肺癌表皮生長因子受體突變分析及其相關訊息傳遞與開發標靶治療藥物之研究

Detection of epidermal growth factor receptor mutations with multiplex PCR concerning its signaling and development of the target therapeutic drugs to lung cancers

林慶雄(Ching-Hsiung Lin)

指導教授 ： 張建國

高雄醫學大學/醫學院/醫學研究所/博士(2011年)

https://doi.org/10.6832/KMU.2011.00018

摘要

背景：表皮生長因子(Epidermal growth factor, EGF)與其接受器(Epidermal growth factor receptor, EGFR)突變是影響肺癌細胞轉移的重要因子，而癌細胞轉移會降低肺癌病患的存活率。因此發展可靠快速方法檢測表皮生長因子接受器突變類型為當務之急。另一方面，慢性阻塞性肺病(Chronic Obstructive Pulmonary Disease, COPD)是一種肺部慢性發炎及氧化及抗氧化平衡失調，近年來的研究發現香菸中的物質會促進腫瘤壞死因子α的產生，增加基質金屬蛋白酶-9活性，導致肺部發炎及癌細胞的侵犯。所以開發標靶治療的藥物是目前醫界一致的努力方向。
材料與方法：GMI（Ganoderma. Microsporum，微孢子靈芝免疫調節蛋白）對肺癌細胞抑制方面以傷口癒合移行實驗(wound-healing assay)及博登轉移實驗(Boyden chamber assay)來分析肺癌細胞移行與侵犯的能力。使用西方點墨法觀察EGFR、AKt與STAT3蛋白的表現。利用沉澱試驗分析Rac1/Cdc42的GTPase活性。以GMI與表皮生長因子處理A549細胞，並以免疫螢光染色後，透過顯微鏡觀察細胞中骨架結構是否受到影響。確認GMI功能後，發展以multiplex PCR 和primer extension方法同時快速檢測表皮生長因子接受器多項突變型。在GMI對於腫瘤壞死因子α的調控方面以Hgelatin zymography 偵測MMP-9蛋白活性，並以RT-RCR及luciferase reporter assay方式分析MMP-9基因轉錄調控。利用in vitro (EMSA)和in vivo (CHIP)結合實驗證實NF-kB結合能力的影響， 再使用西方點墨法及核質分離的方式同步觀察NF-kB調控分子的表現。最後以免疫螢光染色，透過顯微鏡觀察細胞中NF-kB進出細胞核的狀況。
結果：我們使用表皮生長因子當作細胞侵犯的促進劑，藉此觀察GMI如何影響表皮生長因子所造成之A549肺癌細胞株侵犯。從wound-healing assay以及Boyden chamber assay結果中可見，GMI能有效抑制表皮生長因子所誘發之細胞移行與侵犯。並發現GMI能抑制表皮生長因子所誘發之表皮生長因子接受器磷酸化及AKt訊息傳遞路徑活化。此外，表皮生長因子所促使之Cdc42 GTPase活化，亦可受GMI所抑制。而表皮生長因子所促使之Rac1 GTPase活化，則可些微受GMI影響。我們也發現GMI能抑制表皮生長因子所誘發之微絲解聚合作用。另外，我們在此研究使用多重PCR 方式分析得表皮生長因子接受器基因啟動子-216區域及exons 18-21 的位置codon 719, 746, 750, 790及858突變，分析結果有81位病人檢體其中26位具突變性。而另一方面，GMI也可以抑制NF-kB進入細胞核與MMP-9的啟動子結合，降低由TNF-α所誘發的MMP-9轉錄活性，同步地也可以看見因此細胞侵犯能力顯著地受到抑制。
結論：本研究探討GMI對於抑制肺癌轉移之分子機制，為GMI在抗癌功能上之新發現，未來於臨床上可能搭配化療藥物共同使用，達到治療肺癌的效果。而發展多重快速PCR檢測EGFR突變方式有利於標靶藥物的使用選擇。而臨床上慢性阻塞性肺病人氣道發炎的主要刺激因子TNF-α，也同步受到GMI抑制其反應的分子訊息途徑。

