間質蛋白酶在表皮生長因子與K-Ras所誘發之大腸癌細胞侵襲移動能力中扮演角色

Abstract

表皮生長因子受器(EGFR)訊息傳遞被認為與大腸癌的侵襲和轉移是有著莫大關係。而在臨床的分析發現，若將病患施予表皮生長因子受器(EGFR)抑制劑作標靶治療，對於具K-Ras 基因發生突變的病人，其治療效果通常是不佳的。然而表皮生長因子受器與K-Ras 促使大腸直腸癌惡化之進程、侵襲與轉移的詳細機制，仍有許多不了解。在本篇研究中，我們利用一組人類大腸癌惡化轉移細胞模式，包括KM12C, KM12-L4, KM12-SM細胞，其中KM12-L4、 KM12-SM由細胞KM12C經轉移篩選而來，相較KM12C細胞具更強的侵襲與轉移能力。由我們的結果顯示, 表皮生長因子受器的蛋白質表達量、磷酸化程度，以及間質蛋白酶的活性，在具高轉移侵襲力的大腸癌KM-L4和KM12-SM細胞中，與KM12C相較下均明顯的增加，並與細胞的侵襲能力呈現正相關。再者, 利用基因剔除的實驗，發現表皮生長因子受器及間質蛋白酶，均參與EGF所誘發增加的大腸癌細胞侵襲能力。同時，進一步由抑制劑的實驗發現，EGF可以透過活化PI3K/AKT而非MEK/Erk1/2之訊息路徑，進而活化間質蛋白酶及促進大腸癌細胞侵襲力的上升。然而, 本研究也進一步探討表皮生長因子受器下游K-Ras蛋白在大腸癌侵襲能力扮演之角色。我們利用PCR方法得到wild-type K-Ras, 並經 site-directed mutagenesis技術, 建立了2個持續活化的mutant K-Ras (G12D and G13D). 結果顯示大量表達的K-Ras 的組別 (wild-type, G12D and G13D) 可以促進KM12C 細胞的侵襲能力及間質蛋白酶的活化。同時我們由 PI3K 或 MEK 抑制劑的實驗結果發現, 暗示著PI3K 或 MEK 訊息路徑皆參與K-Ras 誘導之大腸癌細胞侵襲能力。有趣的是，具持續活化的mutant K-Ras (G12D and G13D) 細胞在表皮生長因子受器抑制劑afatinib的處理下, 對於細胞侵襲能力以及間質蛋白酶活性的抑制效果具有抵抗之能力, 暗示著K-Ras 誘導間質蛋白酶的活化 可能對於抗表皮生長因子受器治療之抗藥性扮演角色。綜合上述結果，表皮生長因子受器與K-Ras 透過PI3K/AKT 訊息活化間質蛋白酶進而促使大腸癌細胞侵襲能力增加。而我們的結果也進一步暗示, 或許以細胞周圍間絲胺酸蛋白酶為治療標的, 可以為抗表皮生長因子受器治療具抗藥性的病人, 提供一個新的治療策略。

EGFR signaling has been proposed to play an important role in colorectal cancer progression and metastasis. The clinical results have further shown that EGFR target therapies have no significant effect on the colorectal cancer patients with K-Ras mutations. However, how EGFR and K-Ras signaling promotes colorectal cancer progression, cell invasion and metastasis remains largely unknown. In this study, the roles of EGFR, K-Ras and membrane-anchored serine protease matriptase in colorectal cancer cell invasion were further addressed, using a human colorectal cancer metastasis progression model (KM12C, KM12-L4 and KM12-SM cells). KM12-L4 and KM12-SM cells are two cell lines generated from KM12C cells using in vivo metastatic selection, and exhibit higher invasion and metastasis potentials than KM12C cells. The results showed that the protein and tyrosine phosphorylation levels of EGFR were increased in KM12-L4 and KM12-SM cells compared to KM12C cells, which were concurrent with the activated levels of matriptase, an oncogenic pericellular membrane-anchored serine protease. Moreover, the results from knockdown approaches indicated that EGFR and matriptase were involved in colorectal cancer cell invasion. The results further showed that EGF could induce colorectal cancer cell invasion and matriptase activation, via PI3K/AKT but not MEK/Erk1/2 signaling. To further address the role of K-Ras, a downstream molecule of EGFR, in colorectal cancer cell invasion, I used PCR to isolate the coding region of wild-type K-Ras and utilized site-directed mutagenesis to construct two constitutively active mutants of K-Ras (G12D and G13D). The results showed that K-Ras-overexpression (wild-type, G12D and G13D) could enhance KM12C cell invasion and induce matriptase activation in the cells. The inhibition of PI3K or MEK signaling could reduce K-Ras-overexpression-induced colorectal cancer cell invasion, suggesting that both of the signal pathways are involved in K-Ras-induced cancer cell invasion. Interestingly, the KM12C cells with G12D or G13D K-Ras overexpression resisted to the inhibitory effect of afatinib, an EGFR inhibitor, on the cell invasion and matriptase activation, suggesting that K-Ras-induced matriptase activation may play a role in drug resistance for anti-EGFR therapy. The data together indicate that both EGFR signaling- and K-Ras-increased colorectal cancer cell invasion are via inducing PI3K/AKT signaling and matriptase activation. The results further suggest that targeting pericellular serine protease may provide a strategy for those colorectal cancer patients with a resistance to anti-EGFR therapy.