Interleukin-6 (IL-6)是一個具有多種功能的Cytokine，調節體內許多的生理反應，也和許多人類的疾病有相關。除了一些發炎、感染與自體免疫的疾病之外，在許多腫瘤的生長當中，包括肺癌在內，IL-6也都扮演了一個重要的角色。從過去的研究已經知道，血清中IL-6濃度較高的肺癌病人，往往有較末期的疾病以及對治療反應較差，但是IL-6濃度的高低對於預後上的重要性，卻一直未有一致的定論。在此研究當中，我們利用當初收集在Genetic Epidemiological Study of Lung AdenoCarcinoma (GELAC)當中的肺癌病人，來分析「肺癌病人血清中IL-6濃度的高低與存活時間的關係」，從Kaplan-Meier和Log-Rank test分析的結果發現：血清中IL-6濃度高、中、低三組不同的肺癌病人，其存活的時間上是有統計學上的差異的！而在使用Cox proportional regression model以及利用分層的方式來將可能的干擾因子去除後，一樣可以看到存活時間上的差異，由此更加證實了IL-6對於肺癌的重要性。
我們在證實臨床上IL-6對肺癌的重要性之後，試著在肺癌細胞株上，尋找可能抑制肺癌細胞分泌IL-6的方式。由於IL-6會促進肺癌細胞的生長、肺癌細胞還可以透過IL-6 autocrine的方式促進細胞的存活與增生、再加上肺癌細胞調控IL-6 autocrine的三個重要訊息傳遞路徑，其中的訊息傳遞分子都是熱休克蛋白九十所保護的蛋白質(client protein)，因此我們選擇「熱休克蛋白九十」為抑制目標，試圖利用熱休克蛋白九十抑制劑(Hsp90 inhibitor)來抑制熱休克蛋白九十以及透過組織蛋白去乙醯酶抑制劑(HDACs inhibitor)來影響熱休克蛋白九十的乙醯化程度與功能，進一步影響其保護的蛋白：STAT3、Akt、和MEK而達成抑制肺癌細胞分泌IL-6的目標。研究結果顯示：熱休克蛋白九十的抑制劑：NYP-AUY922與NYP-HSP990，可以有效的抑制熱休克蛋白九十以及與調控肺癌IL-6 autocrine的JAK2/STAT3、PI3K/Akt、Ras/Raf/MEK/ERK的訊息傳遞，不過對於肺癌細胞分泌IL-6的抑制效果，則不甚理想；其他與IL-6分泌相關的訊息傳遞路徑，包括NF-κB、p38 MAPK、與GSK-3的路徑，也並沒有在熱休克蛋白九十抑制劑的使用狀況下被活化。肺癌細胞是藉由什麼樣的機制來繼續分泌IL-6，還需要往後更進一步的研究。使用組織蛋白去乙醯酶的抑制劑，則可以對肺癌細胞分泌IL-6有很好的抑制效果，但是組織蛋白去乙醯酶抑制劑影響的目標並不專一，是否一定是藉由影響熱休克蛋白九十而抑制肺癌細胞分泌IL-6，同樣有待後續的研究來解答。

Interleukin-6 is a pleiotropic cytokine that modulates a variety of physiological functions. It is also associated with many human diseases, such as infection, inflammation, autoimmune diseases, and cancers. IL-6 is involved in the growth of a variety of cancers and acts as a growth factor for many tumors, including lung cancer. Clinically, elevated plasma IL-6 levels have been described in patients with lung cancer, and higher serum levels of IL-6 are related to disseminated disease, tumor progression, and unresponsive to chemotherapy. However, the correlation between serum IL-6 and patient survival does not have the consistent results. In this study, we try to investigate the correlation between the plasma IL-6 level and survival of lung cancer patients by analyzing the cohort of lung cancer patients in the Genetic Epidemiological Study of Lung AdenoCarcinoma (GELAC). The result of Kaplan-Meier survival analyses and Log-Rank test show that there is statistically significant difference in survival of lung cancer patients across three IL-6 level groups. Results of Cox proportional regression model adjusted for age, sex, smoking history, histologic type and stage of lung cancer also demonstrates significant relationship between IL-6 level and survival time.
