Annexin A4 Enhances Cell Migration Ability through Triggering Endoplasmic Reticulum Stress Signaling

Annexin家族廣泛存在於各物種內，是一群於C端具有獨特重複片段 (annexin repeat)的蛋白質，能藉由重複片段與鈣離子結合，並由鈣離子作為橋梁，可與膜上特定帶負電的磷脂質結合；而成員間的差異在於N端序列的變異性高，這使它們各自具有獨特的功能，參與了多樣的生理功能，包括：胞器運送、訊息傳遞、細胞分裂、細胞生長調節、細胞凋亡等。許多已發表的研究結果指出不同的annexin成員會於特定的腫瘤細胞中表現量上升，除了可作為生物標誌的可能性外，也暗示著annexin可能參與了腫瘤生成與發展的過程。根據先前我們實驗室發表的研究報告指出，annexin家族成員之一的annexin A4在受到胃幽門螺旋桿菌 (Helicobacter pylori)感染的胃癌病人組織及胃癌細胞株中皆會有表現量上升的現象，而抑制annexin A4表現則會減少因幽門螺旋桿菌感染而導致的interleukin-8分泌。為了探討annexin A4於胃癌細胞中的功能及其表現量增加後對胃癌細胞產生的影響，我們運用了蛋白質二維膠體電泳合併質譜儀的方法，偵測annexin A4大量表現後胃癌細胞AGS的蛋白質體變化；我們總共鑑定出36個表現量出現明顯差異的蛋白質，經由生物資訊資料庫軟體Ingenuity Pathway Analysis的輔助分析，推測細胞中產生了內質網壓力反應。在已發表的文獻中指出內質網壓力能促進細胞遷移能力，於是我們利用西方墨點法、創傷癒合實驗、胃癌細胞遷移能力測試及微陣列資料分析，證實了在annexin A4表現上升的胃癌細胞中，內質網壓力反應相關的蛋白質表現量有上升的趨勢，其下游的轉錄因子Nuclear factor kappa B (NF-kB)及c-myc的表現量上升，且同時促進Akt的活化，這些變化誘導真核起始因子4E (eukaryotic initiation factor 4E, EIF4E)的表現增加，導致胃癌細胞遷移的能力提高的結果。而給予細胞內質網壓力反應抑制劑8-(N,N-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8)則能回復這個現象。我們的結果提供了一個可能的作用機制，以解釋為何annexin A4表現量增加會導致胃癌細胞遷移的能力，這將為胃癌的治療提供有用的資訊。The annexin family is ubiquitous proteins capable of binding to membrane with their C-terminal annexin repeats in a Ca2+-dependent manner. The variances of N-terminal of each annexin member contribute to their wide variety of functions including vesicle trafficking, signaling, cell division, growth regulation and apoptosis. Their differentially expressions in distinct tumors are considered as biomarkers and may imply the participation in tumorgenesis. Our previous study showed that annexin A4, a member of annexin family, was overexpressed in both gastric tumor tissues and host cancer cells infected by Helicobacter pylori (H. pylori). Meanwhile, the production of IL-8, an indicator of infection, decreased after annexin A4 knockdown. To investigate the role of annexin A4 in gastric cancer cells, proteomics and network analysis were performed. 36 differentially expressed proteins and a network analysis related to protein folding were identified using Ingenuity Pathway Analysis software. Furthermore, we found ER stress pathway which may increase cell migration ability was very important in annexin A4-overexpressed cells. Therefore, we used western blotting, wound healing assay, cell migration ability assay to elucidate the expression levels of proteins involved in the response to ER stress and migration ability in annexin A4-overexpressed gastric cancer cells. Here, we show that annexin A4 overexpression not only increases ER stress related protein expression and migration ability but also activates two transcription factors, Nuclear factor kappa B (NF-kB) and c-myc, and triggers Akt phosphorylation. These changes might lead to a substantial rise in the expression of the eukaryotic initiation factor 4E (eIF4E) gene, ultimately promoting the migratory ability of gastric cancer cells. The phenomenon was reversed by treating cells with ER stress signaling inhibitor, 8-(N,N-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8). Our results demonstrated a plausible mechanism to explain the phenomena that migratory ability increased in annexin A4-overexpressed gastric cancer cells. Eventually, these findings provide useful information in gastric cancer therapy