大腸直腸癌是全球第二大常見且致死率位居第三名的癌症。抗藥性是癌症治療的一大挑戰。根據實驗室先前研究結果發現在oxaliplatin抗藥性細胞株有較高細胞遷移、侵襲以及癌症幹細胞的特性。本論文確認了在oxaliplatin抗藥性的大腸直腸癌細胞中，Wnt/β-catenin訊息傳遞途徑有過度活化的現象，並且其下游基因的表現量增加；其中，由大腸直腸癌臨床檢體分析的結果得知，Wnt/β-catenin訊息傳遞途徑下游產物MMP-7在復發病患的血漿中有增加的趨勢。另外，Dicer蛋白的表現量在抗藥性細胞中增加；降低Dicer後能增加抗藥性細胞對於oxaliplatin的敏感性，以及抑制抗藥性細胞的生長特性。Wnt/β-catenin訊息傳遞路徑在細胞的恆定以及胚胎的發育當中扮演著重要調控者的角色，許多文獻報導也證明Wnt/β-catenin訊息傳遞路徑能參與在癌症的發生和化療的抗藥性，並且在大腸直腸癌有過度活化的現象，說明Wnt/β-catenin訊息傳遞路徑的異常活化可能與大腸直腸癌的抗藥性有很大的關聯。然而，本論文透過小分子抑制劑RPI-724去抑制Wnt/β-catenin訊息傳遞路徑，卻無法增加抗藥性細胞株對oxaliplatin的敏感性。本論文認為，Dicer是導致大腸直腸癌細胞後天的oxaliplatin抗藥性的重要因子；而Wnt/β-catenin訊息傳遞途徑的活化可能參與在大腸直腸癌的惡化過程而不是oxaliplatin抗藥性。

Colorectal cancer (CRC) is the second most common cancer and has the third highest mortality rate in Taiwan. Chemoresistance is one of the major challenges for cancer therapy. In previous results, we found that chemoresistant cells possessed higher abilities in migration, invasion and oncosphere formation. In this study, we focused on investigating novel therapeutic strategies which can overcome oxaliplatin resistance in resistant CRC cell lines. By cDNA microarray analysis, the results suggested that the Wnt/β-catenin signaling pathway may be involved in oxaliplatin resistance. Both reporter assay and RT-qPCR confirmed that the Wnt/β-catenin signaling pathway is over-activated and the expression level of its downstream targets is increased in oxaliplatin resistant CRC cells. By MMP-7 Elisa kit, MMP-7 concentration is increased in resustant CRC patient plasma. Interestingly, Dicer protein is overexpressed in drug-resistant cells; the knock-down of Dicer increased oxaliplatin sensitivity and reduced the ability of migration, invasion and oncosphere formation in oxaliplatin-resistant cells. However, inhibition of the Wnt/β-catenin signaling pathway by PRI-724 was not able to overcome oxaliplatin resistance. Our results suggested that Dicer is an important factor for acquired oxaliplatin resistance and contributes to migration, invasion and sphere formation in resistant CRC cells; as such, activation of the Wnt/β-catenin signaling pathway might play a role in colorectal cancer progression but not oxaliplatin resistance.

林勵娟，研究Dicer以及Aurora-A蛋白對大腸直腸癌化療抗藥性的影響及其機轉，國立成功大學生物資訊與訊息傳遞研究所碩士論文，2017。

Adida, C., Haioun, C., Gaulard, P., Lepage, E., Morel, P., Briere, J., Dombret, H., Reyes, F., Diebold, J., Gisselbrecht, C., Salles, G., Altieri, D.C., and Molina, T.J. Prognostic significance of survivin expression in diffuse large B-cell lymphomas. Blood 96, 1921-1925, 2000.

Allen, K.E., and Weiss, G.J. Resistance may not be futile: microRNA biomarkers for chemoresistance and potential therapeutics. Molecular Cancer Therapeutics 9, 3126-3136, 2010.