After the importance of IL-6 in lung cancer patients is confirmed clinically, we try to find ways to suppress IL-6 secretion of lung cancer cells. IL-6 could promote tumor growth through an “autocrine” mechanism. Because the JAK2/STAT3, PI3K/Akt and Ras/Raf/MEK/ERK signaling pathways all contribute to IL-6 autocrine in lung cancer cells, and STAT3, Akt, and MEK are all client proteins of heat shock protein 90 (Hsp90), we plan to suppress IL-6 secretion of lung cancer cells via inhibiting Hsp90 by Hsp90 inhibitors and histone deacetylase (HDAC) inhibitors. Our study shows that synthetic Hsp90 inhibitors, NYP-AUY922 and NYP-HSP990, could inhibit Hsp90 and block JAK2/STAT3, PI3K/Akt and Ras/Raf/MEK/ERK signaling pathways in lung cancer cells. However, they do not suppress IL-6 secretion of lung cancer cells obviously. We try to identify possible activation of signaling pathways associated with IL-6 secretion, including NF-κB, p38 MAPK, and GSK-3 signaling pathways, but no one is up-regulated under the use of Hsp90 inhibitors. The mechanism of failed blockade of IL-6 secretion by Hsp90 inhibitors is worth to be further investigated. In contrast, HDAC inhibitors could suppress IL-6 secretion of lung cancer patients well. However, the HDAC inhibitor (LBH589) we used is a pan-HDAC inhibitor which could not only disrupt the chaperone function of Hsp90 but also induce acetylation of histone H3 and H4. So, the definite mechanism of suppression of IL-6 secretion by HDAC inhibitors in lung cancer cells is also waiting to be clarified.

Ara, T. and Y. A. Declerck "Interleukin-6 in bone metastasis and cancer progression." Eur J Cancer 46(7): 1223-31, (2010).
Banerji, U., M. Walton, F. Raynaud, R. Grimshaw, L. Kelland, M. Valenti, I. Judson and P. Workman "Pharmacokinetic-pharmacodynamic relationships for the heat shock protein 90 molecular chaperone inhibitor 17-allylamino, 17-demethoxygeldanamycin in human ovarian cancer xenograft models." Clin Cancer Res 11(19 Pt 1): 7023-32, (2005).
Baselga, J. and J. Albanell "Targeting epidermal growth factor receptor in lung cancer." Curr Oncol Rep 4(4): 317-24, (2002).
Chang, C. H., C. F. Hsiao, G. C. Chang, Y. H. Tsai, Y. M. Chen, M. S. Huang, W. C. Su, W. S. Hsieh, P. C. Yang, C. J. Chen and C. A. Hsiung "Interactive effect of cigarette smoking with human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) polymorphisms on the risk of lung cancer: a case-control study in Taiwan." Am J Epidemiol 170(6): 695-702, (2009).
Chauhan, D., H. Uchiyama, Y. Akbarali, M. Urashima, K. Yamamoto, T. A. Libermann and K. C. Anderson "Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B." Blood 87(3): 1104-12, (1996).
Clarke, P. A., I. Hostein, U. Banerji, F. D. Stefano, A. Maloney, M. Walton, I. Judson and P. Workman "Gene expression profiling of human colon cancer cells following inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, an inhibitor of the hsp90 molecular chaperone." Oncogene 19(36): 4125-33, (2000).
David, S. E., A. Wallace and B. Hossein "NCCN Guidelines Version 3.2011 Non-Small Cell Lung Cancer." NCCN Clinical Practice Guilindes in Oncology, (2010).
De Vita, F., M. Orditura, A. Auriemma, S. Infusino, A. Roscigno and G. Catalano "Serum levels of interleukin-6 as a prognostic factor in advanced non-small cell lung cancer." Oncol Rep 5(3): 649-52, (1998).
