探討ARMS的生物功能：(I)參與黑色素細胞癌腫瘤生成機制之探討及(II)KIF3導引之神經軸突運送及神經突生長

Characterization of the biological functions of ARMS (ankyrin repeat-rich membrane spanning) in melanoma tumorigenesis and KIF3-mediated axonal transport and neurite outgrowth

廖怡華(Yi-Hua Liao)

指導教授 ： 許世明

國立臺灣大學/醫學院/病理學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02581

摘要

ARMS (Ankyrin repeat-rich membrane spanning)，又名Kidins220 (kinase D-interacting substrate of 220 kDa)，是一個不隨演化改變，經細胞膜的蛋白分子，主要分布於神經組織及神經內分泌細胞。ARMS可和Trk 或Eph受器作用，是神經趨化因子(neurotrophin)/Trk及ephrin/Eph路徑的下游標的分子。ARMS因擁有多個蛋白質交互作用區域，可作為蛋白質交互作用時的建構平台，是一具有重要生物功能的分子，但許多ARMS扮演的生物角色仍未完全明瞭。
皮膚黑色素細胞癌是由黑色素細胞轉型產生，是由神經嵴衍生而來的惡性腫瘤，所以在黑色素細胞癌生成期，轉型細胞常會重新表現神經發育相關分子。因為神經趨化因子及ephrin調控的訊息傳遞和黑色素細胞癌腫瘤生成及進展有關，我們進而想探究ARMS是否參與黑色素細胞癌之腫瘤生成。我們發現ARMS在病患黑色素細胞癌檢體及人類黑色素細胞癌細胞株皆有過量表現。以RNA干擾方式降低ARMS在B16F0黑色素細胞癌細胞株的表現，會有意義降低癌細胞株的接觸獨立生長 (anchorage-independent growth)，並抑制接種於SCID小鼠皮下之腫瘤生長。更重要的是，減少ARMS在黑色素細胞癌細胞的表現，會促進紫外線B光誘發之細胞凋亡，其作用機轉是透過MEK/ERK傳遞路徑之抑制，此路徑在多數亞洲人常見的肢端小痣型黑色素細胞癌並無自身活化之現象。我們的研究顯示ARMS單方面的過量表現，可藉由MEK/ERK路徑抑制壓力誘發之細胞凋亡，進而促進黑色素細胞癌的形成。
雖然最近的研究已使我們對ARMS在神經趨化因子引導的神經分化中扮演的角色有進一步了解，但有關ARMS如何被導引至神經突末端之機制仍為未知。 KIF3 (kinesin超家族蛋白質3; kinesin superfamily protein 3) 屬於異三聚體運輸蛋白(KIF3A-KIF3B-KAP或KIF3A-KIF3C-KAP3)，負責膜性囊泡順式轉運(anterograde)的軸突傳導。由酵母菌雙雜交法、GST pull-down法、免疫共同沉澱法及免疫螢光共軛焦顯微鏡的研究顯示，ARMS的C端區段可個別和KIF3複合體的任一次單元(即KIF3A，KIF3B，KIF3C或KAP3A)作用。ARMS在神經突末端之定位需仰賴微管(microtubule)，並需要ARMS的C端區段和有功能的KIF3複合體。除此之外，ARMS的 C端區段之過量表現或降低KIF3的表現及功能，皆會在接受神經生長因子刺激的PC12細胞中減少其神經突外長。由免疫分離及免疫電子顯微鏡的實驗顯示， ARMS和KIF3一同位於低電子密度、直徑約100至220 nm的囊泡上。我們的研究顯示ARMS位於膜性囊泡上，藉由KIF3複合體進行神經軸突傳導定位，此過程並可影響神經突生長。

關鍵字

ARMS ； 黑色素細胞癌 ； 細胞凋亡 ； MEK/ERK路徑 ； KIF3 ； 神經軸突傳導 ； 神經突生長

並列摘要

Ankyrin repeat-rich membrane spanning (ARMS), also known as kinase D-interacting substrate of 220 kDa (Kidins220), is an evolutionarily conserved transmembrane protein mainly expressed in the neural tissues and neuroendocrine cells. ARMS functions as a downstream and interacting molecule in the neurotrophin/Trk- and the ephrin/Eph- pathways, and it also acts as a scaffold molecule for protein-protein interaction because of the presence of multiple protein-protein interaction domains. Many of the biologic functions of ARMS remain unknown.
Malignant melanoma is a malignancy derived from neural crest. Because both neurotrophin- and ephrin-mediated signaling pathways have been demonstrated to be involved in melanoma tumorigenesis and progression, we thus wonder whether ARMS also participates in the carcinogenesis of malignant melanoma. We demonstrated overexpression of ARMS in human melanoma specimens and melanoma cell lines. Down-regulation of ARMS by RNA interference in B16F0 melanoma cells resulted in significant inhibition of anchorage-independent growth and restrictive growth of melanoma inoculated subcutaneously in SCID mice. Importantly, depletion of ARMS facilitated UVB-induced apoptosis in melanoma cells through inactivation of MEK/ERK pathway, which is not constitutively activated in most cases of acral lentiginous melanoma, the melanoma subtype found mostly in Asians. Our study suggests that overexpression of ARMS per se serves as one mechanism to promote melanoma formation by preventing stress-induced apoptotic death mediated by the MEK/ERK signaling pathway, especially in acral lentiginous melanoma
Although recent studies have elucidated the role of ARMS in neurotrophin-induced neuronal differentiation, the mechanism how ARMS is targeted at neurite tips remains unknown. KIF3 (kinesin superfamily protein 3) is the heterotrimeric motor (KIF3A-KIF3B-KAP3 or KIF3A-KIF3C-KAP3) responsible for anterograde axonal transport for membranous vesicles. Here, we demonstrated a direct molecular interaction of the carboxy (C)-terminal region of ARMS with each subunit of KIF3 (KIF3A, KIF3B, KIF3C, or KAP3A, respectively) via yeast two-hybrid assay, GST pull-down, coimmunoprecipitation, and immunofluorescent confocal colocalization. Localization of ARMS at the tips of neurites was microtubule-dependent and required the presence of the C-terminal region of ARMS as well as functional KIF3 complexes. Furthermore, overexpression of the C-terminal region of ARMS or functionally knockdown of KIF3 impaired neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. Immunoisolation and immunoelectron microscopy demonstrated the colocalization of ARMS and KIF3 on the same electron-lucent vesicles ranging from 100 to 220 nm in diameter. Our study suggests that ARMS resides in membranous vesicles and relies on KIF3 complexes for their axonal targeting, a finding which plays an important role in neurite outgrowth.

並列關鍵字

ARMS ； melanoma ； apoptosis ； MEK/ERK pathway ； KIF3 ； axonal transport ； neuronal outgrowth

參考文獻

Chapter I

Bedogni B, O'Neill MS, Welford SM, et al. Topical treatment with inhibitors of the phosphatidylinositol 3'-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways reduces melanoma development in severe combined immunodeficient mice. Cancer Res 2004;64:2552-60.

Bevona C, Sober AJ. Melanoma incidence trends. Dermatol Clin 2002;20:589-95.

Boucher MJ, Morisset J, Vachon PH, Reed JC, Laine J, Rivard N. MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem 2000;79:355-69.

Bulavin DV, Saito S, Hollander MC, et al. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J 1999;18:6845-54.