快速生長分裂的癌細胞相較於正常細胞需要合成更大量的蛋白質，而在調控蛋白質合成的速率上，RNA polymerase III (RNA pol III) 的活性扮演了重要的角色。過去有一些證據指出，癌細胞會提高 RNA Pol III的轉錄作用，然而RNA Pol III在癌細胞當中詳細的調控機轉卻還尚待探討，因此，本篇研究中，我們想要探討RNA Pol III轉錄物在癌症發展的過程中是否扮演重要的角色，並進一步評估RNA Pol III轉錄物是否能作為癌症治療的標的。考量到目前臨床上，三重陰性 (triple negative) 乳癌病患的治療仍面臨挑戰，因此我們選擇一株正常的乳腺上皮細胞H184B5F5/M10 ，與三株惡性的乳癌細胞MCF-7 、MDA-MB-468和MDA-MB-231作為實驗模式，其中MDA-MB-468和MDA-MB-231為三陰性細胞株。首先，我們分析 RNA Pol III 基因在這四種細胞株之間的表現是否有差異，結果顯示，RNA Pol III轉錄物的在惡性細胞中有較高的表現。接下來，我們繼續探討哪些機轉誘發RNA Pol III 的轉錄作用，由ChIP 的結果顯示，惡性細胞不是透過改變組蛋白上修飾程度改變而增加RNA Pol III 的轉錄作用 此外我們也發現，惡性細胞中，RNA Pol III中重要的轉錄因子Brf-1 表現量上升，且RNA Pol III 抑制蛋白Maf-1表現量下降。接下來，我們以 MCF-7 建立Maf-1 knockdown細胞株，藉以了解Maf-1在癌細胞惡化過程中的重要性，然而卻發現，對於轉錄作用扮演抑制角色的Maf-1被抑制後，反而使細胞生長速度更為緩慢，暗示著Maf-1對於細胞的影響，勢必有更複雜的調控機制參與在其中。接著，我們在乳癌臨床檢體中分析Maf-1的表現，意外的發現Maf-1在腫瘤組織中過度表現，而與細胞株的結果相反，我們更看到了在惡性細胞株中，Maf-1大部分都累積在細胞核而非細胞質，這些結果暗示著我們，一些與Maf-1相關的因子可能產生了變異，導致Maf-1的功能產生的改變。最後總結，我們發現惡性細胞中，可以透過提高Brf-1表現且降低Maf-1表現來活化RNA Pol III的轉錄作用，這些惡性細胞中過度表現的轉錄物可能在乳癌惡化的過程中扮演重要的角色。

Rapidly growing cancer cells need more proteins than normal cells and protein synthesis highly depends on RNA polymerase III (RNA Pol III) activities. Some evidence shows that RNA pol III transcription is implicated in cancer. However, few results address the full significance of this issue. Therefore, we want to investigate whether RNA Pol III transcripts play important roles in cancer progression and whether RNA Pol III transcripts could be therapeutic targets in cancer therapy. For breast cancer therapy, it is still hard to treat triple negative breast cancer. Thus, we chose one normal breast epithelial cell, H184B5F5/M10 and three malignant breast cancer cell MCF-7, MDA-MB-468 and MDA-MB-231 as experimental cell models. In addition, MDA-MB-468 and MDA-MB-231 are triple negative cells. First, we evaluated expression level of RNA Pol III related genes including 5S-rRNA and tRNAs between these four cell lines, and found that expression level of RNA Pol III transcripts were increased in malignant cell lines. Next, we want to know what mechanisms participate in RNA Pol III transcription induction. According to ChIP assay data, we did not detect histone protein modification change on active sites in malignant cells. In addition, the key transcription factor Brf-1 was up-regulated, and RNA Pol III repressor Maf-1 was down-regulated in malignant cells at protein level. Next, we knockdown Maf-1 in MCF-7 cells to determine the importance of Maf-1 in breast cancer progression. We found that cell growth was inhibited after knockdown Maf-1. Maf-1 is a negative factor for cell growth, but Maf-1 knockdown transfectants reveal slower growth rate. There are more complicated mechanisms involved in Maf-1 regulation. Next, we detected Maf-1 expression pattern in clinical breast cancer specimen. Interestingly, Maf-1 was not deceased in tumor part comparing to normal part, and these data were not consistent with in vitro cell line data. We further observed Maf-1 in malignant cells is accumulated in nuclear rather than cytosol. These data reflect that there may be some mutations in Maf-1 related factors. In conclusion, we found that high expression of RNA Pol III related genes was associated with higher expression of Brf-1 and lower expression of Maf-1 in malignant breast cancer cell lines in vitro. Over-produced RNA Pol III transcripts may play important roles in breast cancer progression.

