根據2012年的台灣常見癌症排名，膀胱癌在男人和女人分別位居第9和第16名，而且人數有逐年升高的趨勢。而實驗室先前的研究統計發現，在泌尿上皮癌中，穿膜蛋白EGFR與RON蛋白會有高度表現與協同作用。近期亦發現RON在環境壓力下，例如:缺乏血清和缺氧的情況下，可以直接進入細胞核並轉錄調控下游的基因。透過實驗與軟體分析，碳水化合物反應元件結合蛋白轉錄因子(ChREBP)即是RON與EGFR所調控的下游基因其中之一。 ChREBP在膀胱癌及肝癌細胞中，可藉由葡萄糖、生長因子和環境壓力(缺乏血清及缺氧)刺激進而由細胞質進入細胞核中，調節脂質生成和糖解作用。除了RT4分化良好的癌細胞，泌尿上皮癌細胞皆有表現ChREBP蛋白，其中以TSGH8301癌細胞表現量最低，而J82癌細胞最高。另外，ChREBP蛋白在泌尿上皮癌的表現，與EGFR的表現趨勢一致。在J82癌細胞中，轉染RON的突變基因、FL(全長基因)、或TM(去除與入核相關的基因)，會使得ChREBP的表現量上升。反之，當J82癌細胞轉染RON的突變基因K(酪氨酸激酶的基因)，會導致ChREBP表現量下降，此結果證實酪氨酸激酶活性對於活化ChREBP表現的重要性。當TSGH8301癌細胞在血清缺乏的環境時，不管是相對於載體的控制組或是有血清的控制組，皆能促進ChREBP促進子的活性(顯著差異分別<0.01和0.001)。同理，RON蛋白對於ChREBP促進子的調控能力，也在HEK293細胞的轉染實驗證實。另外，當分別抑制TSGH8301癌細胞的RON或EGFR蛋白的表現時，在培養液中加入血清，RON與EGFR會扮演調控ChREBP promoter的重要腳色；而在無血清的情況下，RON與EGFR的影響則會被抑制。分別在293T或TSGH8301癌細胞中大量表現ChREBP的情況下，會促進癌細胞增生、轉移與非貼附性的生長；反之，在Hep3B 與 J82癌細胞中降低ChREBP的蛋白表現，則會降低癌細胞增生、轉移與非貼附性的生長，因此，我們的資料，確認ChREBP在膀胱癌細胞中扮演致癌基因的角色。根據先前的健保統計，第二型糖尿病的病患罹患膀胱癌的機率相對提高。然而，有服用降血脂藥物metformin的糖尿病病患，相對於沒有服用過的，能夠降低罹患膀胱癌的風險。 而metformin除了降低血糖，亦能降低ChREBP的蛋白表現。 隨Metformin濃度增高，會抑制泌尿上皮癌細胞的生長，且其半致死率會低於肝癌細胞。而且，在J82癌細胞轉染RON的突變基因TM和K，會增加J82癌細胞對於藥物Metformin的敏感性，反之，轉染RON全長基因，則會增加對Metformin的抗藥性。而轉染在自身的ChREBP含量多的Hep3B肝癌細胞，則可以促進其對Metformin的抗藥性。所以綜合來說，Metformin可以老藥新用來治療膀胱癌，並成為未來治療ChREBP所調控相關生物機制的抗癌藥物。

Bladder cancer is the most common urinary tract cancer in the world and ranked the 9th and 16th cancer incidence in the male and female in Taiwan, respectively, in 2012. We have demonstrated co-expression and intimately cross-talk between EGFR and RON in human bladder cancer. Moreover, transmembrane receptor tyrosine kinase-RON was recently discovered to function as an alternative transcription factor in response to serum starvation or hypoxic stress. The carbohydrate responsive element binding protein (ChREBP) was proved to be one of target genes for nuclear RON. ChREBP could regulate lipogenesis and glycolysis in liver cancer cells in vitro through translocation from cytosol to the nucleus by glucose, serum starvation, hypoxic stress or growth factor stimulation. Except for well differentiated RT4 bladder cancer cells, the remaining uroepithelial cell lines showed universal expression of ChREBP, with the lowest and highest level observed in the TSGH8301 and J82 cancer cells, respectively. Pattern of ChREBP expression parallels with that of EGFR. Transfection of FL and TM domain deletion mutant of RON upregulated ChREBP expression in J82 cells, while deletion of tyrosine kinase suppressed the ChREBP expression, supporting the importance of tyrosine kinase domain in the activation of ChREBP. Consistent with our hypothesis, serum starvation further enhanced the ChREBP promoter activity in TSGH8301 cancer cells compared with serum treatment or vector control, respectively (P < 0.01 & 0.001). The regulatory effect of RON on ChREBP promoter activity was confirmed in HEK293 cell transfection experiment. Using TSGH8301 cells, knocking down experiments showed that RON and EGFR have comparable transcriptional activation of ChREBP, irrespective of serum concentration. Overexpression ChREBP in 293T and TSGH8301 cancer cell