口腔鱗狀上皮細胞癌幹細胞特性依賴MAT2A表現

MAT2A is required for stemness in oral squamous cell carcinoma

黃雲佳(Yun-Jia Huang)

指導教授 ： 郭彥彬

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102421

摘要

根據我國衛福部108年國人十大死因統計，惡性腫瘤為十大死因中的首位，而口腔癌為十大癌症發生率的第五位。在台灣，口腔癌的發生與抽菸、喝酒、嚼食檳榔有密切的關係，檳榔中所含的檳榔鹼Arecoline是造成口腔癌主要的致病因子，但其致病機轉仍未詳細闡述。文獻指出甲硫胺酸腺苷轉移酶2A (methionine adenosyltransferase 2A (MAT2A))在許多癌症中都有過度表現的情況，包括肝癌、乳癌、結腸癌等，但其在口腔癌中的相關表現及機轉均還未被探討。MAT2A可催化生物體中S-adenosylmethionine (SAM)的合成，參與甲硫胺酸循環，於生物體代謝至關重要。實驗室初步研究發現口腔癌檢體有MAT2A過度表現。 本研究中發現Arecoline會誘導口腔癌SAS及CA922細胞株中的MAT2A表現。TGF-β1中和抗體、ALK5抑制劑 (SB431542)及Smad3抑制劑(SIS3)可降低Arecoline所誘導的SAS及CA922細胞中MAT2A表現，顯示Arecoline是經由TGF-β1訊息傳遞路徑誘導口腔上皮細胞中MAT2A的表現。MAT2A siRNA抑制MAT2A表現量高的SAS細胞株的MAT2A表現，發現抑制MAT2A 會使SAS細胞轉移與侵襲性降低，並下調由TGF-β1所引起的癌幹細胞標誌(stemness marker)上升。文獻指出，癌幹細胞會參與腫瘤維持、轉移及復發，是使其對化療、放療產生抗性的原因之一，而聚球體(sphere)細胞是一種能表現較多幹細胞的特性的細胞，本研究針對SAS細胞及SAS sphere細胞進行比較，結果顯示SAS sphere細胞有較高的MAT2A表現量。通過sphere forming assay觀察MAT2A siRNA抑制的 SAS sphere細胞，可發現SAS sphere細胞形成聚球體的能力降低，且同時會下調部分癌幹細胞標誌，包括: CD133、CD44、KLF4、OCT4A和Nanog的表現。本實驗也以MAT2A抑制劑FIDAS-5處理SAS細胞，並透過transwell migration assay與invasion assay觀察，可發現其侵襲與轉移能力均降低。若是使用FIDAS-5處理SAS sphere，可發現其也可降低SAS sphere細胞形成聚球體的能力，並下調癌幹細胞標誌，包括包括: CD133、CD44、KLF4、OCT4A、SOX2和Nanog的表現，顯示FIDAS-5具有治療口腔癌的潛力。

關鍵字

口腔癌 ； 甲硫胺酸腺苷轉移酶2A ； 檳榔鹼 ； 轉化生長因子 ； FIDAS-5 ； 癌幹性

並列摘要

According to the statistics of the Ministry of Health and Welfare from Taiwan in 2018, oral cancer is ranked the fifth leading cause of cancer-related deaths. In Taiwan, alcohol consumption, smoking and areca nut (AN) chewing are related to development of oral cancer. Methionine adenosyltransferase 2A (MAT2A) is an enzyme for synthesis of S-adenosylmethionine (SAM), playing important role of cell metabolic. MAT2A overexpression has been found in several human epithelial tumors, including liver, breast and colon cancer. Our preliminary study showed MAT2A is overexpressed in oral cancer. However, the mechanisms are still not clear. The present study showed arecoline, a main alkaloid of areca nut, induced the expression of MAT2A protein in oral epithelial cell line SAS, Ca922. Pretreatment with TGF-β neutralizing antibody, SB431542 and smad3 inhibitor SIS3 suppressed the arecoline-induced MAT2A expression, indicating arecoline-induced MAT2A expression is mediated by TGF-β1 signaling pathway in SAS and Ca922 cells. We next used siRNA to knockdown MAT2A in SAS cell line. We found that it could not only downregulate the expression of stemness markers induced by TGF-β1 but also suppress the ability of migration and invasion. Cancer stem cells are involved in tumor maintenance, metastasis and recurrence, which is one of the reasons that make them resistant to chemotherapy and radiotherapy. Sphere cells are a kind of cells that can show the characteristics of stemness. SAS sphere cells had higher expression of MAT2A as compare to SAS parent cells. We found knockdown MAT2A in SAS sphere decreased the SAS sphere formation and downregulate the expression of stemness markers, such as CD133, CD44, KLF4, OCT4A and Nanog. Furthermore, we observed that the MAT2A inhibitor FIDAS-5 could also reduce the ability of migration and invasion in SAS cell lines. FIDAS-5 also decreased the SAS sphere formation and downregulated the expression of stemness markers, including CD133、CD44、KLF4、OCT4A、SOX2 and Nanog. These results indicate that FIDAS-5 potentially qualified as a useful reagent for the therapy of oral cancer treatment.

並列關鍵字

Oral cancer ； MAT2A ； Arecoline ； TGF-β1 ； FIDAS-5 ； Cancer stemness

參考文獻

1. Weatherspoon, D.J., et al., Oral cavity and oropharyngeal cancer incidence trends and disparities in the United States: 2000-2010. Cancer Epidemiol, 2015. 39(4): p. 497-504.

2. Sankaranarayanan, R., et al., Effect of screening on oral cancer mortality in Kerala, India: a cluster-randomised controlled trial. Lancet, 2005. 365(9475): p. 1927-33.

3. Swango, P.A., Cancers of the oral cavity and pharynx in the United States: an epidemiologic overview. J Public Health Dent, 1996. 56(6): p. 309-18.

4. Neville, B.W., et al., Oral and maxillofacial pathology. 2015: Elsevier Health Sciences.

5. Silverman, S., Oral cancer. Vol. 1. 2003: PMPH-USA.