Fiskus, W., Y. Ren, A. Mohapatra, P. Bali, A. Mandawat, R. Rao, B. Herger, Y. Yang, P. Atadja, J. Wu and K. Bhalla "Hydroxamic acid analogue histone deacetylase inhibitors attenuate estrogen receptor-alpha levels and transcriptional activity: a result of hyperacetylation and inhibition of chaperone function of heat shock protein 90." Clin Cancer Res 13(16): 4882-90, (2007).
Fukuoka, M., S. Yano, G. Giaccone, T. Tamura, K. Nakagawa, J. Y. Douillard, Y. Nishiwaki, J. Vansteenkiste, S. Kudoh, D. Rischin, R. Eek, T. Horai, K. Noda, I. Takata, E. Smit, S. Averbuch, A. Macleod, A. Feyereislova, R. P. Dong and J. Baselga "Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected]." J Clin Oncol 21(12): 2237-46, (2003).
Gabai, V. L., K. R. Budagova and M. Y. Sherman "Increased expression of the major heat shock protein Hsp72 in human prostate carcinoma cells is dispensable for their viability but confers resistance to a variety of anticancer agents." Oncogene 24(20): 3328-38, (2005).
Gao, S. P., K. G. Mark, K. Leslie, W. Pao, N. Motoi, W. L. Gerald, W. D. Travis, W. Bornmann, D. Veach, B. Clarkson and J. F. Bromberg "Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas." J Clin Invest 117(12): 3846-56, (2007).
Gotschel, F., C. Kern, S. Lang, T. Sparna, C. Markmann, J. Schwager, S. McNelly, F. von Weizsacker, S. Laufer, A. Hecht and I. Merfort "Inhibition of GSK3 differentially modulates NF-kappaB, CREB, AP-1 and beta-catenin signaling in hepatocytes, but fails to promote TNF-alpha-induced apoptosis." Exp Cell Res 314(6): 1351-66, (2008).
Gregoretti, I. V., Y. M. Lee and H. V. Goodson "Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis." J Mol Biol 338(1): 17-31, (2004).
Haura, E. B., S. Livingston and D. Coppola "Autocrine interleukin-6/interleukin-6 receptor stimulation in non-small-cell lung cancer." Clin Lung Cancer 7(4): 273-5, (2006).
Hideshima, T., K. Podar, D. Chauhan and K. C. Anderson "Cytokines and signal transduction." Best Pract Res Clin Haematol 18(4): 509-24, (2005).
Hirano, T. "Interleukin 6 and its receptor: ten years later." Int Rev Immunol 16(3-4): 249-84, (1998).
Hodge, D. R., E. M. Hurt and W. L. Farrar "The role of IL-6 and STAT3 in inflammation and cancer." Eur J Cancer 41(16): 2502-12, (2005).
Hoffman, P. C., A. M. Mauer and E. E. Vokes "Lung cancer." Lancet 355(9202): 479-85, (2000).
Huang, W. L., H. H. Yeh, C. C. Lin, W. W. Lai, J. Y. Chang, W. T. Chang and W. C. Su "Signal transducer and activator of transcription 3 activation up-regulates interleukin-6 autocrine production: a biochemical and genetic study of established cancer cell lines and clinical isolated human cancer cells." Mol Cancer 9: 309, (2010).
Jemal, A., R. Siegel, J. Xu and E. Ward "Cancer statistics, 2010." CA Cancer J Clin 60(5): 277-300, (2010).
Jeong, H. J., S. H. Hong, R. K. Park, T. Shin, N. H. An and H. M. Kim "Hypoxia-induced IL-6 production is associated with activation of MAP kinase, HIF-1, and NF-kappaB on HEI-OC1 cells." Hear Res 207(1-2): 59-67, (2005).
Kamimura, D., K. Ishihara and T. Hirano "IL-6 signal transduction and its physiological roles: the signal orchestration model." Rev Physiol Biochem Pharmacol 149: 1-38, (2003).
Kaminska, J., M. Kowalska, B. Kotowicz, M. Fuksiewicz, M. Glogowski, E. Wojcik, M. Chechlinska and J. Steffen "Pretreatment serum levels of cytokines and cytokine receptors in patients with non-small cell lung cancer, and correlations with clinicopathological features and prognosis. M-CSF - an independent prognostic factor." Oncology 70(2): 115-25, (2006).