lines could stimulate the proliferation, migration and colony formation in vitro. On the contrary, knocking down of ChREBP in Hep3B or J82 cells inhibited the proliferation, migration and colony formation. Taken together, ChREBP functions as an oncogene in bladder cancer. According to National Health Insurance Research Database, incidence of bladder cancer is significantly higher in type 2 diabetic patients, but declines in patients that have been treated with glucose-lowering drug-metformin for a while. The metformin was demonstrated to exhibit a dose-dependent suppressor effect on urothelial carcinoma in vitro, with mean IC50 values lower than that of hepatoma cells. Transfection of TM or tyrosine kinase domain deletion RON mutant sensitized J82 cells to metformin treatment in vitro compared with FL RON. Activation of ChREBP also appears to increase the sensitivity of Hep3B hepatoma cells to metformin treatment in vitro. Together, metformin may have potential as an anti-cancer agent for ChREBP-mediated biological effects.

1. Hung C-F, Yang C-K, Ou Y-C. Urologic cancer in Taiwan. Japanese Journal of Clinical Oncology. 2016;46(7):605-9.
2. Chang HY, Liu HS, Lai MD, Tsai YS, Tzai TS, Cheng HL, et al. Hypoxia promotes nuclear translocation and transcriptional function in the oncogenic tyrosine kinase RON. Cancer Res. 2014;74(16):4549-62.
3. Chiang CJ, Lo WC, Yang YW, You SL, Chen CJ, Lai MS. Incidence and survival of adult cancer patients in Taiwan, 2002-2012. J Formos Med Assoc. 2016;115(12):1076-88.
4. Saint-Jacques N, Parker L, Brown P, Dummer TJ. Arsenic in drinking water and urinary tract cancers: a systematic review of 30 years of epidemiological evidence. Environmental Health. 2014;13(1):44.
5. Konety BR, Allareddy V, Herr H. Complications after radical cystectomy: analysis of population-based data. Urology. 2006;68(1):58-64.
6. Gstraunthaler G, Lindl T, Van Der Valk J. A plea to reduce or replace fetal bovine serum in cell culture media. Cytotechnology. 2013;65(5):791-3.
7. Chen M, Huang J, Yang X, Liu B, Zhang W, Huang L, et al. Serum Starvation Induced Cell Cycle Synchronization Facilitates Human Somatic Cells Reprogramming. PLOS ONE. 2012;7(4):e28203.
8. Hsu P-Y, Liu H-S, Cheng H-L, Tzai T-S, Guo H-R, Ho C-L, et al. Collaboration of RON and Epidermal Growth Factor Receptor in Human Bladder Carcinogenesis. Journal of Urology. 2006;176(5):2262-7.
9. Liu HS, Hsu PY, Lai MD, Chang HY, Ho CL, Cheng HL, et al. An unusual function of RON receptor tyrosine kinase as a transcriptional regulator in cooperation with EGFR in human cancer cells. Carcinogenesis. 2010;31(8):1456-64.
10. Matsubara T, Ikeda M, Kiso Y, Sakuma M, Yoshino K-I, Sakane F, et al. c-Abl Tyrosine Kinase Regulates Serum-induced Nuclear Export of Diacylglycerol Kinase α by Phosphorylation at Tyr-218. 2012;287(8):5507-17.
11. Du Z, Lovly CM. Mechanisms of receptor tyrosine kinase activation in cancer. Mol Cancer. 2018;17(1):58.
12. Song S, Rosen KM, Corfas G. Biological function of nuclear receptor tyrosine kinase action. Cold Spring Harb Perspect Biol. 2013;5(7).
