The anti-cancer mechanism of dasatinib against head and neck squamous cell carcinoma (HNSCC)

本論文探討dasatinib引發頭頸癌細胞凋亡的藥理機轉。頭頸部鱗狀細胞癌(簡稱頭頸癌)是一個病程快速且預後不佳的世界性疾病。表皮生長因子受體(EGFR)會促進頭頸癌細胞的生長分裂，而其大量表現與頭頸癌的臨床預後不佳有關。雌激素受體(estrogen receptor)是一個作用廣泛的細胞核受體，能直接引發基因表現或是非基因表現的訊息傳遞。在頭頸癌中，雌激素受體會與表皮生長因子受體產生協同作用以促進細胞的生長與侵犯能力。近年來是標靶治療的時代，表皮生長因子受體的單株抗體cetuximab在頭頸癌成功的展現療效；儘管如此，cetuximab的效果仍然有限，頭頸癌整體治療成績仍然不佳，因此開發新的治療是重要課題。Dasatinib是一個對於Bcr-abl及Src均有抑制能力的磷酸激酶抑制劑(kinase inhibitor)，已被核准用於慢性骨髓性白血病的臨床治療，也被認為對於固態腫瘤具有治療潛力。然而在dasatinib於頭頸癌的臨床試驗中，雖然僅有少數病人得到效果，然而Src的抑制與療效之間並無關連，推測應有Src以外的機轉決定dasatinib的療效。本研究的第一部分指出，表皮生長因子的分解是一個決定dasatinib是否引發頭頸癌細胞凋亡的關鍵步驟，而此步驟是經由溶小體(lysosome)所完成。此外，雌激素受體也參與表皮生長因子受體調控dasatinib引發的細胞凋亡。進一步在動物模式中証實，dasatinib的確會藉由負調控(down-regulation)表皮生長因子及雌激素受體而抑制頭頸癌的生長。 本研究的第二部分則是進一步發現，dasatinib引發之表皮生長因子分解是來自於AMPK引發的內質網壓力(ER stress)。Dasatinib會引發內質網壓力，再經由c-cbl的作用促進表皮生長因子受體之分解。Dasatinib所誘發的AMPK活化可能是來自於PDK4增加導致細胞內之ATP下降。metformin引發AMPK活化則會增強dasatinib在細胞模式及動物模式中抑制頭頸癌的效果，在人類頭頸癌的檢體也發現AMPK活化與表皮生長因子受體表現的相關性。本研究指出dasatinib是經由AMPK-dependent ER stress導致表皮生長因子受體分解及頭頸癌細胞凋亡，藉由活化AMPK會進一步加強dasatinib對頭頸癌的療效，為頭頸癌的治療帶來進步。The mechanism of dasatinib-induced apoptosis of head and neck squamous cell carcinoma (HNSCC) cells was investigated in this study. HNSCC is a worldwide disease with aggressive course and dismal outcome. The expression of epidermal growth factor receptor (EGFR) promotes cell growth and proliferation and is associated with clinical poor outcome of HNSCC. Estrogen receptor is a nuclear receptor which can exert both genomic and non-genomic actions. In HNSCC, estrogen receptor signaling is found to work in concert with EGFR to enhance cell growth and invasion. In the era of molecular targeted therapy, the emergence of cetuximab, an monoclonal antibody against EGFR, has led to a progress in HNSCC, but the efficacy is modest. Thus, new therapies are needed. Dasatinib is a Bcr-abl and Src inhibitor which has been approved for chronic myeloid leukemia and is expected to have activity against solid tumors. However, few patients benefit from dasatinib in clinical trials despite consistent Src inhibition, implicating that there are mechanisms beyond Src inhibition responsible for the efficacy of dasatinib. The first part of our study disclosed that EGFR degradation was the key event to mediate dasatinib-induced apoptosis in HNSCC cells. This event was through lysosome degradation. In addition, estrogen receptor was found to be associated with EGFR in dasatinib-induced apoptosis. Furthermore, xenograft model showed that dasatinib inhibited HNSCC tumor growth through in vivo down-regulation of EGFR and estrogen receptor. In the second part of the study, we further investigated the mechanism of dasatinib-induced EGFR degradation and showed that AMPK-dependent endoplasmic reticulum (ER) stress is responsible for this event. Dasatinib induced ER stress which mediated EGFR degradation in a c-cbl-dependent manner. AMPK activation induced by dasatinib might be due to ATP decrease through the up-regulation of pyruvate dehydrogenase kinase 4 (PDK4). Furthermore, activation of AMPK by metformin sensitized dasatinib-induced in vitro and in vivo anti-cancer effect. The correlation of AMPK activation and EGFR expression was seen in HNSCC cells and human tumor specimens. Our results disclose that AMPK-dependent ER stress-mediated EGFR degradation plays a crucial role in the anti-cancer effect of dasatinib in HNSCC. Activation of AMPK by metformin might enhance dasatinib efficacy in HNSCC treatment.Abbreviation............................................2 中文摘要................................................4 Abstract in English.....................................5 Chapter I. Introduction 1. Head and neck squamous cell carcinoma (HNSCC)..6 2. Epidermal growth factor receptor (EGFR).......10 3. Src.......................................... 15 4. Estrogen receptor.............................19 5. BCL-2 family proteins and apoptosis...........22 6. Endoplasmic reticulum (ER) stress.............24 7. AMP-activated protein kinase (AMPK)...........26 8. Dasatinib and experimental rationale..........30 Chapter II. Materials and Methods......................31 Chapter III. Results Part I. Degradation of epidermal growth factor receptor (EGFR) mediates dasatinib-induced apoptosis in head and neck squamous cell carcinoma cells.....................37 Part II. Metformin sensitizes anticancer effect of dasatinib in head and neck squamous cell carcinoma cells through AMPK-dependent ER stress.......................57 Chapter IV. Conclusion and perspective.................75 Reference..............................................78 Publications and Honors................................9