Kishimoto, T. "Interleukin-6: from basic science to medicine--40 years in immunology." Annu Rev Immunol 23: 1-21, (2005).
Kovacs, J. J., P. J. Murphy, S. Gaillard, X. Zhao, J. T. Wu, C. V. Nicchitta, M. Yoshida, D. O. Toft, W. B. Pratt and T. P. Yao "HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor." Mol Cell 18(5): 601-7, (2005).
Kris, M. G., R. B. Natale, R. S. Herbst, T. J. Lynch, Jr., D. Prager, C. P. Belani, J. H. Schiller, K. Kelly, H. Spiridonidis, A. Sandler, K. S. Albain, D. Cella, M. K. Wolf, S. D. Averbuch, J. J. Ochs and A. C. Kay "Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial." JAMA 290(16): 2149-58, (2003).
Kurashina, R., J. H. Ohyashiki, C. Kobayashi, R. Hamamura, Y. Zhang, T. Hirano and K. Ohyashiki "Anti-proliferative activity of heat shock protein (Hsp) 90 inhibitors via beta-catenin/TCF7L2 pathway in adult T cell leukemia cells." Cancer Lett 284(1): 62-70, (2009).
Lane, A. A. and B. A. Chabner "Histone deacetylase inhibitors in cancer therapy." J Clin Oncol 27(32): 5459-68, (2009).
Lemoine, M. and A. Younes "Histone deacetylase inhibitors in the treatment of lymphoma." Discov Med 10(54): 462-70, (2010).
Li, Y. S., E. Davidson, C. N. Reid and A. P. McHale "Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: potential applications for gene therapy of cancer." Cancer Lett 273(1): 62-9, (2009).
Lynch, T. J., D. W. Bell, R. Sordella, S. Gurubhagavatula, R. A. Okimoto, B. W. Brannigan, P. L. Harris, S. M. Haserlat, J. G. Supko, F. G. Haluska, D. N. Louis, D. C. Christiani, J. Settleman and D. A. Haber "Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib." N Engl J Med 350(21): 2129-39, (2004).
Mahalingam, D., R. Swords, J. S. Carew, S. T. Nawrocki, K. Bhalla and F. J. Giles "Targeting HSP90 for cancer therapy." Br J Cancer 100(10): 1523-9, (2009).
Markel, T. A., P. R. Crisostomo, M. Wang, C. M. Herring, T. Lahm, K. K. Meldrum, K. D. Lillemoe, F. J. Rescorla and D. R. Meldrum "Iron chelation acutely stimulates fetal human intestinal cell production of IL-6 and VEGF while decreasing HGF: the roles of p38, ERK, and JNK MAPK signaling." Am J Physiol Gastrointest Liver Physiol 292(4): G958-63, (2007).
Martin, F., F. Santolaria, N. Batista, A. Milena, E. Gonzalez-Reimers, M. J. Brito and J. Oramas "Cytokine levels (IL-6 and IFN-gamma), acute phase response and nutritional status as prognostic factors in lung cancer." Cytokine 11(1): 80-6, (1999).
Mok, T. S., Y. L. Wu, S. Thongprasert, C. H. Yang, D. T. Chu, N. Saijo, P. Sunpaweravong, B. Han, B. Margono, Y. Ichinose, Y. Nishiwaki, Y. Ohe, J. J. Yang, B. Chewaskulyong, H. Jiang, E. L. Duffield, C. L. Watkins, A. A. Armour and M. Fukuoka "Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma." N Engl J Med 361(10): 947-57, (2009).
Paez, J. G., P. A. Janne, J. C. Lee, S. Tracy, H. Greulich, S. Gabriel, P. Herman, F. J. Kaye, N. Lindeman, T. J. Boggon, K. Naoki, H. Sasaki, Y. Fujii, M. J. Eck, W. R. Sellers, B. E. Johnson and M. Meyerson "EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy." Science 304(5676): 1497-500, (2004).
Powers, M. V., P. A. Clarke and P. Workman "Dual targeting of HSC70 and HSP72 inhibits HSP90 function and induces tumor-specific apoptosis." Cancer Cell 14(3): 250-62, (2008).