關鍵字

none

並列摘要

Dysregulation of human epidermal growth factor receptor pathways by over-expression or constitutive activation can promote lung tumor processing including angiogenesis and metastasis and is associated with poor prognosis in non-small cell lung cancers. Ganoderma also known as Lingzhi has been one of the most popular chemoprevention mushrooms in East Asia for centuries. Among many bioactive components identified from Ganoderma, an immunomodulatory protein is the major ingredient for the treatment of lung cancer. Recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. However, knowledge on the pharmacological and molecular mechanisms in suppressing EGF-mediated tumor invasion and metastasis is poorly understood. The goal of the study is investigate in suppressing tumor invasion and metastasis activity of GMI.
Our Results show GMI exhibited an inhibitory effect on EGF-induced migration and invasion. GMI treatment with EGF presented the most potent anti-migration and anti-invasion properties on Boyden chamber assay. GMI inhibited EGF-induced phosphorylation and activation of EGFR and AKt pathway kinases in a dose-dependent manner. Additionally, the EGF-induced activation of Cdc42 GTPase was inhibited by GMI, while GMI had little effect on the EGF-induced activation of Rac1 GTPase. GMI also inhibited the EGF-induced microfilament depolymerization. These findings are the first to reveal the novel functions of GMI in tumor anti-metastasis and the molecular basis for its anticancer action.
On the other hand, epidermal growth factor receptor (EGFR) kinase domain mutations hyper-activate the kinase function and confer kinase addiction of the non-small-cell lung cancer (NSCLC) tumor cells. Almost all of these mutations are located within exons 18-21. The -216 single nucleotide polymorphism in the promoter region is associated with increased EGFR production. We present a method for detecting these common mutations in 81 cases of NSCLC. The protocol is based on the multiplex amplification of promoter region and exons 18-21 of the EGFR genes in a single tube, followed by primer extension of the PCR products using various sizes of primers to detect base changes at -216 promoter region and codons 719, 746-750, 790, 858 of the EGFR gene. We compared the results with that from direct sequencing for detecting EGFR mutations in 81 cases of NSCLC. The two methods identified the same 26 mutations, but our method is superior to direct sequencing in terms of the amount of work and time required. We presented a simple and fast method to detect mutations of EGFR genes in NSCLC.
Matrix metalloproteinase 9 (MMP-9) has been implicated in airway injury in chronic obstructive pulmonary disease (COPD), lung inflammation, and lung cancer and plays a major role in tumor necrosis factor-α (TNF-α)-stimulated tumor invasion and lung inflammation. MMP-9 activity is promoted by the pro-inflammatory cytokine TNF-α.
GMI, cloned from Ganoderma microsporum and purified, is one of the recombinant fungal immunomodulatory proteins. To understand the molecular mechanisms involved in the suppression of TNF-α-mediated tumor invasion and inflammation, GMI modulation of this pathway was investigated in human alveolar epithelial A549 cells in this study.
Our results show GMI exhibited an inhibitory effect on TNF-α-induced invasion, with GMI treatment and TNF-α exposure presenting the most anti-invasive properties on Boyden chamber assay. GMI reduced TNF-α-induced MMP-9 activities on gelatin zymography assay through inhibition of MMP-9 transcriptional activity. RT-PCR and MMP-9 promoter luciferase analysis revealed that GMI inhibits the transcription of MMP-9 mRNA. Moreover, in vitro and in vivo binding assay, an electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation assay (ChIP) demonstrated that GMI suppresses DNA binding of nuclear factor (NF)-κB transcription factors to MMP-9 promoter. Western blot analysis indicated that GMI blocks the phosphorylation and degradation of IκBα, which in turn leads to suppression of the phosphorylation and nuclear translocation of p65. Thus, our results indicated that GMI mediates antitumor invasion and anti-inflammatory effects through modulation of NF-κB/MMP-9 pathways.

並列關鍵字

Immunomoduatory protein ； GMI ； tumor necrosis factor-α ； NF-kB ； MMP-9 ； invasion

參考文獻

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