Almendro, V., Ametller, E., García Recio, S., Collazo, O., Casas, I., Augé, J.M., Maurel, J., and Gascón, P. The role of MMP7 and its cross-talk with the FAS/FASL system during the acquisition of chemoresistance to oxaliplatin. Public Library of Science 4, e4728, 2009.

Anastas, J.N., and Moon, R.T. WNT signalling pathways as therapeutic targets in cancer. Nature Reviews Cancer 13, 11-26, 2013.

Annunziata, C.M., Stavnes, H.T., Kleinberg, L., Berner, A., Hernandez, L.F., Birrer, M.J., Steinberg, S.M., Davidson, B., and Kohn, E.C. Nuclear factor kappaB transcription factors are coexpressed and convey a poor outcome in ovarian cancer. Cancer 116, 3276-3284, 2010.

Bahubeshi, A., Tischkowitz, M., and Foulkes, W.D. miRNA processing and human cancer: DICER1 cuts the mustard. Science Translational Medicine 3, 111ps146, 2011.

Behrens, J., von Kries, J.P., Kühl, M., Bruhn, L., Wedlich, D., Grosschedl, R., and Birchmeier, W. Functional interaction of β-catenin with the transcription factor LEF-1. Nature 382, 638-642, 1996.

Bhanot, P., Brink, M., Samos, C.H., Hsieh, J.C., Wang, Y., Macke, J.P., Andrew, D., Nathans, J., and Nusse, R. A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225-230, 1996.

Brenner, H., Kloor, M., and Pox, C.P. Colorectal cancer. Lancet 383, 1490-1502, 2014.

Carvajal Rodríguez, A. Myriads: P-value-based multiple testing correction. Bioinformatics 34, 1043-1045, 2017.

Chen, H.H., Yu, H.I., Yang, M.H., and Tarn, W.Y. DDX3 Activates CBC-eIF3-Mediated Translation of uORF-Containing Oncogenic mRNAs to Promote Metastasis in HNSCC. Cancer Research 78, 4512-4523, 2018.

Chen, S., Guttridge, D.C., You, Z., Zhang, Z., Fribley, A., Mayo, M.W., Kitajewski, J., and Wang, C.Y. Wnt-1 signaling inhibits apoptosis by activating beta-catenin/T cell factor-mediated transcription. Journal of Cell Biology 152, 87-96, 2001.

Cheng, X., Xu, X., Chen, D., Zhao, F., and Wang, W. Therapeutic potential of targeting the Wnt/beta-catenin signaling pathway in colorectal cancer. Biomed Pharmacother 110, 473-481, 2019.

Chomczynski, P., and Sacchi, N. The single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction: twenty something years on. Nature Protocols 1, 581-585, 2006.

Clevers, H. Wnt/beta-catenin signaling in development and disease. Cell 127, 469-480, 2006.

de Gramont, A., Figer, A., Seymour, M., Homerin, M., Hmissi, A., Cassidy, J., Boni, C., Cortes Funes, H., Cervantes, A., Freyer, G., Papamichael, D., Le Bail, N., Louvet, C., Hendler, D., de Braud, F., Wilson, C., Morvan, F., and Bonetti, A. Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. Journal of Clinical Oncology 18, 2938-2947, 2000.

Douillard, J.Y., Cunningham, D., Roth, A.D., Navarro, M., James, R.D., Karasek, P., Jandik, P., Iveson, T., Carmichael, J., Alakl, M., Gruia, G., Awad, L., and Rougier, P. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet 355, 1041-1047, 2000.

Dow, L.E., O'Rourke, K.P., Simon, J., Tschaharganeh, D.F., van Es, J.H., Clevers, H., and Lowe, S.W. Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer. Cell 161, 1539-1552, 2015.

El Khoury, F., Corcos, L., Durand, S., Simon, B., and Le Jossic-Corcos, C. Acquisition of anticancer drug resistance is partially associated with cancer stemness in human colon cancer cells. International Journal of Oncology 49, 2558-2568, 2016.