13. Mitsudomi T, Yatabe Y. Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer. The FEBS Journal. 2010;277(2):301-8.
14. Chow N-H, Chan S-H, Tzai T-S, Ho C-L, Liu H-S. Expression Profiles of ErbB Family Receptors and Prognosis in Primary Transitional Cell Carcinoma of the Urinary Bladder. Clinical Cancer Research. 2001;7(7):1957-62.
15. Wang MH, Padhye SS, Guin S, Ma Q, Zhou YQ. Potential therapeutics specific to c-MET/RON receptor tyrosine kinases for molecular targeting in cancer therapy. Acta Pharmacol Sin. 2010;31(9):1181-8.
16. Cheng HL, Liu HS, Lin YJ, Chen HH, Hsu PY, Chang TY, et al. Co-expression of RON and MET is a prognostic indicator for patients with transitional-cell carcinoma of the bladder. Br J Cancer. 2005;92(10):1906-14.
17. Iizuka K. The transcription factor carbohydrate-response element-binding protein (ChREBP): A possible link between metabolic disease and cancer. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2017;1863(2):474-85.
18. 丁雅柔. 碳水化合物反應元件結合蛋白轉錄因子在細胞壓力反應下的研究. 台南市: 國立成功大學; 2013.
19. Rojas LBA, Gomes MB. Metformin: an old but still the best treatment for type 2 diabetes. Diabetology & Metabolic Syndrome. 2013;5(1):6.
20. <American Diabetes Association Summary of revisions to the 2011 clinical practice recommendations..pdf>.
21. Summary of Revisions to the 2011 Clinical Practice Recommendations. Diabetes Care. 2011;34(Supplement_1):S3-S.
22. Zhang T, Guo P, Zhang Y, Xiong H, Yu X, Xu S, et al. The antidiabetic drug metformin inhibits the proliferation of bladder cancer cells in vitro and in vivo. Int J Mol Sci. 2013;14(12):24603-18.
23. Li X, Kover KL, Heruth DP, Watkins DJ, Moore WV, Jackson K, et al. New Insight Into Metformin Action: Regulation of ChREBP and FOXO1 Activities in Endothelial Cells. Mol Endocrinol. 2015;29(8):1184-94.
24. Tavaluc RT, Hart LS, Dicker DT, El-Deiry WS. Effects of low confluency, serum starvation and hypoxia on the side population of cancer cell lines. Cell Cycle. 2007;6(20):2554-62.
25. Shi Y, Felley-Bosco E, Marti TM, Orlowski K, Pruschy M, Stahel RA. Starvation-induced activation of ATM/Chk2/p53 signaling sensitizes cancer cells to cisplatin. BMC cancer. 2012;12:571-.
26. Braun F, Bertin-Ciftci J, Gallouet A-S, Millour J, Juin P. Serum-nutrient starvation induces cell death mediated by Bax and Puma that is counteracted by p21 and unmasked by Bcl-x(L) inhibition. PloS one. 2011;6(8):e23577-e.
27. Ward PS, Thompson CB. Signaling in control of cell growth and metabolism. Cold Spring Harbor perspectives in biology. 2012;4(7):a006783-a.
28. Zhu Z, Wang X, Shen Z, Lu Y, Zhong S, Xu C. Risk of bladder cancer in patients with diabetes mellitus: an updated meta-analysis of 36 observational studies. BMC Cancer. 2013;13(1):310.
29. Zhu Z, Zhang X, Shen Z, Zhong S, Wang X, Lu Y, et al. Diabetes mellitus and risk of bladder cancer: a meta-analysis of cohort studies. PloS one. 2013;8(2):e56662-e.
30. Tseng CH. Metformin may reduce bladder cancer risk in Taiwanese patients with type 2 diabetes. Acta Diabetol. 2014;51(2):295-303.
31. Mackenzie TA, Zaha R, Smith J, Karagas MR, Morden NE. Diabetes Pharmacotherapies and Bladder Cancer: A Medicare Epidemiologic Study. Diabetes Ther. 2016;7(1):61-73.
32. Okubo K, Isono M, Asano T, Sato A. Metformin Augments Panobinostat's Anti-Bladder Cancer Activity by Activating AMP-Activated Protein Kinase. Transl Oncol. 2019;12(4):669-82.