口腔癌是一種頭部和頸部常見的癌症，它是全球第六大常見的惡性腫瘤，而舌頭、頰黏膜與唇是口腔癌的好發次部位。自噬作用在許多癌症中，對於腫瘤的生長和發展都扮演重要的角色，在腫瘤細胞中，增加自噬作用有可能促進腫瘤細胞存活但也有可能促進腫瘤細胞死亡。本研究探討LC3、p62、p65和EGFR蛋白表現與口腔癌的腫瘤新生、臨床病理結果和存活之間的關係，共收納498位口腔鱗狀細胞癌患者 (包含181位頰黏膜鱗狀細胞癌、244舌頭鱗狀細胞癌及73位唇鱗狀細胞癌) ，此外，76位因患有睡眠呼吸中止症而進行懸雍垂切除手術的患者也收案為對照組。我們利用免疫組織化學染色法評估在組織微陣列上的三種不同組織中 (包括正常組織、腫瘤鄰近正常組織和腫瘤組織) ， LC3、p62、細胞核中p65 (p65-N)、細胞質中p65 (p65-C) 、 細胞膜中EGFR (EGFR-M)和細胞質中EGFR (EGFR-C)的蛋白表現量。在口腔鱗狀細胞癌及其三個原發次部位中，LC3、p62、p65-C和EGFR的蛋白表現程度皆顯著高於正常組織和腫瘤鄰近正常組織 (p<0.001)。而p65-N的蛋白表現除了在唇鱗狀細胞癌中沒有差異之外，在口腔鱗狀細胞癌及其他兩個原發次部位皆是顯著下降 (p<0.001)。在頰黏膜鱗狀細胞癌患者中，細胞分化差 (p=0.015) 和有淋巴結轉移 (p=0.033) 的病人，p62表現量較高。然而，沒有淋巴結轉移的病人，EGFR-M (p=0.031)反而表現量較高。在舌鱗狀細胞癌患者中，細胞分化較差的病人， LC3 (p=0.045)、p62 (p=0.040) 及p65-C (p<0.001) 表現量較高。然而，AJCC病理分期晚 (p<0.001)、腫瘤分期晚 (p=0.003)、及淋巴結轉移的病人 (p=0.013)，EGFR-C表現量較高。在唇鱗狀細胞癌患者中，細胞分化較差的病人，p62會有較高的表現 (p=0.003)。而在年老的患者 (>50歲) 中， p65-C容易有高表現 (p=0.009)。此外，在口腔鱗狀細胞癌患者中，LC3、p65-N和p65-C的表現量在口腔癌的三個不同原發次部位皆具顯著差異 (p <0.001)。高表現的p62 (p=0.001) 和p65-C (p<0.001) 會有較差的細胞分化。然而，AJCC病理分期晚 (p<0.001) 及腫瘤分期晚 (p=0.006) 的病人其癌組織EGFR-C表現量較高。有淋巴結轉移的病人， p65-C (p=0.012)及EGFR-C (p=0.012)表現較高，但EGFR-M (p=0.019) 表現較低。最後，經單變量和多變量存活分析發現，p62高表現和較差的口腔鱗狀細胞癌患者的特定疾病存活和無疾病存活具顯著相關，尤其是特定疾病存活於頰黏膜鱗狀細胞癌患者, 無疾病存活於唇鱗狀細胞癌患者。在口腔鱗狀細胞癌 (包括三個原發次部位) 中，LC3、p62、p65和EGFR可作為腫瘤新生之生物標誌。此外，p62可能是口腔鱗狀細胞癌，尤其是唇鱗狀細胞癌和頰黏膜鱗狀細胞癌一個具潛力的預後因子。

