氧化壓力誘導生長抑制蛋白OSGIN1在二十二碳六烯酸調控人類乳癌細胞凋亡、自噬作用中扮演之角色

乳癌是全球女性最常見的癌症，在台灣女性中，所有癌症罹患率排第3位。依照不同基因圖譜表現，分類為luminal A、luminal B、HER2+/ER-、basal-like和normal-like等不同亞型。表現不同類型的受體乳腺癌在預後和治療反應方面存在明顯差異。氧化壓力誘導生長抑制蛋白(Oxidative stress induced growth inhibitor 1, OSGIN1)，是著名的抑癌蛋白，被認為是治療癌症的目標之一。相較於非腫瘤組織的OSGIN1高表現，人類乳腺癌、腎癌和肝細胞惡性腫瘤組織的OSGIN1表現量低。已知誘導OSGIN1表現可促進癌細胞凋亡與抑制癌細胞增生，然而缺乏OSGIN1蛋白將加速腫瘤生長。乳癌的發生與進展與基因、飲食和日常生活方式息息相關。高熱量、高糖、高飽和脂肪飲食，和低-3脂肪酸、抗氧化維生素與膳食纖維攝取與乳癌發生呈現正相關。OSGIN1已被證實可誘發細胞凋亡及自噬作用，OSGIN1是否在-3和-6多元不飽和脂肪酸可以被誘發，及誘發後影響癌細胞凋亡和自噬作用中扮演之角色，仍不清楚。因此，本實驗利用MCF-7與Hs578T人類乳癌細胞比較-3和-6多元不飽和脂肪酸對OSGIN1表現之影響可能作用機制，再進一步釐清OSGIN1在DHA調控乳癌細胞凋亡中及自噬作用中的角色，最後，測試OSGIN1大量表現是否可增強化療藥物-順鉑(Cisplatin)的功效，以改善乳癌預後。本研究發現，在眾多多元不飽和脂肪酸中，僅有二十二碳六烯酸(DHA)可顯著誘發OSGIN1蛋白質表現，這暗示著OSGIN1可能參與DHA抗乳癌效應。DHA可誘發活性氧生成及Nrf2核內累積。Nrf2 knockdown減弱DHA誘導的OSGIN1表達。預處理N-acetyl-L-cysteine降低DHA 誘導的Akt磷酸化、Nrf2核內累積和OSGIN1的表達。DHA可增加Bax/Bcl-2比率、粒線體OSGIN1和p53累積及細胞色素c的釋放，而 OSGIN1 knockdown後則減弱這些效應。結果說明DHA可能透過ROS介導的PI3K/Akt/Nrf2訊號誘發OSGIN1表現，進而促進乳腺癌細胞凋亡。 目前已知 DHA 可調節癌細胞中的自噬作用。我們將進一步探討OSGIN1是否參與了乳腺癌細胞中DHA對自噬作用的調節與其可能機制。結果發現，DHA上調 OSGIN1、LC3-II 和 SQSTM1/p62的蛋白質表達，而mTOR與p-mTORS2448的表達下降。在GFP/RFP-LC3螢光染色中我們看到，DHA處理下，自噬作用的信號表現與DHA劑量呈依賴性增加。接著用OSGIN1過表達來模擬DHA處理，也證明LC3-II和GFP/RFP-LC3信號以及p-AMPKαT172和p-RaptorS792的表達顯著增加，而mTOR、p-mTORS2448和p-ULK1S757 表達降低。此外，OSGIN1過表達造成OSGIN1在線粒體積累與粒線體ROS增加，隨後增加了p-AMPKαT172和p-RaptorS792的表達。用粒線體 ROS的抑制劑Mito-TEMPO預處理後，這些結果被逆轉。總之，這些結果表明 DHA 可以通過激活p-AMPKαT172/p-Raptor S792和失活p-mTORS2448/p-ULK1Ser757信號通路，短暫提高線粒體中ROS產生並促進自噬小體的形成，這些影響透過OSGIN1蛋白調控在MCF-7 細胞中。 最後，我們進一步結果發現，高表達OSGIN1在乳腺癌細胞中，會通過提高細胞內ROS產生和膜電位的破壞，以及誘導裂解caspase 3/7和PARP，來增強順鉑的細胞毒性，進而增加MCF-7和Hs578T細胞凋亡。總結，DHA部分通過 PI3K/Akt/Nrf2信號路徑活化上調OSGIN1表達，進而誘導的乳腺癌細胞凋亡和不完全自噬，以及OSGIN1的誘導可能增強乳腺癌細胞的化學敏感性。

Breast cancer is the most commonly occurring cancer among women worldwide, and is the third common death caused cancer in Taiwan. Breast cancer is classified into luminal A, luminal B, HER2+/ER-, basal-like and normal-like subtypes base on differences in gene expression profiling. These different types of receptors in breast cancer with distinct differences in prognosis and response to therapy. Oxidative stress induced growth inhibitor 1 (OSGIN1), a well-known tumor suppressor, regarded as a therapeutic target for cancer. Compared with the high expression of OSGIN1 in nontumor tissues, expression of OSGIN1 is significantly lower in breast, renal, and hepatocellular carcinoma tissues. Induction of OSGIN1 is known to promote apoptosis and inhibit proliferation in cancer cells, whereas the lack of OSGIN1 protein is able to accelerate tumor growth. The occurrence and progression of breast cancer are closely related to genetic inheritance, diet and lifestyle. High-calorie, high-sugar, high-saturated fat diet, and low intake of -3 polyunsaturated fatty acids (PUFAs), antioxidant vitamins and dietary fiber are positively correlated to breast cancer incidence. Although induction of OSGIN1 has been reported to induce apoptotic cell death and autophagy, whether OSGIN1 plays an essential role in the cancer cell apoptosis and autophagy induced by -3 and -6 PUFAs is still not understood. Thus, the MCF-7 and Hs578T human breast cancer cells were used to