Bates, S., and G. Peters. 1995. Cyclin D1 as a cellular proto-oncogene. Semin Cancer Biol. Vol. 6. 73-82.

Brooks, R.A., L. Mitchell, C. O'Connor, and G.D. Chiro. 1981. On the relationship between computed tomography numbers and specific gravity. Phys Med Biol. 26:141-147.

Daly, N.L., D.A. Arvanitis, J.A. Fairley, N. Gomez-Roman, J.P. Morton, S.V. Graham, D.A. Spandidos, and R.J. White. 2004. Deregulation of RNA polymerase III transcription in cervical epithelium in response to high-risk human papillomavirus. Oncogene. 24:880-888.

Dang, C.V. 1999. c-Myc target genes involved in cell growth, apoptosis, and metabolism. Mol Cell Biol. 19:1-11.

Derenzini, M., D. Trere, A. Pession, L. Montanaro, V. Sirri, and R.L. Ochs. 1998. Nucleolar function and size in cancer cells. Am J Pathol. 152:1291-1297.

Dieci, G., G. Fiorino, M. Castelnuovo, M. Teichmann, and A. Pagano. 2007. The expanding RNA polymerase III transcriptome. Trends Genet. 23:614-622.

dos Reis, M., R. Savva, and L. Wernisch. 2004. Solving the riddle of codon usage preferences: a test for translational selection. Nucleic Acids Res. 32:5036-5044.

Felton-Edkins, Z.A., and R.J. White. 2002. Multiple mechanisms contribute to the activation of RNA polymerase III transcription in cells transformed by papovaviruses. J Biol Chem. 277:48182-48191.

Gomez-Roman, N., C. Grandori, R.N. Eisenman, and R.J. White. 2003. Direct activation of RNA polymerase III transcription by c-Myc. Nature. 421:290-294.

Guttman, M., and J.L. Rinn. 2012. Modular regulatory principles of large non-coding RNAs. Nature. 482:339-346.
Hunter, T. 1994. Cyclins and cancer II: cyclin D and CDK inhibitors come of age. Cell. 79:573-582.

Johnson, D.L. 2009. The JNKs differentially regulate RNA polymerase III transcription by coordinately modulating the expression of all TFIIIB subunits. Proc Natl Acad Sci 106:12682-12687.

Johnson, S.A.S., L. Dubeau, and D.L. Johnson. 2008. Enhanced RNA polymerase III-dependent transcription is required for oncogenic transformation. J Biol Chem. 283:19184-19191.

Killander, D., and A. Zetterberg. 1965. Quantitative cytochemical studies on interphase growth. I determine of DNA, RNA and mass content of age determined mouse fibroblast in vitro and of intercellular variation generation time. Exp Cell Res. 38:272-284.

Larminie, C.G.C., C.A. Cairns, R. Mital, K. Martin, T. Kouzarides, S.P. Jackson, and R.J. White. 1997. Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein. EMBO J. 16:2061-2071.

Lee, J.H., R.D. Moir, and I.M. Willis. 2009. Regulation of RNA polymerase III transcription involves SCH9-dependent and SCH9-independent branches of the target of rapamycin (TOR) pathway. J Biol Chem. . 284:12604-12608.

Lehmann, B.D., J.A. Bauer, X. Chen, M.E. Sanders, A.B. Chakravarthy, Y. Shyr, and J.A. Pietenpol. 2011. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 121:2750-2767.

Marshall, L., N.S. Kenneth, and R.J. White. 2008. Elevated tRNA Synthesis Can Drive Cell Proliferation and Oncogenic Transformation. Cell. 133:78-89.

Marshall, L., and R.J. White. 2008. Non-coding RNA production by RNA polymerase III is implicated in cancer. Nat Rev Cancer. 8:911-914.

Mayer, C., and I. Grummt. 2006. Ribosome biogenesis and cell growth: mTOR coordinates transcription by all three classes of nuclear RNA polymerases. Oncogene. 25:6384-6391.

Murawski, M., B. Szcześniak, T. Zoładek, A. Hopper, N. Martin, and M. Boguta. 1994. maf1 mutation alters the subcellular localization of the Mod5 protein in yeast. Acta Biochim Pol. 41:441-448.

Oficjalska-Pham, D., O. Harismendy, W.J. Smagowicz, A. Gonzalez de Peredo, M. Boguta, A. Sentenac, and O. Lefebvre. 2006. General repression of RNA polymerase III transcription is triggered by protein phosphatase type 2A-mediated dephosphorylation of Maf1. Mol Cell. 22:623-632.

