過去研究指出在許多的疾病或腫瘤可以觀察到內質網壓力如肝癌或和糖尿病，許多的因子如突變蛋白、病毒感染、低氧、低葡萄糖或化學物質可能干擾內質網內正確的蛋白質折疊。當大量累積未正常摺疊的蛋白質所造成的內質網壓力會透過活化 ATF6、PERK 及Ire1 訊息傳遞路徑。最近的研究顯示Nucleobindin-2 (NUCB-2) 在臨床腫瘤樣本中發現有過量表現的情形如胃癌、前列腺癌和乳癌。在高NUCB-2 表現的癌症病人其存活率比低NUCB-2 的癌症病人低，然而NUCB-2 在腫瘤細胞過量表的機制和影響尚未釐清。所以在本計劃我們想要去確認NUCB-2 在腫瘤細胞中過量表現的新 奇的角色及機制。我們的初步結果指出tunicamycin 及brefledin A 這兩個內質網壓力誘導劑可以增加Huh-7、MCF-7 及HepG2 細胞株和動物模式下NUCB-2 的表現。利用RT-PCR 及real-time PCR 確認內質網壓力誘導NUCB-2 mRNA 的表現量，並且在內質網壓力下NUCB-2 蛋白質表現量也明顯的增加。更進一步的也觀察到在內質網壓力 NUCB-2 有入核的情形。我們也去探討NUCB-2 在內質網壓力誘導細胞凋亡所扮演的角色，利用NUCB-2 shRNA來降低Huh-7 細胞NUCB-2表現量可以促進內質網壓力所誘導細胞凋亡情形。除此之外，我們利用在Huh-7 細胞表現B型肝炎表面突變蛋白 (pre-S2)來誘導內質網壓力，結果顯示透過pre-S2所誘導的內質網壓力其NUCB-2 表現量也有意義的增加，進一步我們在人類肝腫瘤組織裡也可觀察到NUCB-2 表現量與內質網壓力有關係。在本計畫中我們將著重於三個主軸: (1) 分析NUCB-2 在內質網壓力所扮演的角色。 (2) 確認在內質網壓力下NUCB-2 的誘導機制。 (3) 探討NUCB-2 過量表現對於化學治療要處理之影響。我們希望利用內質網壓力的環境來探討NUCB-2與腫瘤發展的關係，這樣的結果可以提供一個重要的資訊可以提供未來應用在腫瘤生成及化學治療方面。總結而論，這樣的機制研究可以提供一個分子基礎來研發新的癌症治療藥物給這一群高NUCB-2 表現的病人。 Previous studies have indicated that the induction of endoplasmic reticulum stress was observed in many disease or tumors such as hepatocellular carcinoma or diabetes. Many factors may interfere with proper protein folding activity in the endoplasmic reticulum (ER) such as mutant proteins, viral infection, hypoxia, low glucose or chemical compounds. Accumulation of unfolded protein will activate ATF-6, PERK and Ire1 signaling pathways in response to ER stress. Recently many studies have indicated that overexpression of nucleobindin-2 (NUCB-2) is observed in clinical tumor samples such as gastric cancer, prostate cancer and breast cancer. The survival rate of high-level expression of NUCB-2 in cancer patients was lower than cancer patients with low-level expression of NUCB-2. However, the effect of NUCB-2 overexpression on tumor cells is still unclear. In this project, we want to identify the novel role and mechanisms of NUCB-2 overexpression in hepatoma cells. Our preliminary results indicated that two ER stress inducers, tunicamycin or brefeldin A, increased NUCB-2 expression in three cell lines (Huh-7/MCF-7/HepG2) and in vivo. NUCB-2 mRNA expression level was induced by ER stress as determined with RT-PCR and real-time PCR, and induction of NUCB-2 protein level was significantly increased by ER stress. Furthermore, nuclear translocation of NUCB-2 was observed during ER stress. The role of NUCB-2 in mediating tunicamycin-induced apoptosis was also investigated, and downregulation of NUCB-2 expression by NUCB-2 shRNA significantly promoted tunicamycin-induced apoptosis in Huh-7 cells. In addition, induction of ER stress by HBV large surface mutant protein pre-S2 was observed in Huh-7 cells. The result has shown that increased ER stress by pre-S2 protein was significantly enhanced NUCB-2 expression. We also observed that the overexpression of NUCB-2 was corrected with ER stress in human hepatocellular carcinoma tumor samples. In this project, we will focus on three aims: (1) To analyze the role of NUCB-2 in response to ER stress. (2) To determine the mechanism of induction of NUCB-2 overexpression by ER stress. (3) To investigate the effect of NUCB-2 overexpression on the action of chemotherapeutic agents. We hope to identify the relationship between NUCB-2 and tumor development in response to ER stress, and these results could provide important insight in tumorigenesis and chemotherapy. Consequently, this mechanistic research may provide a molecular basis to develop a novel cancer chemotherapeutic agent for high level expression of NUCB-2 in cancer patients.
