臺灣博碩士論文加值系統

在西方國家中，前列腺癌是男性最常見的癌症。其進展由早期的良性增生形成前列腺上皮腫瘤，隨後進一步發展為惡性腫瘤以及晚期癌細胞轉移到身體其他部位。在先前的文獻中指出，P53 基因的突變主要都是發現在癌症的晚期及轉移的病例中，但對於癌症早期或是原發腫瘤的影響仍然未知；而在最近的文獻中指出，前列腺癌的原發及轉移的病理中發現到RAS/MAPK訊息傳遞路徑的活化扮演非常重要的角色。本研究的目的為建立兩種不同基因工程小鼠來探討P53基因的剃除搭配K-rasG12D或是BRAFV600E突變的活化下對於前列腺癌的發展影響。我們發現這兩種動物模型在隨著時間的進展下，展現出高度的增生能力引發腫瘤形成並造成癌細胞的轉移。另外，在搭配前列腺腫瘤組織分離出的老鼠初代前列腺癌細胞下，結合了免疫組織化學染色及西方墨點法的實驗結果，我們觀察到MAPK訊息路徑具有高度的活化。特別的是，K-rasG12D蛋白還調控活化了另外一條Akt/mTOR 訊息傳遞，進一步比較出K-rasG12D與BRAFV600E這兩者之間所調控的分子機制。接著，透過收集前列腺癌小鼠的血清，我們找出了具有潛力的生物標記，mGluR1。當使用了抑制劑Riluzole處理在前列腺初代癌細胞中，能夠降低細胞增殖/存活表現。西方墨點法分析結果也發現了caspase 3活化，證實mGluR1在前列腺癌細胞的重要性。最後，我們成功的建立出新的前列腺癌基因轉殖動物模型，在未來提供一個非常好的工具來找尋新的抗癌藥物，並應用在前列腺癌的治療。

Prostate cancer (PCa) is one of the most common malignant tumors in men and the second leading cause of cancer deaths in male worldwide. P53 tumor suppressor gene is one of the most frequently inactivated genes in advanced prostate lesions, suggesting that aberrations of p53 would be required for PCa progression. On the other hand, activation of the the RAS/MAPK pathway occurred in both primary and metastatic prostate lesions, whereas p-ERK has been shown to regulate androgen signaling by phosphorylating androgen receptor (AR) coactivators, which may play an important role in prostate carcinogenesis. In this study, we developed two genetically engineered mouse (GEM) models that crossed both conditional activate LSL/KrasG12D or mutant BrafV600E and conditional abrogate p53 function in a prostate-specific manor, recapitulating the PCa histopathology, precursor lesions, and clinical behavior of the human disease with high fidelity. These tissue-specific compound mutant mice develop prostate tumors and metastasis. We also generated primary PCa cell lines derived from two mouse models to characterize and compare their different malignant behaviors in vitro. The phenotypic comparison of the Pb-Cre; K-rasG12D; P53L/L mice with the Pb-cre; BrafV600E; P53L/L mice by immunohistochemistry stain (IHC) and western blot analysis showed that not only MAPK pathway, K-ras also activates the Akt-mTOR signaling. Through proteomic analysis of mouse plasma samples, finding the new biomarker in PCa, mGluR1. Inhibition of mGluR1 by Riluzole in primary PCa cells had reduced cell proliferation/viability and increased caspase 3 activity. Moreover, western blot data found that Riluzole suppress signaling through the MAPK and AKT/mTOR pathways. Finally, our PCa models provide a unique tool for searching potential biomarkers and for drug discovery affecting K-ras signaling can be valuable for treating this disease. Anti-mGluR1 may be contributing a new treatment for prostate cancer.

論文審定書……………………………………………i
Abstract in Chinese……………………………………ii
Abstract in English……………………………...….…iii
Abbreviation……………………………………………v
Contents....................................................vi
Figures and Legends.....................................vii
Introduction………………………………………….... 1
Materials and Methods………………………………...8
Results…………………………………………………….16
Discussion…………………………………………………25
Figures and Tables………………………………………. 29
References……………………………………………….. 47

Abate-Shen, C., Shen, M. M., &; Gelmann, E. (2008). Integrating differentiation and cancer: the Nkx3.1 homeobox gene in prostate organogenesis and carcinogenesis. Differentiation, 76(6), 717-727. doi: 10.1111/j.1432-0436.2008.00292.
Amaral, T. M., Macedo, D., Fernandes, I., &; Costa, L. (2012). Castration-resistant prostate cancer: mechanisms, targets, and treatment. Prostate Cancer, 2012, 327253. doi: 10.1155/2012/327253
Ascierto, P. A., Kirkwood, J. M., Grob, J.-J., Simeone, E., Grimaldi, A. M., Maio, M., . . . Mozzillo, N. (2012). The role of BRAF V600 mutation in melanoma. J Transl Med, 10(85), 10.1186.