Emami, K.H., Nguyen, C., Ma, H., Kim, D.H., Jeong, K.W., Eguchi, M., Moon, R.T., Teo, J.L., Kim, H.Y., Moon, S.H., Ha, J.R., and Kahn, M. A small molecule inhibitor of beta-catenin/CREB-binding protein transcription. Proceedings of the National Academy of Sciences of the United States of America 101, 12682-12687, 2004.

Fan, K., Li, N., Qi, J., Yin, P., Zhao, C., Wang, L., Li, Z., and Zha, X. Wnt/beta-catenin signaling induces the transcription of cystathionine-gamma-lyase, a stimulator of tumor in colon cancer. Cell Signal 26, 2801-2808, 2014.

Franken, N.A.P., Rodermond, H.M., Stap, J., Haveman, J., and van Bree, C. Clonogenic assay of cells in vitro. Nature Protocols 1, 2315-2319, 2006.

Friedman, R.C., Farh, K.K., Burge, C.B., and Bartel, D.P. Most mammalian mRNAs are conserved targets of microRNAs. Genome Research 19, 92-105, 2009.

Gao, M., Miao, L., Liu, M., Li, C., Yu, C., Yan, H., Yin, Y., Wang, Y., Qi, X., and Ren, J. miR-145 sensitizes breast cancer to doxorubicin by targeting multidrug resistance-associated protein-1. Oncotarget 7, 59714-59726, 2016.

Giovannetti, E., Erozenci, A., Smit, J., Danesi, R., and Peters, G.J. Molecular mechanisms underlying the role of microRNAs (miRNAs) in anticancer drug resistance and implications for clinical practice. Critical Reviews in Oncology Hematology 81, 103-122, 2012.

Heinemann, V., Douillard, J.Y., Ducreux, M., and Peeters, M. Targeted therapy in metastatic colorectal cancer - an example of personalised medicine in action. Cancer Treatment Reviews 39, 592-601, 2013.

Itamochi, H., Kigawa, J., and Terakawa, N. Mechanisms of chemoresistance and poor prognosis in ovarian clear cell carcinoma. Cancer Science 99, 653-658, 2008.

Ivanyuk, A., Livio, F., Biollaz, J., and Buclin, T. Renal Drug Transporters and Drug Interactions. Clinical Pharmacokinetics 56, 825-892, 2017.

Jensen, N.F., Stenvang, J., Beck, M.K., Hanáková, B., Belling, K.C., Do, K.N., Viuff, B., Nygård, S.B., Gupta, R., Rasmussen, M.H., Tarpgaard, L.S., Hansen, Tine P., Budinská, E., Pfeiffer, P., Bosman, F., Tejpar, S., Roth, A., Delorenzi, M., Andersen, C.L., Rømer, M.U., Brünner, N., and Moreira, J.M.A. Establishment and characterization of models of chemotherapy resistance in colorectal cancer: Towards a predictive signature of chemoresistance. Molecular Oncology 9, 1169-1185, 2015.

Jho, E.h., Zhang, T., Domon, C., Joo, C.K., Freund, J.N., and Costantini, F. Wnt/β-Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway. Molecular and Cellular Biology 22, 1172-1183, 2002.

Jing, L. Real Time Quantitative PCR (for Suspension Cells). Bio-protocol 1, e27, 2011.

Johnson, C.M., Wei, C., Ensor, J.E., Smolenski, D.J., Amos, C.I., Levin, B., and Berry, D.A. Meta-analyses of colorectal cancer risk factors. Cancer Causes Control 24, 1207-1222, 2013.

Kawahara, K., Nakayama, H., Nagata, M., Yoshida, R., Hirosue, A., Tanaka, T., Nakagawa, Y., Matsuoka, Y., Kojima, T., Takamune, Y., Yoshitake, Y., Hiraki, A., and Shinohara, M. A low Dicer expression is associated with resistance to 5-FU-based chemoradiotherapy and a shorter overall survival in patients with oral squamous cell carcinoma. Journal of Oral Pathology and Medicine 43, 350-356, 2014.

