檳榔鹼誘導RREB1表現之訊息傳遞路徑與RREB1在口腔癌變的作用

Abstract

根據衛福部2020年十大癌症統計口腔癌排名第六名，且近年病患有年輕化的趨勢。其中檳榔內含的檳榔鹼 (Arecoline) 被歸類一級致癌物，亦為口腔癌主要致病因子，但其致癌機轉尚未清楚。過去研究指出，Ras-responsive element binding protein 1 (RREB1) 參與MAPK訊息傳遞路徑，在前列腺癌、結直腸癌、泌尿癌扮演重要的作用。但RREB1在口腔癌癌化與癌幹細胞特性上尚未被探討。先前實驗室發現RREB1在口腔癌OSCC患者檢體中有過度表現，且RREB1蛋白於SAS細胞聚球體中表現量增加。本研究發現以檳榔鹼arecoline刺激Ca9-22與SAS細胞株可誘導RREB1表現，加入TGF-?1中和抗體、ALK5抑制劑 (SB431542)、Smad3抑制劑 (SIS3)可降低由檳榔鹼所誘導的RREB1表現。當加入EGFR抑制劑 (AG1478)、MMP抑制劑 (GM6001)、p53抑制劑(Pifithrin-α)、、p38抑制劑 (SB203580) 、MEK1/2抑制劑 (PD98059) 、JNK抑制劑 (SP600125)，可降低由TGF-?1所誘導的RREB1的蛋白表現。以RREB1 siRNA處理RREB1表現量較高的SAS細胞，會降低其Migration與Invasion能力，亦下調由TGF-?1誘導的EMT 相關標誌蛋白。同時RREB1 siRNA可以降低SAS聚球體細胞的形成，並發現CD133、CD44、KLF4、OCT4A、SOX2、Nanog等癌幹細胞標誌表現下降。經過RREB1 overexpression之細胞其proliferation、Migration與Invasion的能力上升，且影響EMT markers，上調N-cadherin、Vimentin、Slug、Snail、Twist表現，下調E-cadherin表現。同時增加聚球體細胞形成並上調CD133、CD44、KLF4、OCT4A、SOX2、Nanog。使用Nod-scid老鼠進行實驗發現RREB1可以促進腫瘤的生長。初步觀察到RREB1 overexpression之細胞會上調p-EGFR、IFIT1、IFIT3的表現，以siIFIT1與siIFIT3處理並降低了RREB1 overexpression細胞的Migration、Invasion能力，下調EMT與癌幹細胞標誌蛋白，同時降低聚球體細胞數量的形成。另以Gefitinib處理72小時RREB1 overexpression細胞後，發現相比Control組具有抑制的效果，顯示Gefitinib藥物具有治療RREB1過表現之口腔癌的潛力。

According to the death statistics of Ministry of Health and Welfare from Taiwan in 2020, oral cancer was ranked 6th of the top ten cancer. Furthermore, patients who had oral cancer were getting younger recently. Arecoline in betel nut is classified as a first-class carcinogen, and it was also the main factor of oral cancer. However, its detailed carcinogenic mechanism is not clear yet. Based on past studies, Ras-responsive element binding protein 1 (RREB1) was involved in the MAPK signaling pathway, and played an important role in prostate cancer, colorectal cancer, and urinary cancer when its expression was unbalanced. Nevertheless, the mechanisms of RREB1 in oral cancer carcinogenesis and cancer stem cells were not been full explored. Previously, our laboratory found that RREB1 was overexpressed in oral cancer OSCC patients, and the expression of RREB1 protein was significantly increased in SAS sphere cells. In this study, arecoline was used to stimulate Ca9-22 and SAS cell lines to induce RREB1 expression, and the additions of TGF-?1 neutralizing antibody, ALK5 inhibitor (SB431542), and Smad3 inhibitor (SIS3) could decrease the expression of RREB1 induced by arecoline. When treating EGFR inhibitor (AG1478), MMP inhibitor (GM6001), p53 inhibitor (Pifithrin-α), MEK1/2 inhibitor (PD98059), p38 inhibitor (SB203580), JNK inhibitor (SP600125), it decreased the protein expression of RREB1 induced by TGF-?1. Treating SAS cells with RREB1 siRNA, it reduced the ability of cell migration, invasion, and also down-regulated EMT-related markers which induced by TGF-?1. Moreover, RREB1 siRNA could reduce the formation of SAS sphere cells, and we found that the expression of CD133, CD44, KLF4, OCT4A, SOX2, Nanog decreased. With RREB1 overexpression in cells , the proliferation of cells increased, and the abilities of migration and invasion were also enhanced, and the expressions of EMT markers like N-cadherin, Vimentin, Slug, Snail, Twist were up-regulated , and the expression of E-cadherin was down-regulated. It also increased the formation of sphere cells and up-regulates stemness marker expressions like CD133, CD44, KLF4, OCT4A, SOX2, Nanog.Moreover, We use Nod-scid mice to test and found that RREB1 can promote tumor growth in vivo.In preliminary experiments ,we observed that RREB1 overexpression of cells may up-regulated the expression of p-EGFR, IFIT1, and IFIT3. When treating cells with siIFIT1 and siIFIT3, it might reduced the migration, invasion, the formation of sphere cells, and down-regulated EMT markers and stemness markers of RREB1 overexpressed cells. In addition, it was found to have an inhibitory effect compared with control group when treating the RREB1 overexpression cells with Gefitinib for 72 hours, indicating that Gefitinib might had potential to be a therapeutic drug with RREB1 overexpressed oral cancer.