Bai, L., &; Zhu, W.-G. (2006). p53: structure, function and therapeutic applications. J Cancer Mol, 2(4), 141-153.
Beltran, H., Yelensky, R., Frampton, G. M., Park, K., Downing, S. R., MacDonald, T. Y., . . . Rubin, M. A. (2013). Targeted next-generation sequencing of advanced prostate cancer identifies potential therapeutic targets and disease heterogeneity. Eur Urol, 63(5), 920-926. doi: 10.1016/j.eururo.2012.08.053
Berney, D., Gopalan, A., Kudahetti, S., Fisher, G., Ambroisine, L., Foster, C., . . . Kattan, M. (2009). Ki-67 and outcome in clinically localised prostate cancer: analysis of conservatively treated prostate cancer patients from the Trans-Atlantic Prostate Group study. British journal of cancer, 100(6), 888-893.
Bostwick, D. G., &; Qian, J. (2004). High-grade prostatic intraepithelial neoplasia. Mod Pathol, 17(3), 360-379. doi: 10.1038/modpathol.3800053
Brose, M. S., Volpe, P., Feldman, M., Kumar, M., Rishi, I., Gerrero, R., . . . Nicholson, A. (2002). BRAF and RAS mutations in human lung cancer and melanoma. Cancer research, 62(23), 6997-7000.
Cairns, P., Okami, K., Halachmi, S., Halachmi, N., Esteller, M., Herman, J. G., . . . Sidransky, D. (1997). Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer research, 57(22), 4997-5000.
Chen, Z., Trotman, L. C., Shaffer, D., Lin, H. K., Dotan, Z. A., Niki, M., . . . Pandolfi, P. P. (2005). Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature, 436(7051), 725-730. doi: 10.1038/nature03918
Cho, N. Y., Choi, M., Kim, B. H., Cho, Y. M., Moon, K. C., &; Kang, G. H. (2006). BRAF and KRAS mutations in prostatic adenocarcinoma. Int J Cancer, 119(8), 1858-1862. doi: 10.1002/ijc.22071
Conn, P. J., &; Pin, J.-P. (1997). Pharmacology and functions of metabotropic glutamate receptors. Annual review of pharmacology and toxicology, 37(1), 205-237.
Daskivich, T. J., &; Oh, W. K. (2006). Recent progress in hormonal therapy for advanced prostate cancer. Current opinion in urology, 16(3), 173-178.
Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., Clegg, S., . . . Bottomley, W. (2002). Mutations of the BRAF gene in human cancer. Nature, 417(6892), 949-954.
Denmeade, S. R., &; Isaacs, J. T. (2002). A history of prostate cancer treatment. Nat Rev Cancer, 2(5), 389-396. doi: 10.1038/nrc801
DeSantis, C. E., Lin, C. C., Mariotto, A. B., Siegel, R. L., Stein, K. D., Kramer, J. L., . . . Jemal, A. (2014). Cancer treatment and survivorship statistics, 2014. CA Cancer J Clin, 64(4), 252-271. doi: 10.3322/caac.21235
Haggman, M. J., Macoska, J. A., Wojno, K. J., &; Oesterling, J. E. (1997). The relationship between prostatic intraepithelial neoplasia and prostate cancer: critical issues. The Journal of urology, 158(1), 12-22.
Hussain, M. R. M., Baig, M., Mohamoud, H. S. A., Ulhaq, Z., Hoessli, D. C., Khogeer, G. S., . . . Al-Aama, J. Y. (2015). BRAF gene: From human cancers to developmental syndromes. Saudi Journal of Biological Sciences, 22(4), 359-373. doi: 10.1016/j.sjbs.2014.10.002
Ittmann, M., Huang, J., Radaelli, E., Martin, P., Signoretti, S., Sullivan, R., . . . Cardiff, R. D. (2013). Animal models of human prostate cancer: the consensus report of the New York meeting of the Mouse Models of Human Cancers Consortium Prostate Pathology Committee. Cancer Res, 73(9), 2718-2736. doi: 10.1158/0008-5472.CAN-12-4213
Kasper, S., &; Matusik, R. J. (2000). Rat probasin: structure and function of an outlier lipocalin. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1482(1), 249-258.
Kaur, S., Kumar, S., Momi, N., Sasson, A. R., &; Batra, S. K. (2013). Mucins in pancreatic cancer and its microenvironment. Nat Rev Gastroenterol Hepatol, 10(10), 607-620. doi: 10.1038/nrgastro.2013.120
Kelly-Spratt, K. S., Kasarda, A. E., Igra, M., &; Kemp, C. J. (2008). A mouse model repository for cancer biomarker discovery. J. Proteome Res, 7(8), 3613-3618.
