靈芝蛋白(GMI)是一種由小孢子靈芝中所萃取出的免疫調節蛋白質，在先前的文獻中發現GMI具有免疫調節的作用以及抗腫瘤的活性。肺癌是目前世界上發生率及致死率皆居於首的癌症。粘著斑激酶（FAK）為一非受體酪氨酸激酶，其在許多癌症中的時常有過度表達及活化的現象，它調控多種訊息傳遞途徑並且往往促進癌細胞的生長以及轉移。細胞自噬作用是細胞內透過分解故障或受損的蛋白質以及胞器，來達到一種生理平衡循環的機制，在本篇中，我們證明了GMI會誘導非小細胞肺癌（NSCLC）產生細胞自噬作用，並降低FAK和Src磷酸化。更進一步，我們發現，處理抑制劑（氯喹）或使用shRNA knockdown系統抑制自噬作用後再處理GMI，癌細胞中的磷酸化Src的量相較於對照組有回升的現象並且增強細胞死亡。這些結果表明，GMI誘導非小細胞肺癌產生自噬作用。而自噬作用通常可以被認為是一細胞自我保護的機制。因此，未來在利用GMI的治療上合併使用自噬作用的抑製劑可能為一策略，能夠提升GMI抗癌的功效。

GMI, an immune-modulatory protein from Ganoderma microsporum, exhibits immunomodulatory and antitumor activity. Lung cancer is the leading cause of cancer incidence and death in the world. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is ubiquitously overexpressed and activated in several solid cancers. It regulates various signaling pathways to promote cancer cells growth and metastasis. Autophagy is an important homeostatic cellular recycling mechanism that involves cells degradation of malfunctioning proteins or damaged organelles. Here we demonstrated that GMI induced autophagy in non-small cell lung cancer (NSCLC), and down-regulated phosphorylation of FAK and Src. Specifically, we found that inhibition of autophagy by inhibitor treatment (chloroquine) or shRNA knockdown system rescued the phosphorylation level of Src and enhanced cell death in cancer cells upon GMI treatment. These results suggest that GMI induces autophagic flux in NSCLC. Autophagic flux generally may be considered as cyto-protective effect. Thus, Combination of autophagy inhibitor with GMI may a strategy to increase GMI anti-cancer efficacy.

誌謝 i
Abbreviations ii
Abstract iii
中文摘要 iv
Introduction 1
Materials and methods 5
Cell lines 5
Preparation of the recombinant GMI protein 5
Reagents and antibodies 6
Western blot analysis 6
Cell viability assay 6
Transfection of shRNAs 7
Statistical analysis 7
Results 8
GMI induces autophagy in NSCLC cell lines 8
GMI inhibits FAK/Src signaling pathway in NSCLC cell lines 8
The turn-over of phospho src is enhanecd in a lysosomal degradation 9
Inhibition of autophagy promotes cancer cell death in GMI treatment 9
Knockdown c-Cbl promotes cancer cell death in GMI treatment 10
Src catalytic activity is needed for autophagic targeting 10
Inhibition of autophagy increase p53 and PARP expression by GMI treatment 10
Discussion 12
References 14
Figure legends 20
Figure 1. Induction of autophagy in NSCLC cells by GMI treatment. 20
Figure 2. GMI inhibits FAK/Src signaling pathway. 24
Figure 3. The turn-over of phospho src is enhanecd in a lysosomal degradation 26
Figure 4. Inhibition of autophagy promotes cancer cell death in GMI treatment. 29
Figure 5. Knockdown c-Cbl promotes cancer cell death in GMI treatment. 32
Figure 6. Src catalytic activity is needed for autophagic targeting. 34
Figure 7. Inhibition of autophagy increase p53 and PARP expression by GMI treatment 37

Brown, M. T. and J. A. Cooper (1996). "Regulation, substrates and functions of src." Biochim Biophys Acta 1287(2-3): 121-149.

Cance, W. G. and V. M. Golubovskaya (2008). "Focal adhesion kinase versus p53: apoptosis or survival?" Sci Signal 1(20): pe22.

Canel, M., A. Serrels, M. C. Frame and V. G. Brunton (2013). "E-cadherin-integrin crosstalk in cancer invasion and metastasis." J Cell Sci 126(Pt 2): 393-401.

Cartwright, C. A., M. P. Kamps, A. I. Meisler, J. M. Pipas and W. Eckhart (1989). "pp60c-src activation in human colon carcinoma." J Clin Invest 83(6): 2025-2033.