Powers, M. V. and P. Workman "Inhibitors of the heat shock response: biology and pharmacology." FEBS Lett 581(19): 3758-69, (2007).
Sehgal, P. B., L. Wang, R. Rayanade, H. Pan and L. Margulies "Interleukin-6-type cytokines." Ann N Y Acad Sci 762: 1-14, (1995).
Shepherd, F. A., J. Rodrigues Pereira, T. Ciuleanu, E. H. Tan, V. Hirsh, S. Thongprasert, D. Campos, S. Maoleekoonpiroj, M. Smylie, R. Martins, M. van Kooten, M. Dediu, B. Findlay, D. Tu, D. Johnston, A. Bezjak, G. Clark, P. Santabarbara and L. Seymour "Erlotinib in previously treated non-small-cell lung cancer." N Engl J Med 353(2): 123-32, (2005).
Solit, D. B. and G. Chiosis "Development and application of Hsp90 inhibitors." Drug Discov Today 13(1-2): 38-43, (2008).
Songur, N., B. Kuru, F. Kalkan, C. Ozdilekcan, H. Cakmak and N. Hizel "Serum interleukin-6 levels correlate with malnutrition and survival in patients with advanced non-small cell lung cancer." Tumori 90(2): 196-200, (2004).
Tas, F., D. Duranyildiz, A. Argon, H. Oguz, H. Camlica, V. Yasasever and E. Topuz "Serum levels of leptin and proinflammatory cytokines in advanced-stage non-small cell lung cancer." Med Oncol 22(4): 353-8, (2005).
Travis, W. D., E. Brambilla, H. K. Muller-Hermelink and C. C. Harris "World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Lung, Pleura,Thymus and Heart." IARC Press: Lyon, (2004).
Trikha, M., R. Corringham, B. Klein and J. F. Rossi "Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: a review of the rationale and clinical evidence." Clin Cancer Res 9(13): 4653-65, (2003).
Tsui, K. H., T. H. Feng, W. C. Hsieh, P. L. Chang and H. H. Juang "Expression of interleukin-6 is downregulated by 17-(allylamino)-17-demethoxygeldanamycin in human prostatic carcinoma cells." Acta Pharmacol Sin 29(11): 1334-41, (2008).
Vanden Berghe, W., L. Vermeulen, G. De Wilde, K. De Bosscher, E. Boone and G. Haegeman "Signal transduction by tumor necrosis factor and gene regulation of the inflammatory cytokine interleukin-6." Biochem Pharmacol 60(8): 1185-95, (2000).
Wandinger, S. K., K. Richter and J. Buchner "The Hsp90 chaperone machinery." J Biol Chem 283(27): 18473-7, (2008).
Whitesell, L., R. Bagatell and R. Falsey "The stress response: implications for the clinical development of hsp90 inhibitors." Curr Cancer Drug Targets 3(5): 349-58, (2003).
Whitesell, L. and S. L. Lindquist "HSP90 and the chaperoning of cancer." Nat Rev Cancer 5(10): 761-72, (2005).
Wojciechowska-Lacka, A., M. Matecka-Nowak, E. Adamiak, J. K. Lacki and B. Cerkaska-Gluszak "Serum levels of interleukin-10 and interleukin-6 in patients with lung cancer." Neoplasma 43(3): 155-8, (1996).
Yano, S., H. Shinohara, R. S. Herbst, H. Kuniyasu, C. D. Bucana, L. M. Ellis, D. W. Davis, D. J. McConkey and I. J. Fidler "Expression of vascular endothelial growth factor is necessary but not sufficient for production and growth of brain metastasis." Cancer Res 60(17): 4959-67, (2000).
Young, J. C., V. R. Agashe, K. Siegers and F. U. Hartl "Pathways of chaperone-mediated protein folding in the cytosol." Nat Rev Mol Cell Biol 5(10): 781-91, (2004).
Zhang, H. and F. Burrows "Targeting multiple signal transduction pathways through inhibition of Hsp90." J Mol Med 82(8): 488-99, (2004).