Koochekpour, S., Majumdar, S., Azabdaftari, G., Attwood, K., Scioneaux, R., Subramani, D., . . . Vessella, R. L. (2012). Serum glutamate levels correlate with Gleason score and glutamate blockade decreases proliferation, migration, and invasion and induces apoptosis in prostate cancer cells. Clin Cancer Res, 18(21), 5888-5901. doi: 10.1158/1078-0432.CCR-12-1308
Le, M. N., Chan, J. L., Rosenberg, S. A., Nabatian, A. S., Merrigan, K. T., Cohen-Solal, K. A., &; Goydos, J. S. (2010). The glutamate release inhibitor Riluzole decreases migration, invasion, and proliferation of melanoma cells. J Invest Dermatol, 130(9), 2240-2249. doi: 10.1038/jid.2010.126
Marin, Y. E., Namkoong, J., Cohen-Solal, K., Shin, S. S., Martino, J. J., Oka, M., &; Chen, S. (2006). Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon. Cell Signal, 18(8), 1279-1286. doi: 10.1016/j.cellsig.2005.10.012
Melamed, J., Einhorn, J. M., &; Ittmann, M. M. (1997). Allelic loss on chromosome 13q in human prostate carcinoma. Clinical cancer research, 3(10), 1867-1872.
Mulholland, D. J., Kobayashi, N., Ruscetti, M., Zhi, A., Tran, L. M., Huang, J., . . . Wu, H. (2012). Pten loss and RAS/MAPK activation cooperate to promote EMT and metastasis initiated from prostate cancer stem/progenitor cells. Cancer Res, 72(7), 1878-1889. doi: 10.1158/0008-5472.CAN-11-3132
Namkoong, J., Shin, S.-S., Lee, H. J., Marín, Y. E., Wall, B. A., Goydos, J. S., &; Chen, S. (2007). Metabotropic glutamate receptor 1 and glutamate signaling in human melanoma. Cancer research, 67(5), 2298-2305.
NAVONE, N. M., LABATE, M. E., TRONCOSO, P., PISTERS, L. L., CONTI, C. J., VON ESCHENBACH, A. C., &; LOGOTHETIS, C. J. (1999). p53 mutations in prostate cancer bone metastases suggest that selected p53 mutants in the primary site define foci with metastatic potential. The Journal of urology, 161(1), 304-308.
Neumann, J., Zeindl-Eberhart, E., Kirchner, T., &; Jung, A. (2009). Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer. Pathology-Research and Practice, 205(12), 858-862.
Nowak, M., Svensson, M. A., Carlsson, J., Vogel, W., Kebschull, M., Wernert, N., . . . Perner, S. (2014). Prognostic significance of phospho-histone H3 in prostate carcinoma. World journal of urology, 32(3), 703-707.
Parisotto, M., &; Metzger, D. (2013). Genetically engineered mouse models of prostate cancer. Mol Oncol, 7(2), 190-205. doi: 10.1016/j.molonc.2013.02.005
Pearson, H. B., Phesse, T. J., &; Clarke, A. R. (2009). K-ras and Wnt signaling synergize to accelerate prostate tumorigenesis in the mouse. Cancer Res, 69(1), 94-101. doi: 10.1158/0008-5472.CAN-08-2895
Petrylak, D. P., Tangen, C. M., Hussain, M. H., Lara Jr, P. N., Jones, J. A., Taplin, M. E., . . . Kohli, M. (2004). Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. New England Journal of Medicine, 351(15), 1513-1520.
Pritchard, C., Carragher, L., Aldridge, V., Giblett, S., Jin, H., Foster, C., . . . Kamata, T. (2007). Mouse models for BRAF-induced cancers. Biochemical Society Transactions, 35(Pt 5), 1329.
Qian, J., Hirasawa, K., Bostwick, D. G., Bergstralh, E. J., Slezak, J. M., Anderl, K. L., . . . Jenkins, R. B. (2002). Loss of p53 and c-myc overrepresentation in stage T2-3N1-3M0 prostate cancer are potential markers for cancer progression. Modern pathology, 15(1), 35-44.
Qian, J., Jenkins, R. B., &; Bostwick, D. G. (1997). Detection of chromosomal anomalies and c-myc gene amplification in the cribriform pattern of prostatic intraepithelial neoplasia and carcinoma by fluorescence in situ hybridization. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc, 10(11), 1113-1119.
Rabiau, N., Dechelotte, P., Guy, L., Satih, S., Bosviel, R., Fontana, L., . . . Bernard-Gallon, D. (2009). Immunohistochemical staining of mucin 1 in prostate tissues. In Vivo, 23(2), 203-207.
Roy, A., Lavrovsky, Y., Song, C., Chen, S., Jung, M., Velu, N., . . . Chatterjee, B. (1998). Regulation of androgen action. Vitamins and hormones, 55, 309-352.