Cecconi, F. (2012). "c-Cbl targets active Src for autophagy." Nat Cell Biol 14(1): 48-49.

Cooper, J. A., K. L. Gould, C. A. Cartwright and T. Hunter (1986). "Tyr527 is phosphorylated in pp60c-src: implications for regulation." Science 231(4744): 1431-1434.

Fan, H., X. Zhao, S. Sun, M. Luo and J. L. Guan (2013). "Function of focal adhesion kinase scaffolding to mediate endophilin A2 phosphorylation promotes epithelial-mesenchymal transition and mammary cancer stem cell activities in vivo." J Biol Chem 288(5): 3322-3333.
Frisch, S. M., M. Schaller and B. Cieply (2013). "Mechanisms that link the oncogenic epithelial-mesenchymal transition to suppression of anoikis." J Cell Sci 126(Pt 1): 21-29.

He, C. and D. J. Klionsky (2009). "Regulation mechanisms and signaling pathways of autophagy." Annu Rev Genet 43: 67-93.
Hsin, I. L., C.-C. Ou, T.-C. Wu, M.-S. Jan, M.-F. Wu, L.-Y. Chiu, K.-H. Lue and J.-L. Ko (2011). "GMI, an immunomodulatory protein from Ganoderma microsporum, induces autophagy in non-small cell lung cancer cells." Autophagy 7(8): 873-882.

Hu, Y. L., A. Jahangiri, M. Delay and M. K. Aghi (2012). "Tumor cell autophagy as an adaptive response mediating resistance to treatments such as antiangiogenic therapy." Cancer Res 72(17): 4294-4299.

Irby, R. B., W. Mao, D. Coppola, J. Kang, J. M. Loubeau, W. Trudeau, R. Karl, D. J. Fujita, R. Jove and T. J. Yeatman (1999). "Activating SRC mutation in a subset of advanced human colon cancers." Nat Genet 21(2): 187-190.

Jinn, T.-R., C.-M. Wu, W.-C. Tu, J.-L. Ko and J. T. C. Tzen (2014). "Functional Expression of FIP-gts, a Fungal Immunomodulatory Protein fromGanoderma Tsugaein Sf21 Insect Cells." Bioscience, Biotechnology and Biochemistry 70(11): 2627-2634.

Kafri, T., H. van Praag, L. Ouyang, F. H. Gage and I. M. Verma (1999). "A packaging cell line for lentivirus vectors." J Virol 73(1): 576-584.

Kroemer, G., G. Marino and B. Levine (2010). "Autophagy and the integrated stress response." Mol Cell 40(2): 280-293.

Li, X. Y., X. Zhou, R. G. Rowe, Y. Hu, D. D. Schlaepfer, D. Ilic, G. Dressler, A. Park, J. L. Guan and S. J. Weiss (2011). "Snail1 controls epithelial-mesenchymal lineage commitment in focal adhesion kinase-null embryonic cells." J Cell Biol 195(5): 729-738.
Lin, C.-H., G.-T. Sheu, Y.-W. Lin, C.-S. Yeh, Y.-H. Huang, Y.-C. Lai, J.-G. Chang and J.-L. Ko (2010). "A new immunomodulatory protein from Ganoderma microsporum inhibits epidermal growth factor mediated migration and invasion in A549 lung cancer cells." Process Biochemistry 45(9): 1537-1542.

Lin, C. H., Y. M. Hsiao, C. C. Ou, Y. W. Lin, Y. L. Chiu, K. H. Lue, J. G. Chang and J. L. Ko (2010). "GMI, a Ganoderma immunomodulatory protein, down-regulates tumor necrosis factor alpha-induced expression of matrix metalloproteinase 9 via NF-kappaB pathway in human alveolar epithelial A549 cells." J Agric Food Chem 58(22): 12014-12021.

Lipfert, L., B. Haimovich, M. D. Schaller, B. S. Cobb, J. T. Parsons and J. S. Brugge (1992). "Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets." J Cell Biol 119(4): 905-912.

Lock, R. and J. Debnath (2008). "Extracellular matrix regulation of autophagy." Curr Opin Cell Biol 20(5): 583-588.

Meng, X. N., Y. Jin, Y. Yu, J. Bai, G. Y. Liu, J. Zhu, Y. Z. Zhao, Z. Wang, F. Chen, K. Y. Lee and S. B. Fu (2009). "Characterisation of fibronectin-mediated FAK signalling pathways in lung cancer cell migration and invasion." Br J Cancer 101(2): 327-334.

