探討CASK在非小細胞肺癌中對表皮生長因子受器作用之調節

Abstract

CASK 屬於一種骨架蛋白，並且已知在調控腦神經元的發育及功能中，扮演關鍵性的角色。最近的研究揭示CASK在其他器官和癌細胞的表達，例如結腸癌、膀胱癌和幽門螺桿菌相關的胃癌，而CASK在這些細胞中的調控大多與細胞增殖和移行有關。除此之外，在秀麗隱桿線蟲的外陰前體細胞，CASK參與EGFR定位的調節。然而，人們對於CASK的作用，以及其與EGFR的關係尚不了解。在此研究中，我們使用轉移性NSCLC細胞株H1299，發現將CASK基因靜默之後，雖然不會改變細胞的增殖和存活率，但會增加EGFR mRNA的表現量，不論細胞是在含有血清或無血清培養基中。此外，CASK的靜默會增加細胞表面EGFR的表現量。值得注意的是，EGF刺激可造成EGFR在3到6小時內的調降作用，而此作用在靜默CASK的細胞中卻明顯的被延遲，但靜默CASK並不影響膜上EGFR在受到EGF刺激1小時內，產生快速內吞至近核區之作用。靜默CASK除了增加EGFR mRNA 的表現量，還會延長EGFR蛋白質的安定性。我們利用共軛焦顯微鏡及免疫沉澱法，證明CASK和EGFR之間有共定位及結合作用。此外，靜默CASK會增加EGF刺激造成的ERK及Akt之磷酸化，亦會增加H1299細胞之移行能力，而此作用和ERK的活化有關，且和EGF的作用沒有加成性。雖然CASK和EGFR都位於粒線體，但靜默CASK並不會影響粒腺體的有氧呼吸作用。我們的結果指出在H1299細胞，CASK是具多面向調控EGFR的一個新的分子。CASK為EGFR的結合蛋白，會降低EGFR的蛋白質安定性和基因表現量，並且抑制細胞移行，但在A549細胞靜默CASK並不會造成細胞移行之改變。總的來說，我們的研究結果提供對CASK在NSCLC中與EGFR有關之功能的新見解，未來仍需要更多的研究來達成對於CASK的功能及分子作用機轉之完整的了解。

Calcium/calmodulin-dependent serine protein kinase (CASK) is a scaffold protein, which has been known to play crucial roles in the regulation of neuron development. Recent studies revealed that CASK is also expressed in other tissues and cancer cells. The role of CASK in these cells is mainly involved in the regulation of cell proliferation and migration. Moreover, CASK has been demonstrated to regulate EGFR localization in the vulval precursor cells of C. elegans. However, it is still poorly understood regarding the role of CASK and its relationship with EGFR in cancer cells. In this study, we primarily used metastatic NSCLC cell line H1299 and found that silencing CASK did not alter cell proliferation and viability. Yet, it increased basal level of EGFR mRNA in cells cultured in either complete medium or serum-free medium. Moreover, silencing CASK increased total EGFR protein and its expression in the plasma membrane. Of note, EGF-induced down-regulation of EGFR within 3-12 h was attenuated in CASK knockdown cells, while EGFR activation was more prolonged within 3-6 h. Nevertheless, silencing CASK did not alter the rapid internalization of surface EGFR nor the trafficking of EGFR to peri-nuclear sites induced by EGF within 1 h. Besides the increased EGFR mRNA, silencing CASK also prolonged the stability of EGFR protein. Studies using confocal microscopy and immunoprecipitation revealed the co-localization and interaction between CASK and EGFR, respectively. In addition, silencing CASK enhanced EGF-elicited ERK and Akt activities, and promoted the migration of H1299 cells which was dependent on ERK and was non-additive to EGF. Although both CASK and EGFR are located in mitochondria, silencing CASK did not alter mitochondrial respiration. All these results suggest that CASK is a novel molecule to regulate EGFR via multifaceted mechanisms in H1299 cells. CASK is a binding protein of EGFR, which can reduce EGFR protein stability and EGFR gene expression, and inhibit cell migration. In contrast to the findings in H1299 cells, silencing CASK failed to change cell migration in A549 NSCLC cell line. In conclusion, our findings provide a new insight into the EGFR-associated actions of CASK in NSCLC and further study is needed to have a full understanding on CASK function and underlying molecular mechanisms in NSCLC.