肺癌轉移相關基因之探討暨臨床預後方法之研發

Abstract

肺癌細胞轉移的分子基礎研究有助於治療方式的改善及發現新治療標的分子。正確的預後評估及治療方式的選擇則決定癌症病人的存活率。因此，本論文的研究目標在於探討肺癌轉移相關基因及研發臨床預後的方法。 首先利用資料庫分析，並以一對具有不同轉移力的肺癌細胞株來探討癌轉移相關基因。經過篩選後，發現一個有趣的基因－人類第八號激肽釋放酶，它是屬於人類組織激肽釋放酶基因家族中的一員。人類第八號激肽釋放酶為絲胺酸蛋白酶，已知為卵巢癌病人預後良好的評估指標。然而，其生物意義卻不清楚。實驗結果顯示藉由轉殖於具有高侵襲力肺癌細胞株中人類第八號激肽釋放酶基因的過量表現，可抑制肺癌細胞的侵襲力。相反地，若利用具專一性的短髮夾核糖核酸來抑制細胞內生性人類第八號激肽釋放酶，則可增加肺癌細胞的侵襲力。根據原位分解技術及細胞附著力偵測的結果顯示，人類第八號激肽釋放酶的剪接產物可分解血清纖維結合蛋白，進而改變細胞外圍環境。去氧核糖核酸微陣列實驗及細胞內肌動蛋白纖維染色結果，顯示了人類第八號激肽釋放酶的剪接產物分解血清纖維結合蛋白後，抑制了整合素的訊息傳遞途徑，並藉由抑制F肌動蛋白的重排而妨礙肺癌細胞的移動能力。此外，以人類Alu序列為標的來偵測並定量小鼠動物實驗中鼠血液內人類肺癌細胞存在多寡的實驗顯示，在活體試驗中人類第八號激肽釋放酶可抑制腫瘤生長及轉移。近一步由臨床研究肺癌病人檢體發現，若肺癌早期病人(第一、二期)的癌組織中測得較高的人類第八號激肽釋放酶基因的表現，則病人有明顯較長的緩和期及較低的復發率。此發現可歸納出人類第八號激肽釋放酶的功能為阻礙腫瘤的轉移，並可利用此基因來當作非小細胞肺癌病人預後的指標。 臨床上最有效於提高療效的方法為早期診斷出癌轉移並施予有效的療程。因此，另一個計畫著重於早期檢測血液中癌細胞的研究。目前肺癌分期及病況評估主要是根據腫瘤影像法。然而此法受限於不夠靈敏，而無法正確地診斷出早期癌轉移的發生。此研究利用數個指標基因來偵測血液循環中的癌細胞，以增加目前肺癌分期及病況評估的正確度，並可用於快速評估藥效。我們利用基因庫來搜尋合適的基因用以偵測血液循環中的癌細胞，經實驗證明其中四個基因可當作標的基因。利用這四個標的基因來偵測五十四個非小細胞肺癌病人血液中癌細胞的存在與否，可達到百分之七十二檢出率。若利用即時定量聚合脢放大偵測法轉換成癌細胞載荷量來評估肺癌病人血液中癌細胞量與臨床結果的關聯性，則病人癌細胞載荷量越高者其治療效果較差且存活時間較短。治療效果不好的病人，其治療後仍可測到血液中癌細胞的存在，並有較短的存活期。藉由此四個標的基因及癌細胞載荷量來反映出肺癌病人血液中癌細胞量，可增強傳統肺癌分期法，進而提高檢測率及快速評估治療效果。此外也可輔佐醫師在治療肺癌病人上給予更適當的治療方式。

Research investigations on the molecular basis of lung carcinoma metastasis are helpful to identify therapeutic targets for metastasis. An accurate prognosis and selection of therapeutic modality determines the survival of cancer patients. Therefore, this thesis aims to characterize metastasis associated genes and develop clinical prognosis assay for lung cancer. Firstly an in silico analysis approach was used to examine metastasis associated genes by a cell line model of human lung adenocarcinoma with different invasive abilities. After screening, one interesting gene was found, human kallikrein 8 (KLK8), a member of human tissue kallikrein gene family. The serine protease KLK8 protein (hK8) is known to be a favorable prognostic marker in ovarian cancer, but the biological basis of this is not understood. The experimental results showed that overexpressing the KLK8 gene in highly invasive lung cancer cell lines suppressed their invasiveness. This role in invasiveness was further confirmed by the fact that inhibition of endogenous KLK8 expression with a specific short hairpin RNA enhanced cancer cell invasiveness. In situ degradation and cell adhesion assays showed that proteins produced from KLK8 splice variants modify the extracellular microenvironment by cleaving fibronectin. DNA microarray experiments and cell staining for actin filaments revealed that the degradation of fibronectin by hK8 suppresses integrin signaling and retards cancer cell motility by inhibiting actin polymerization. In addition, studies in a mouse model coupled with detection of circulating tumor cells by quantitative PCR for the human Alu sequence demonstrated that KLK8 suppresses tumor growth and invasion in vivo. Furthermore, studies of clinical specimens from non-small cell lung cancer (NSCLC) patients showed a 52% recurrence rate for early-stage (stage I & II) patients with low KLK8 expression in their tumor cells and a 23% recurrence rate for patients with high KLK8 expression. Collectively, these findings show that KLK8 retards cancer metastasis and that further investigation of KLK8 as a prognostic marker for NSCLC is warranted. The most promising way to improve prognosis is by means of early metastasis detection. Thus, the other project in this thesis study is focused on detection of disseminated cancer cells of non-small cell lung cancer patients in their peripheral blood. Current lung cancer staging and prognosis methods are based on imaging methods which may not be sensitive enough for early and accurate detection of metastasis. A panel of markers was validated for circulating cancer cell detection to improve the accuracy of cancer staging, prognosis, and as a rapid assessment of therapeutic response. NCI-CGAP database was used to identify potential marker genes for the detection of circulating cancer cells in peripheral blood. A panel of 4 marker genes was identified and experimentally validated. With these marker genes, the results achieved an overall positive detection rate of 72% for circulating cancer cells in the peripheral blood of 54 NSCLC patients. Nested real-time quantitative PCR (qPCR) and a scoring method using cancer cell load, Lc, were employed to correlate the amount of circulating cancer cells with clinical outcomes in NSCLC patients. Patients who had higher Lc values had worse outcomes and shorter survival times. Patients with poor therapeutic response were revealed by positive detection of circulating cancer cells after therapy. The results correlated well with the patients’ survival time. Circulating cancer cell detection by a panel of markers and the Lc scoring method can supplement the current TNM staging method for improved prognosis and for rapid assessment of therapeutic response. Together, they may facilitate the design of better therapeutic strategies for the treatment of NSCLC patients.