Mitra, S. K., D. A. Hanson and D. D. Schlaepfer (2005). "Focal adhesion kinase: in command and control of cell motility." Nat Rev Mol Cell Biol 6(1): 56-68.

Miyoshi, H., U. Blomer, M. Takahashi, F. H. Gage and I. M. Verma (1998). "Development of a self-inactivating lentivirus vector." J Virol 72(10): 8150-8157.

Mizushima, N., B. Levine, A. M. Cuervo and D. J. Klionsky (2008). "Autophagy fights disease through cellular self-digestion." Nature 451(7182): 1069-1075.

Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma and D. Trono (1996). "In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector." Science 272(5259): 263-267.

Okada, M. (2012). "Regulation of the SRC family kinases by Csk." Int J Biol Sci 8(10): 1385-1397.
Pallis, A. G. (2012). "A Review of Treatment in Non-small-cell Lung Cancer." EUROPEAN ONCOLOGY &; HAEMATOLOGY 8(4): 5.

Parsons, J. T. (2003). "Focal adhesion kinase: the first ten years." J Cell Sci 116(Pt 8): 1409-1416.
Paterson, R. R. (2006). "Ganoderma - a therapeutic fungal biofactory." Phytochemistry 67(18): 1985-2001.

Roskoski, R. (2004). "Src protein–tyrosine kinase structure and regulation." Biochemical and Biophysical Research Communications 324(4): 1155-1164.

Roskoski, R., Jr. (2005). "Src kinase regulation by phosphorylation and dephosphorylation." Biochem Biophys Res Commun 331(1): 1-14.

Sandilands, E., B. Serrels, D. G. McEwan, J. P. Morton, J. P. Macagno, K. McLeod, C. Stevens, V. G. Brunton, W. Y. Langdon, M. Vidal, O. J. Sansom, I. Dikic, S. Wilkinson and M. C. Frame (2012). "Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling." Nat Cell Biol 14(1): 51-60.

Schaller, M. D. (2001). "Biochemical signals and biological responses elicited by the focal adhesion kinase." Biochim Biophys Acta 1540(1): 1-21.

Sharma, S. V., D. W. Bell, J. Settleman and D. A. Haber (2007). "Epidermal growth factor receptor mutations in lung cancer." Nat Rev Cancer 7(3): 169-181.

Shibue, T., M. W. Brooks, M. F. Inan, F. Reinhardt and R. A. Weinberg (2012). "The outgrowth of micrometastases is enabled by the formation of filopodium-like protrusions." Cancer Discov 2(8): 706-721.

Siegel, R., J. Ma, Z. Zou and A. Jemal (2014). "Cancer statistics, 2014." CA Cancer J Clin 64(1): 9-29.

Sui, X., R. Chen, Z. Wang, Z. Huang, N. Kong, M. Zhang, W. Han, F. Lou, J. Yang, Q. Zhang, X. Wang, C. He and H. Pan (2013). "Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment." Cell Death Dis 4: e838.

Sulzmaier, F. J., C. Jean and D. D. Schlaepfer (2014). "FAK in cancer: mechanistic findings and clinical applications." Nat Rev Cancer 14(9): 598-610.

Thomas, S. M. and J. S. Brugge (1997). "Cellular functions regulated by Src family kinases." Annu Rev Cell Dev Biol 13: 513-609.

Verbeek, B. S., T. M. Vroom, S. S. Adriaansen-Slot, A. E. Ottenhoff-Kalff, J. G. Geertzema, A. Hennipman and G. Rijksen (1996). "c-Src protein expression is increased in human breast cancer. An immunohistochemical and biochemical analysis." J Pathol 180(4): 383-388.

Yokouchi, M., T. Kondo, A. Sanjay, A. Houghton, A. Yoshimura, S. Komiya, H. Zhang and R. Baron (2001). "Src-catalyzed phosphorylation of c-Cbl leads to the interdependent ubiquitination of both proteins." J Biol Chem 276(37): 35185-35193.

Zou, Z., Z. Yuan, Q. Zhang, Z. Long, J. Chen, Z. Tang, Y. Zhu, S. Chen, J. Xu, M. Yan, J. Wang and Q. Liu (2012). "Aurora kinase A inhibition-induced autophagy triggers drug resistance in breast cancer cells." Autophagy 8(12): 1798-1810.