探討α5菸鹼型乙醯膽鹼受體促進口腔癌細胞之放射線治療抗性的分子機轉

Studying on the molecular mechanism underlying the cholinergic receptor nicotinic alpha 5 subunit-promoted radioresistance in oral cancer

林哲玄(Che-Hsuan Lin)

指導教授 ： 張榮善

共同指導教授 ： 王家儀(Jia-Yi Wang)

臺北醫學大學/醫學院/醫學科學研究所/博士(2020年)

摘要

口腔癌是一種頭頸部惡性腫瘤，癌變病灶可能起源於口腔內的任何部位，隨著組織學上的不同而有不同的分類。而口腔鱗狀細胞癌（oral squamous cell carcinoma, OSCC）是最主要的類型，佔所有口腔癌的90％以上。在腫瘤控制上，放射線治療（external beam radiotherapy, EBRT）在頭頸部癌症的治療中扮演著重要的腳色。然而，目前沒有發表任何關於放射線治療敏感性預測的臨床研究，若能預知腫瘤對放射線的敏感性，了解放射抗性潛在的分子機制可能有助於開發新的抗癌藥物或恢復腫瘤對於放射敏治療的敏感性。
先前的研究已經證明了菸鹼乙酰膽鹼受體（Nicotinic acetylcholine receptors, nAChRs）在腫瘤發生或癌症進展（例如轉移或藥物抗性）中的作用。透過分析The Cancer Genome Atlas（TCGA）資料庫中頭頸部癌症病人資料，初步統計顯示，編碼nicotinic acetylcholine receptor 5的基因CHRNA5，相較於正常組織，在原發性腫瘤中有顯著的高度表現。此外我們發現在OSCC病人中， CHRNA5高表現的族群呈現較差的存活率，特別是在接受放射治療的OSCC患者中更具顯著差異;我們更進一步發現透過抑制CHRNA5的表現，可有效的提高OSCC對於放射線治療的敏感性。此外我們利用the Gene Set Enrichment Analysis針對特定族群(口腔癌患者接受放射線治療後仍復發者)進行分析，我們發現E2F的訊號路徑與CHRNA5的表現呈現高度正相關。透過實驗，我們驗證了抑制CHRNA5的表現會顯著的下降E2F的活性且減少phosphorylated Rb蛋白；反之，若給予尼古丁的刺激則可以顯著增加E2F的活性及增加Rb蛋白的磷酸化。在統計上我們也發現，集合高E2F活性與高CHRNA5表現這兩種不同的基因信號，在口腔癌患者並接受放射治療的族群中有著較差的存活率。
我們的發現驗證CHRNA5不僅可作為預測放射治療效度的有用生物標誌，而且在治療口腔癌時，還可成為治療靶標以增強OSCC對於放射線治療的敏感性。

關鍵字

放射線治療 ； 口腔癌 ； α5菸鹼型乙醯膽鹼受體 ； E2F

並列摘要

Oral squamous cell carcinoma (OSCC) is the most predominant type in oral cancers. External Beam Radiotherapy (EBRT) is the major strategy to treat OSCC in clinics. Nevertheless, a subpopulation of OSCC patients who are insensitive to EBRT showed relatively poorer prognosis. Therefore, the identification of biomarkers for predicting EBRT sensitivity is urgently needed in OSCC patients.
Previous studies have demonstrated the role of nicotinic acetylcholine receptors in tumorigenesis or cancer progression (e.g. metastasis or drug resistance). An in silico analysis against the Cancer Genome Atlas (TCGA) database revealed that CHRNA5, a gene encoding nicotinic acetylcholine receptor 5, is highly expressed in primary tumors compared to normal adjacent tissues derived from OSCC patients. Moreover, our data showed that the levels of CHRNA5 transcript are causally associated with the effective dosage of irradiation in a panel of OSCC cell lines. By silencing the expression of CHRNA5, it can effectively improve the radiosensitivity of OSCC. The Gene Set Enrichment Analysis demonstrated that E2F signaling pathway is highly activated in OSCC tissues with high-level CHRNA5 and derived from patients with cancer recurrence after radiotherapy. CHRNA5 knockdown predominantly suppressed the E2F activity and decreased the phosphorylation of Rb protein, whereas nicotine treatment dramatically promoted E2F activity and increased Rb phosphorylation, which was mitigated after CHRNA5 knockdown in OSCC cells. Notably, the signature of combining the higher mRNA levels of CHRNA5 and E2F signaling gene set associated with a worse recurrence-free survival probability in OSCC patients who were recorded to receive radiotherapy.
Our findings suggest that CHRNA5 not only serves as a useful biomarker to predict the effectiveness of radiotherapy but also a druggable target to enhance the cancericidal effect of irradiation on OSCC.

並列關鍵字

radiotherapy ； oral squamous cell carcinoma ； CHRNA5 ； nicotinic acetylcholine receptor ； E2F

參考文獻

1. Albuquerque, E. X., Pereira, E. F. R., Alkondon, M., &Rogers, S. W. (2009). Mammalian nicotinic acetylcholine receptors: from structure to function. Physiological Reviews, 89(1), 73–120.

2. Araki, K., Ahmad, S. M., Li, G., Bray, D. A., Saito, K., Wang, D., …Li, D. (2008). Retinoblastoma RB94 enhances radiation treatment of head and neck squamous cell carcinoma. Clinical Cancer Research, 14(11), 3514–3519.

3. Arneric, S. P., Holladay, M., &Williams, M. (2007). Neuronal nicotinic receptors: a perspective on two decades of drug discovery research. Biochemical Pharmacology, 74(8), 1092–1101.

4. Bagan, J., Sarrion, G., &Jimenez, Y. (2010). Oral cancer: clinical features. Oral Oncology, 46(6), 414–417.

5. Brejc, K., vanDijk, W. J., Klaassen, R.V, Schuurmans, M., van derOost, J., Smit, A. B., &Sixma, T. K. (2001). Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature, 411(6835), 269.