Kawamoto, S.A., Thompson, A.D., Coleska, A., Nikolovska Coleska, Z., Yi, H., and Wang, S. Analysis of the interaction of BCL9 with beta-catenin and development of fluorescence polarization and surface plasmon resonance binding assays for this interaction. Biochemistry 48, 9534-9541, 2009.

Keum, N., and Giovannucci, E. Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nature Reviews Gastroenterology and Hepatology 16, 713-732, 2019.

Kim, S.A., Kim, I., Yoon, S.K., Lee, E.K., and Kuh, H.J. Indirect modulation of sensitivity to 5-fluorouracil by microRNA-96 in human colorectal cancer cells. Archives of Pharmacal Research 38, 239-248, 2015.

Lee, C.H., Yu, C.C., Wang, B.Y., and Chang, W.W. Tumorsphere as an effective in vitro platform for screening anti-cancer stem cell drugs. Oncotarget 7, 1215-1226, 2016.

Li, Q., Yang, G., Feng, M., Zheng, S., Cao, Z., Qiu, J., You, L., Zheng, L., Hu, Y., Zhang, T., and Zhao, Y. NF-κB in pancreatic cancer: Its key role in chemoresistance. Cancer Letters 421, 127-134, 2018.

Li, T., Gao, F., and Zhang, X.P. miR-203 enhances chemosensitivity to 5-fluorouracil by targeting thymidylate synthase in colorectal cancer. Oncology Reports 33, 607-614, 2015.
Li, X., Lewis, M.T., Huang, J., Gutierrez, C., Osborne, C.K., Wu, M.F., Hilsenbeck, S.G., Pavlick, A., Zhang, X., Chamness, G.C., Wong, H., Rosen, J., and Chang, J.C. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. Journal of the National Cancer Institute 100, 672-679, 2008.

Li, Y., Ahmed, F., Ali, S., Philip, P.A., Kucuk, O., and Sarkar, F.H. Inactivation of nuclear factor kappaB by soy isoflavone genistein contributes to increased apoptosis induced by chemotherapeutic agents in human cancer cells. Cancer Research 65, 6934-6942, 2005.

Li, Z., Huang, Q., Chen, H., Lin, Z., Zhao, M., and Jiang, Z. Interferon Regulatory Factor 7 Promoted Glioblastoma Progression and Stemness by Modulating IL-6 Expression in Microglia. Journal of Cancer 8, 207-219, 2017.

Longley, D.B., and Johnston, P.G. Molecular mechanisms of drug resistance. The Journal of Pathology 205, 275-292, 2005.

Lund, E., and Dahlberg, J.E. Substrate selectivity of exportin 5 and Dicer in the biogenesis of microRNAs. Cold Spring Harbor Symposia on Quantitative Biology 71, 59-66, 2006.

Ma, H., Nguyen, C., Lee, K.S., and Kahn, M. Differential roles for the coactivators CBP and p300 on TCF/beta-catenin-mediated survivin gene expression. Oncogene 24, 3619-3631, 2005.

Mahmood, T., and Yang, P.C. Western blot: technique, theory, and trouble shooting. North American Journal of Medical Sciences 4, 429-434, 2012.

Mapletoft, J.P.J., St Onge, R.J., Guo, B., Butler, P., Masilamani, T.J., D'Costa, L., Pritzker, L.B., and Parissenti, A.M. The RNA disruption assay is superior to conventional drug sensitivity assays in detecting cytotoxic drugs. Scientific Reports 10, 8671-8683, 2020.

Mukherji, S., Ebert, M.S., Zheng, G.X., Tsang, J.S., Sharp, P.A., and van Oudenaarden, A. MicroRNAs can generate thresholds in target gene expression. Nature Genetics 43, 854-859, 2011.