Oler, A.J., and B.R. Cairns. 2012. PP4 dephosphorylates Maf1 to couple multiple stress conditions to RNA polymerase III repression. EMBO J. 31(6):1440-1452.

Pavon-Eternod, M., S. Gomes, R. Geslain, Q. Dai, M.R. Rosner, and T. Pan. 2009. tRNA over-expression in breast cancer and functional consequences. Nucleic Acids Res. 37:7268-7280.

Pluta, K., O. Lefebvre, N.C. Martin, W.J. Smagowicz, D.R. Stanford, S.R. Ellis, A.K. Hopper, A. Sentenac, and M. Boguta. 2001. Maf1p, a Negative Effector of RNA Polymerase III inSaccharomyces cerevisiae. Mol Cell Biol. 21:5031-5040.

Reina, J.H., T.N. Azzouz, and N. Hernandez. 2006. Maf1, a new player in the regulation of human RNA polymerase III transcription. PLoS One. 1:e134.

Schlotter, C.M., U. Vogt, U. Bosse, B. Mersch, and K. Waßmann. 2003. C-myc, not HER-2/neu, can predict recurrence and mortality of patients with node-negative breast cancer. Breast Cancer Res. 5:R30-36.

Schramm, L., and N. Hernandez. 2002. Recruitment of RNA polymerase III to its target promoters. Genes Dev. 16:2593-2620.

Schwartz, L.B., V.E.F. Sklar, J.A. Jaehning, R. Weinmann, and R.G. Roeder. 1974. Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the mouse myeloma, MOPC 315. J Biol Chem. 249:5889-5897.

Scott, M., K.H. Westphal, and P. Rigby. 1983. Activation of mouse genes in transformed cells. Cell. 34:557-567.

Shor, B., J. Wu, Q. Shakey, L. Toral-Barza, C. Shi, M. Follettie, and K. Yu. 2010. Requirement of the mTOR kinase for the regulation of Maf1 phosphorylation and control of RNA polymerase III-dependent transcription in cancer cells. J Biol Chem. 285:15380-15392.

Subik, K., J.F. Lee, L. Baxter, T. Strzepek, D. Costello, P. Crowley, L. Xing, M.C. Hung, T. Bonfiglio, and D.G. Hicks. 2010. The expression patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by immunohistochemical analysis in breast cancer cell lines. Breast Cancer Res. 4:35-41.

Vousden, K.H. 1995. Regulation of the cell cycle by viral oncoproteins. Semin Cancer Biol. Vol. 6.109-116.

Wang, H.D., A. Trivedi, and D.L. Johnson. 1997. Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway. Mol Cell Biol. 17:6838-6846.

Wei, Y., C.K. Tsang, and X.F.S. Zheng. 2009. Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J. 28:2220-2230.

Wei, Y., and X.F.S. Zheng. 2009. Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell cycle. 8:4085-4090.

Wei, Y., and X.F.S. Zheng. 2010. Maf1 regulation: A model of signal transduction inside the nucleus. Nucleus. 1:162-165.

White, R. 2004. RNA polymerase III transcription—a battleground for tumour suppressors and oncogenes. Eur J Cancer. 40:21-27.

White, R.J. 2011. Transcription by RNA polymerase III: more complex than we thought. Nat Rev Genet. 12:459-463.

Williamson, D., Y.J. Lu, C. Fang, K. Pritchard‐Jones, and J. Shipley. 2006. Nascent pre‐rRNA overexpression correlates with an adverse prognosis in alveolar rhabdomyosarcoma. Genes Chromosomes Cancer. 45:839-845.

Winter, A.G., G. Sourvinos, S.J. Allison, K. Tosh, P.H. Scott, D.A. Spandidos, and R.J. White. 2000. RNA polymerase III transcription factor TFIIIC2 is overexpressed in ovarian tumors. Proc Natl Acad Sci U S A. 97:12619-12624.

Woiwode, A., S.A.S. Johnson, S. Zhong, C. Zhang, R.G. Roeder, M. Teichmann, and D.L. Johnson. 2008. PTEN represses RNA polymerase III-dependent transcription by targeting the TFIIIB complex. Mol Cell Biol. 28:4204-4214.

Wullschleger, S., R. Loewith, and M.N. Hall. 2006. TOR signaling in growth and metabolism. Cell. 124:471-484.

Xu, Y., F. Zhao, Z. Wang, Y. Song, Y. Luo, X. Zhang, L. Jiang, Z. Sun, Z. Miao, and H. Xu. 2011. MicroRNA-335 acts as a metastasis suppressor in gastric cancer by targeting Bcl-w and specificity protein 1. Oncogene. 31: 1398-1407