結締組織生長因子於乳癌轉移及抗藥性之調控角色

Abstract

癌細胞轉移以及抗藥性ㄧ直是癌症治療上急需突破之重要課題，已知結締組織生長因子在晚期乳癌患者組織中表現量較高，然其對於癌細胞功能之調控機制仍屬未知。本論文分為兩部份，第一部分(第一章)探討CTGF對於乳癌細胞移動暨轉移能力之影響，我們使用CTGF plasmid將其過度表現於MCF-7細胞株 (MCF-7/CTGF)，發現細胞移動能力之明顯增加，同時細胞外型也產生顯著的紡錘型化改變；反之使用antisense CTGF抑制MDA231細胞株之CTGF表現後 (MDA231/AS)，發現細胞之移動能力更為顯著下降，其外型也轉變為上皮型。以integrin alphavbeta3 中和抗體處理後可發現CTGF所造成的ERK1/2活化受到阻斷，可見CTGF是透過integrin alphavbeta3調控ERK1/2之訊息傳導路徑。此外，我們以cDNA microarray分析與轉移相關之分子，並發現其中與癌細胞轉移密切相關之S100A4基因其mRNA表現之顯著差異。以AS-S100A4抑制MCF-7/CTGF 細胞S100A4表現後，發現CTGF促進之細胞移動受到阻斷；而將S100A4過度表現在MDA231/AS 細胞則回復了因抑制CTGF所造成細胞能力之下降，同樣的結果也顯示在動物的乳癌轉移模式實驗中，可知S100A4對於CTGF促進癌細胞轉移之關鍵角色。我們也使用MEK1 plasmid以及ERK1/2抑制劑進一步證明CTGF是透過ERK1/2調控S100A4。最後，我們以real-time RT-PCR分析並發現臨床上，乳癌組織中CTGF與S100A4之mRNA表現量呈現高度相關。本部份證明了CTGF會透過integrin alphavbeta3/ERK1/2/S100A4之路徑調控乳癌細胞之轉移。此外，由於大部分的晚期癌症轉移通常伴隨著腫瘤抗藥性產生，我們在第二部份(第二章)的研究中也進一步探討CTGF對於癌細胞抗藥性的影響。我們以IHC分析並發現病患乳癌組織中CTGF蛋白之表現量與其化療之效果具有高度反相關。在細胞實驗中，以doxorubicin及paclitaxel處理，可發現CTGF過度表現(MCF-7/CTGF)顯著地增加癌細胞之clonogenic ability，存活能力，以及apoptosis之下降；反之，CTGF表現受到阻斷時(MDA231/AS)則導致相反之結果。我們進一步發現CTGF表現會正向調控Bcl-xL及cIAP1表現。在MCF-7/CTGF細胞中，以siRNA抑制Bcl-xL或cIAP1皆可阻斷CTGF促進抗藥性之效果；反之，利用plasmid轉殖表現因CTGF下降而受抑制之Bcl-xL或cIAP1則可回復其抗藥性之下降。處理integrin alphavbeta3中和抗體或ERK1/2抑制劑則阻斷CTGF調控之Bcl-xL及cIAP1高表現，顯示integrin alphavbeta3/ERK1/2 同樣對於CTGF於抗藥性之調控功能相當重要。以IHC分析發現臨床上CTGF與 Bcl-xL之蛋白表現量具有顯著正相關。本部份證明CTGF對於乳癌細胞之抗藥性之重要性，也找出其中調控Bcl-xL/cIAP1之機制。

Connective tissue growth factor (CTGF) expression is elevated in advanced stages of breast cancer, but the regulatory role of CTGF in invasive breast cancer cell phenotypes is unclear. In the first part (Chapter 1), we found that over-expression of CTGF in MCF-7 cells (MCF-7/CTGF) enhanced cellular migratory ability and spindle-like morphological alterations, as evidenced by actin polymerization and focal adhesion complex aggregation. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) impaired cellular migration and promoted a change to an epithelial-like morphology. A neutralizing antibody to integrin alphavbeta3 significantly attenuated CTGF-mediated ERK1/2 activation and cellular migration, indicating that integrin alphavbeta3/ERK1/2 signaling pathway was critical in mediating CTGF function. Moreover, the cDNA microarray analysis revealed CTGF-mediated regulation of the critical prometastatic gene S100A4. Transfection of MCF-7/CTGF cells with AS-S100A4 reversed the CTGF-induced cellular migratory ability, while over-expression of S100A4 in MDA231/AS cells restored their high migratory ability. Genetic and pharmacological manipulations suggested that the CTGF–mediated S100A4 up-regulation was dependent on ERK1/2 activation, with expression levels of CTGF and S100A4 being closely correlated with human breast tumors. We conclude that CTGF plays a critical role in the migratory/invasive processes in human breast cancer by a mechanism involving the activation of the integrin alphavbeta3/ERK1/2/S100A4 pathway. In addition, since most metastatic disease only responds transiently to chemotherapy and most cancer patients die of metastatic disease. In the second part (Chapter 2), we examined whether CTGF expression could confer drug resistance in human breast cancer. In breast cancer patients who received neoadjuvant chemotherapy, CTGF expression inversely associated with chemotherapy response. Overexpression of CTGF in MCF-7 cells enhanced clonogenic ability, cell viability and resistance to apoptosis upon exposure to doxorubicin and paclitaxel. Reducing the CTGF level in MDA-MB-231 cells decreased these effects. CTGF overexpression resulted in resistance to doxorubicin- and paclitaxel-induced apoptosis by up-regulation of Bcl-xL and cIAP1. Knockdown of Bcl-xL or cIAP1 with specific siRNAs abolished the CTGF-mediated resistance to apoptosis induced by the chemotherapeutic agents in MCF-7/CTGF cells. Inhibition of ERK1/2 effectively reversed the resistance to apoptosis as well as the up-regulation of Bcl-xL and cIAP1 in MCF-7/CTGF cells. A neutralizing antibody against integrin alphavbeta3 significantly attenuated CTGF-mediated ERK1/2 activation and up-regulation of Bcl-xL and cIAP1, indicating that the integrin alphavbeta3/ERK1/2 signaling pathway is essential for CTGF functions. The Bcl-xL level also correlated with the CTGF level in breast cancer patients. We concluded that CTGF expression could confer resistance to chemotherapeutic agents through ERK1/2-mediated Bcl-xL/cIAP1 up-regulation of a survival pathway.