Nagel, R., le Sage, C., Diosdado, B., van der Waal, M., Oude Vrielink, J.A., Bolijn, A., Meijer, G.A., and Agami, R. Regulation of the adenomatous polyposis coli gene by the miR-135 family in colorectal cancer. Cancer Research 68, 5795-5802, 2008.

Natsume, H., Sasaki, S., Kitagawa, M., Kashiwabara, Y., Matsushita, A., Nakano, K., Nishiyama, K., Nagayama, K., Misawa, H., Masuda, H., and Nakamura, H. Beta-catenin/Tcf-1-mediated transactivation of cyclin D1 promoter is negatively regulated by thyroid hormone. Biochemical and Biophysical Research Communications 309, 408-413, 2003.

Novellasdemunt, L., Antas, P., and Li, V.S. Targeting Wnt signaling in colorectal cancer. A Review in the Theme: Cell Signaling: Proteins, Pathways and Mechanisms. American Journal of Physiology: Cell Physiology 309, 511-521, 2015.

Pahl, H.L. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18, 6853-6866, 1999.

Peach, M., Marsh, N., Miskiewicz, E.I., and MacPhee, D.J. Solubilization of proteins: the importance of lysis buffer choice. Methods in Molecular Biology 1312, 49-60, 2015.

Rodriguez Salas, N., Dominguez, G., Barderas, R., Mendiola, M., Garcia Albeniz, X., Maurel, J., and Batlle, J.F. Clinical relevance of colorectal cancer molecular subtypes. Critical Reviews in Oncology/Hematology 109, 9-19, 2017.

Rubinfeld, B., Souza, B., Albert, I., Muller, O., Chamberlain, S., Masiarz, F., Munemitsu, S., and Polakis, P. Association of the APC gene product with beta-catenin. Science 262, 1731-1734, 1993.

Salz, T., Li, G., Kaye, F., Zhou, L., Qiu, Y., and Huang, S. hSETD1A regulates Wnt target genes and controls tumor growth of colorectal cancer cells. Cancer Research 74, 775-786, 2014.

Sampietro, J., Dahlberg, C.L., Cho, U.S., Hinds, T.R., Kimelman, D., and Xu, W. Crystal structure of a beta-catenin/BCL9/Tcf4 complex. Molecular Cell 24, 293-300, 2006.

Shin, S.H., Lim, D.Y., Reddy, K., Malakhova, M., Liu, F., Wang, T., Song, M., Chen, H., Bae, K.B., Ryu, J., Liu, K., Lee, M.H., Bode, A.M., and Dong, Z. A Small Molecule Inhibitor of the beta-Catenin-TCF4 Interaction Suppresses Colorectal Cancer Growth In Vitro and In Vivo. EBioMedicine 25, 22-31, 2017.

Siemens, H., Jackstadt, R., Kaller, M., and Hermeking, H. Repression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness. Oncotarget 4, 1399-1415, 2013.

Smith, D., Ballal, M., Hodder, R., Soin, G., Selvachandran, S.N., and Cade, D. Symptomatic presentation of early colorectal cancer. Annals of the Royal College of Surgeons of England 88, 185-190, 2006.

Song, J.L., Nigam, P., Tektas, S.S., and Selva, E. microRNA regulation of Wnt signaling pathways in development and disease. Cell Signal 27, 1380-1391, 2015.

Su, L., Vogelstein, B., and Kinzler, K. Association of the APC tumor suppressor protein with catenins. Science 262, 1734-1737, 1993.

Sun, Y., Campisi, J., Higano, C., Beer, T.M., Porter, P., Coleman, I., True, L., and Nelson, P.S. Treatment-induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B. Nature Medicine 18, 1359-1368, 2012.

Tsuji, S., Midorikawa, Y., Takahashi, T., Yagi, K., Takayama, T., Yoshida, K., Sugiyama, Y., and Aburatani, H. Potential responders to FOLFOX therapy for colorectal cancer by Random Forests analysis. British Journal of Cancer 106, 126-132, 2012.

van de Merbel, A.F., van der Horst, G., Buijs, J.T., and van der Pluijm, G. Protocols for Migration and Invasion Studies in Prostate Cancer. Methods in Molecular Biology 1786, 67-79, 2018.

van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A.P., Tjon-Pon-Fong, M., Moerer, P., van den Born, M., Soete, G., Pals, S., Eilers, M., Medema, R., and Clevers, H. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111, 241-250, 2002.