Scher, H. I., &; Sawyers, C. L. (2005). Biology of progressive, castration-resistant prostate cancer: directed therapies targeting the androgen-receptor signaling axis. Journal of Clinical Oncology, 23(32), 8253-8261.
SEER Cancer Statistics Factsheets:National Cancer Institute. Bethesda, MD, http://seer.cancer.gov/statfacts/html. Accessed in June 2014.
Sharifi, N., Gulley, J. L., &; Dahut, W. L. (2005). Androgen deprivation therapy for prostate cancer. Jama, 294(2), 238-244.
Shen, M. M., &; Abate-Shen, C. (2010). Molecular genetics of prostate cancer: new prospects for old challenges. Genes Dev, 24(18), 1967-2000. doi: 10.1101/gad.1965810
Shin, S. S., Martino, J. J., &; Chen, S. (2008). Metabotropic glutamate receptors (mGlus) and cellular transformation. Neuropharmacology, 55(4), 396-402. doi: 10.1016/j.neuropharm.2008.04.021
Shin, S. S., Namkoong, J., Wall, B. A., Gleason, R., Lee, H. J., &; Chen, S. (2008). Oncogenic activities of metabotropic glutamate receptor 1 (Grm1) in melanocyte transformation. Pigment cell &; melanoma research, 21(3), 368-378.
Siegel, R. L., Miller, K. D., &; Jemal, A. (2015). Cancer statistics, 2015. CA Cancer J Clin, 65(1), 5-29. doi: 10.3322/caac.21254
Smit, V. T., Boot, A. J., Smits, A. M., Fleuren, G. J., Cornelisse, C. J., &; Bos, J. L. (1988). KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Acids Research, 16(16), 7773-7782.
SWEAT, S. D., PACELLI, A., BERGSTRALH, E. J., SLEZAK, J. M., &; BOSTWICK, D. G. (1999). Androgen receptor expression in prostatic intraepithelial neoplasia and cancer. The Journal of urology, 161(4), 1229-1232.
Taylor, B. S., Schultz, N., Hieronymus, H., Gopalan, A., Xiao, Y., Carver, B. S., . . . Gerald, W. L. (2010). Integrative genomic profiling of human prostate cancer. Cancer Cell, 18(1), 11-22. doi: 10.1016/j.ccr.2010.05.026
Wang, J., Kobayashi, T., Floc''h, N., Kinkade, C. W., Aytes, A., Dankort, D., . . . Abate-Shen, C. (2012). B-Raf activation cooperates with PTEN loss to drive c-Myc expression in advanced prostate cancer. Cancer Res, 72(18), 4765-4776. doi: 10.1158/0008-5472.CAN-12-0820
Wang, S., Gao, J., Lei, Q., Rozengurt, N., Pritchard, C., Jiao, J., . . . Nelson, P. S. (2003). Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell, 4(3), 209-221.
Wang, X., Kruithof-de Julio, M., Economides, K. D., Walker, D., Yu, H., Halili, M. V., . . . Shen, M. M. (2009). A luminal epithelial stem cell that is a cell of origin for prostate cancer. Nature, 461(7263), 495-500. doi: 10.1038/nature08361
Wojno, K. J., &; Epstein, J. I. (1995). The Utility of Basal Cell-Specific Anti-Cytokeratin Antibody (34 [beta] E12) in the Diagnosis of Prostate Cancer: A Review of 228 Cases. The American journal of surgical pathology, 19(3), 251-260.
Wu, X., Wu, J., Huang, J., Powell, W. C., Zhang, J., Matusik, R. J., . . . Roy-Burman, P. (2001). Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation. Mechanisms of development, 101(1), 61-69.
Yonezawa, S., Higashi, M., Yamada, N., Yokoyama, S., &; Goto, M. (2010). Significance of mucin expression in pancreatobiliary neoplasms. J Hepatobiliary Pancreat Sci, 17(2), 108-124. doi: 10.1007/s00534-009-0174-7
Yonezawa, S., Higashi, M., Yamada, N., Yokoyama, S., Kitamoto, S., Kitajima, S., &; Goto, M. (2011). Mucins in human neoplasms: clinical pathology, gene expression and diagnostic application. Pathol Int, 61(12), 697-716. doi: 10.1111/j.1440-1827.2011.02734.x
Zhang, C., Yuan, X. R., Li, H. Y., Zhao, Z. J., Liao, Y. W., Wang, X. Y., . . . Liu, Q. (2015). Anti-cancer effect of metabotropic glutamate receptor 1 inhibition in human glioma U87 cells: involvement of PI3K/Akt/mTOR pathway. Cell Physiol Biochem, 35(2), 419-432. doi: 10.1159/000369707