮演促進細胞凋亡與抑制沾附能力的角色，而造成腹腔轉移的抑制。在第三章中，我們分析出高表現結締組織生長因子的大腸直腸癌細胞株，在細胞離體實驗中，對無貼狀態引起的細胞凋亡較明顯，而且其沾附能力也隨之降低。我們亦進行動物腫瘤生長的實驗來證明結締組織生長因子確能抑制腹腔轉移的現象。 總結來說，本論文提供了結締組織生長因子可抑制人類大腸直腸腺癌遠端轉移及腹腔生長的證據；作為一個癌細胞侵襲與沾附抑制蛋白，無貼附狀態細胞凋亡促進者，結締組織生長因子作為臨床治療的蛋白質類藥物，是相當具潛力而可期待的。

Colorectal cancer is the third leading cause of cancer death in men and women in Taiwan and the second cause in the United States. The major cause of cancer death in patients with colorectal cancer is local recurrence and distal metastasis after curative surgical treatment, intensive chemotherapy and/or radiotherapy. So, it is important for these patients to find the possible risk factors and underlying molecular mechanism of local recurrence and metastasis. Over the last decade, a new family of structurally related proteins has been described that comprises cysteine-rich 61(CYR61; CCN1), connective tissue growth factor (CTGF; CCN2), nephroblastoma over- expressed (NOV; CCN3), Wnt-induced secreted protein-1 (WISP-1; CCN4), WISP -2 (CCN5), and WISP-3 (CCN6). Collectively, CCN proteins appear to be important regulators of diverse cellular functions including cell cycle progression, division, chemotaxis, differentiation, apoptosis and adhesion and angiogenesis. Because I am a clinical surgeon, the research direction would focus on the clinical meanings and the underlying possible mechanism between CTGF and the major causes of cancer death in patients with CRC ----- distal metastasis and peritoneal seeding. In our data, Stages II and III CRC patients whose tumors displayed high CTGF expression in the immunohistochemical staining had a significantly higher overall survival and a disease-free advantage over CRC patients with a low CTGF expression (P < 0.001). In in vitro invasion assays, alterations in the CTGF level in CRC cell lines modulated their invasive ability with an inverse correlation. In addition, a reduction in the CTGF level of CT-26 cells after stable transfection with antisense CTGF resulted in increased liver metastasis in BALB/c mice. The activity of β-catenin/Tcf signaling pathway in these CTGF-transfected cells was strongly attenuated at the level of transcription. Besides, peritoneal dissemination is an important metastatic mode of intra-abdominal cancers such as gastric, colorectal, and ovarian cancer. At initial diagnosis of colorectal cancer, the peritoneal surface is involved by tumor in 10 to 15% patients. Next to the liver, peritoneal surfaces are the most common sites for cancer recurrence (25% to 35%) after so-called curative resection of colorectal cancer. Because this mode of metastasis severely affects the prognosis of cancer patients (median survival 5-6 months), colorectal peritoneal carcinomatosis has previously been considered a pre-terminal condition suitable for palliative treatment. The steps of peritoneal metastasis include detaching from the primary tumor mass, surviving in the abdominal fluid (ascites) and then adhering to the peritoneum, proliferating in the seeding sites. In order to understand whether CTGF is involved in anoikis and adhesion regulation, we analyzed that several colorectal cancer cell lines which expressed different levels of CTGF undergo apoptosis in anoikis condition and own adhesion ability. In chapter 3, we demonstrated that highly CTGF expressed cells showed increased sensitivity to anoikis and reduced adhesion capacity in vitro. We further demonstrated that CTGF inhibited in vivo tumor growth and peritoneal seeding by animal models. In summary, these studies provide evidences for the tumor metastasis suppressing effect of CTGF in the colorectal adenocarcinoma. Herein, as an invasion blocker, anoikis promoter and adhesion inhibitor that specifically targets cancer cells, CTGF has a unique potential for colorectal adenocarcinoma treatment.