Veeman, M.T., Axelrod, J.D., and Moon, R.T. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Developmental Cell 5, 367-377, 2003a.

Veeman, M.T., Slusarski, D.C., Kaykas, A., Louie, S.H., and Moon, R.T. Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Current Biology 13, 680-685, 2003b.

Vrijens, K., Bollati, V., and Nawrot, T.S. MicroRNAs as potential signatures of environmental exposure or effect: a systematic review. Environ Health Perspect 123, 399-411, 2015.

Wang, X., Chen, H., Wen, Y., Yang, X., Han, Q., Jiang, P., Huang, Z., Cai, J., and Wang, Z. Dicer affects cisplatin mediated apoptosis in epithelial ovarian cancer cells. Molecular Medicine Reports 18, 4381-4387, 2018.

Wang, Z.X., Lu, B.B., Wang, H., Cheng, Z.X., and Yin, Y.M. MicroRNA-21 modulates chemosensitivity of breast cancer cells to doxorubicin by targeting PTEN. Archives of Medical Research 42, 281-290, 2011.

Wei, T.T., Lin, Y.T., Tang, S.P., Luo, C.K., Tsai, C.T., Shun, C.T., and Chen, C.C. Metabolic targeting of HIF-1α potentiates the therapeutic efficacy of oxaliplatin in colorectal cancer. Oncogene 39, 414-427, 2020.

Wu, D., Raafat, A., Pak, E., Clemens, S., and Murashov, A.K. Dicer-microRNA pathway is critical for peripheral nerve regeneration and functional recovery in vivo and regenerative axonogenesis in vitro. Experimental Neurology 233, 555-565, 2012.

Wu, X., Luo, F., Li, J., Zhong, X., and Liu, K. Tankyrase 1 inhibitior XAV939 increases chemosensitivity in colon cancer cell lines via inhibition of the Wnt signaling pathway. International Journal of Oncology 48, 1333-1340, 2016.

Xia, Z., Guo, M., Liu, H., Jiang, L., Li, Q., Peng, J., Li, J.D., Shan, B., Feng, P., and Ma, H. CBP-dependent Wnt/beta-catenin signaling is crucial in regulation of MDR1 transcription. Current Cancer Drug Targets 15, 519-532, 2015.

Yamada, T., Takaoka, A.S., Naishiro, Y., Hayashi, R., Maruyama, K., Maesawa, C., Ochiai, A., and Hirohashi, S. Transactivation of the multidrug resistance 1 gene by T-cell factor 4/beta-catenin complex in early colorectal carcinogenesis. Cancer Research 60, 4761-4766, 2000.

You, L., Wang, H., Yang, G., Zhao, F., Zhang, J., Liu, Z., Zhang, T., Liang, Z., Liu, C., and Zhao, Y. Gemcitabine exhibits a suppressive effect on pancreatic cancer cell growth by regulating processing of PVT1 to miR1207. Molecular Oncology 12, 2147-2164, 2018.

Yu, Y., Kanwar, S.S., Patel, B.B., Oh, P.S., Nautiyal, J., Sarkar, F.H., and Majumdar, A.P. MicroRNA-21 induces stemness by downregulating transforming growth factor beta receptor 2 (TGFβR2) in colon cancer cells. Carcinogenesis 33, 68-76, 2012.

Zhan, T., Rindtorff, N., and Boutros, M. Wnt signaling in cancer. Oncogene 36, 1461-1473, 2017.

Zhang, X., and Hao, J. Development of anticancer agents targeting the Wnt/β-catenin signaling. American Journal of Cancer Research 5, 2344-2360, 2015.