study the individual effects of -3 and -6 PUFAs on OSGIN1 expression and the possible mechanisms involved in this gene regulation. Moreover, we further clarified whether OSGIN1 is involved in the regulation of apoptosis and autophagy by DHA in breast cancer cells. Finally, we also examined whether OSGIN1 overexpression can enhance the efficacy of the chemotherapy drugs-cisplatin in breast cancer cells.Among n-3 and n-6 PUFAs, only docosahexaenoic acid (DHA) significantly increased OSGIN1 expression in MCF-7 breast cancer cells, implying OSGIN1 may be involved in anti-cancer effects of DHA in breast cancer cells. DHA triggered reactive oxygen species (ROS) generation and nuclear accumulation of Nrf2. Nrf2 knockdown attenuated DHA-induced OSGIN1 expression. N-Acetyl-L-cysteine, a ROS scavenger, abrogated the DHA-induced increases in Akt phosphorylation, Nrf2 nuclear accumulation, and OSGIN1 expression. DHA induced the Bax/Bcl-2 ratio, mitochondrial accumulation of OSGIN1 and p53, and cytochrome c release, whereas knockdown of OSGIN1 diminished these effects. These results suggest that induction of OSGIN1 may be involved in DHA-induced apoptosis through ROS-mediated PI3K/Akt/Nrf2 signaling in breast cancer cells.DHA is known to regulate autophagy in cancer cells. We explored whether OSGIN1 is involved in the regulation of autophagy by DHA in breast cancer cells and the possible mechanisms involved. The results showed that DHA upregulated the levels of OSGIN1, LC3-II and SQSTM1/p62, whereas the levels of mTOR and p-mTORS2448 expression were decreased. Using GFP/RFP-LC3 fluorescence staining, treatment with DHA showed a dose-dependent response in autophagic signals. OSGIN1 Overexpression mimicked DHA treatment in that LC3-II and GFP/RFP-LC3 signals as well as the expression of p-AMPKαT172 and p-RaptorS792 were significantly increased, whereas mTOR, p-mTORS2448, and p- ULK1S757 expression were decreased. Moreover, OSGIN1 overexpression transiently elevated the mitochondrial accumulation of OSGIN1 and ROS and subsequently increased p-AMPKαT172 and p-RaptorS792 expression. Upon pretreatment with Mito-TEMPO, a scavenger of mitochondrial ROS, these outcomes were reversed. Taken together, these results suggest that DHA can transiently elevate the generation of ROS in mitochondria and promote autophagosome formation through activation of the p-AMPKαT172/p-Raptor S792 and inactivation of the p-mTORS2448/p-ULK1Ser757 signaling pathways, and these effects depend on OSGIN1 protein in MCF-7 cells.We Finally, the results showed that the breast cancer cells with high expression of OSGIN1 enhanced the cytotoxicity of cisplatin through elevation of intracellular ROS generation and disruption of membrane potential as well as induction of cleaved caspase 3/7 and cleaved PARP, contributing apoptosis in MCF-7 and Hs578T cells. In conclusion, DHA induced OSGIN1 expression is mediated at least in part through the PI3K/Akt/Nrf2 signaling pathway. Additionally, induction of OSGIN1 is involved DHA-induced apoptosis and incomplete autophagy in breast cancer cells. Induction of OSGIN1 may enhance the chemo-sensitivity of breast cancer cells.