Oral cancer, a common type of head and neck cancer, is the sixth most common malignant tumors in the world. Tongue, buccal mucosa, and lip are the top three most common subsites of oral cavity. Autophagy plays an important role on tumor growth and progression in many cancers. Upregulation of autophagy in cancer can be either prosurvival or prodeath for tumor cells. The associations of expression levels of LC3, p62, p65, and EGFR with the tumorigenesis, clinicopathologic outcomes, and survival for three primary subsites of oral cancer were investigated. A total of 498 oral squamous cell carcinoma (OSCC) patients were recruited, including 181 buccal mucosal SCC (BMSCC), 244 tongue SCC (TSCC), and 73 lip SCC (LSCC) patients. In addition, another 76 sleep apnea patients for uvula excision were also recruited as controls. The expression levels of LC3, p62, p65 in the nuclear, p65 in the cytoplasm , EGFR in the Membrane (EGFR-M) and EGFR in the cytoplasm (EGFR-C) in three kinds of tissues, including normal, tumor, and corresponding tumor adjacent normal (TAN) were evaluated by immunohistochemistry using tissue microarray. An increased expression of LC3, p62, p65-C, EGFR-M, and EGFR-C was found in OSCC tissues and three subsites as compared to those in TAN and normal tissues (all p<0.001). However, p65-N expression was found significantly decreased in OSCC (all p<0.002), except in LSCC (p=0.119). In BMSCC patients, the higher expression of p62 was associated with poor cell differentiation (p=0.015) and lymph node metastasis (p=0.033). However, the expression of EGFR in N0 was higher than that in lymph node metastasis (p=0.031). In TSCC patients, the higher expressions of LC3 (p=0.045), p62 (p=0.040), and p65-C (p<0.001) were associated with poor cell differentiation. Moreover, higher expressions of EGFR-C are associated with advanced AJCC pathological stage (p<0.001), higher T classification (p=0.003), and lymph node metastasis patients (p=0.013). In LSCC patients, an increased expression of p62 was associated with poor cell differentiation (p=0.003) and p65-C expression was higher in older patients (>50 yrs, p=0.009). In OSCC patients, expression levels of LC3, p65-N, and p65-C were significantly different between three subsites (p<0.001). An increased expression of p62 (p=0.001) and p65-C p<0.001) was found in those with poor cell differentiation. The expressions of EGFR-C was accordingly associated in advanced AJCC pathological stage (p<0.001) and high T classification (p=0.006). An increased expression of p65-C (p=0.012) and EGFR-C (p=0.012) but a reduced expression of EGFR-M (p=0.019) were found in those with lymph node metastasis. Finally, the univariate and multivariate analysis of survival showed that a higher expression level of p62 was associated with a poor disease-specific survival (DSS) and disease-free survival (DFS) of OSCC patients, especially DSS for BMSCC patients) and DFS for LSCC. The LC3, p62, p65, and EGFR could be biomarkers for tumorigenesis in OSCC, including three primary subsites. Moreover, p62 is a potential candidate factor for prognosis in OSCC, especially for BMSCC and LSCC.

Contents
審定書------------------------------------------------------------------------------------------------i
誌謝--------------------------------------------------------------------------------------------------ii
Abstract in Chinese--------------------------------------------------------------------------------iii
Abstract in English---------------------------------------------------------------------------------v
Contents-------------------------------------------------------------------------------------------viii
Abbreviayions---------------------------------------------------------------------------------------x
Introduction------------------------------------------------------------------------------------------1
1. Oral Cancer------------------------------------------------------------------------------------1
2. Autophagy and cancer-----------------------------------------------------------------------3
3. Microtubule-associated protein light chain 3 (LC3)-------------------------------------6
4. SQSTM1/ p62 (p62)--------------------------------------------------------------------------7
5. NF-κB/Rel A(p65)----------------------------------------------------------------------------8
6. Epidermal growth factor receptor (EGFR) ----------------------------------------------10
Specific Aims--------------------------------------------------------------------------------------13
Materials and Methods---------------------------------------------------------------------------14
Results----------------------------------------------------------------------------------------------19
1. Comparisons of LC3, p62, p65, and EGFR expressions in the normal tissue, TAN tissue, tumor tissue of oral SCC-----------------------------------------------------------19
2. The correlation of LC3, p62, p65, and EGFR in oral SCC patients------------------22
3. Expressions of LC3, p62, p65, EGFR and clinicopathologic outcomes in BMSCC patients----------------------------------------------------------------------------------------24
4. Expressions of LC3, p62, p65, EGFR and clinicopathologic outcomes in TSCC patients----------------------------------------------------------------------------------------25
5. Expressions of LC3, p62, p65 and clinicopathologic outcomes in LSCC patients----------------------------------------------------------------------------------------26
6. Expressions of LC3, p62, p65, EGFR and clinicopathologic outcomes in OSCC patients----------------------------------------------------------------------------------------27
7. Expression levels of LC3, p62, p65, and EGFR and disease-specific survival of oral SCC patient-----------------------------------------------------------------------------28
8. Expression levels of LC3, p62, p65, and EGFR and disease-free survival of oral SCC patients---------------------------------------------------------------------------------29
Discussion------------------------------------------------------------------------------------------31
Conclusion-----------------------------------------------------------------------------------------49
References------------------------------------------------------------------------------------------50
Tables-----------------------------------------------------------------------------------------------73
Figures----------------------------------------------------------------------------------------------84

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