Abstract
Nuclear factor-κB (NF-κB) is a core regulator in multiple tumorigenic pathways. Its activation is mediated by IκB kinase β (IKKβ). Protein phosphatase PPM1B is reported to dephosphorylate IKKβ, thereby terminating IKKβ-mediated NF-κB activation. However, the role of PPM1B in bladder cancer is unclear. The aim of this study was to determine the expression patterns and molecular mechanisms of PPM1B in bladder cancer. Comparative analyses were conducted in six bladder cancer cell lines, a normal urinary epithelial cell line, and adjacent non-tumorous bladder epithelia. Searches were conducted through publicly available algorithms and The Cancer Genome Atlas. HT-1376 and RT4 cells were transduced to stably overexpress PPM1B and its predicted regulator miR-186. Subsequent in vitro studies included 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), colony formation, anchorage-independent growth ability, luciferase reporter assays, and flow cytometric cell cycle analyses. A xenograft model was established in nude mice to evaluate the effect of PPM1B in bladder tumors in vivo. The results revealed that PPM1B was frequently downregulated in bladder cancer cells at both protein and messenger RNA (mRNA) levels, whereas miR-186 was upregulated. Further analyses showed that miR-186 promoted G1-S transition by targeting PPM1B at its 3′-untranslated region (3′UTR). Conversely, ectopic expression of PPM1B significantly suppressed proliferation and tumorigenicity in bladder cancer cells in vitro and in vivo, thereby neutralizing the oncogenic effect of miR-186. This study has identified PPM1B and miR-186 as potential diagnostic markers in bladder cancer. Promotion of PPM1B and suppression of miR-186 may offer effective therapeutic strategies in the treatment of bladder cancer.
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References
Mostafid AH, Palou Redorta J, Sylvester R, Witjes JA. Therapeutic options in high-risk non-muscle-invasive bladder cancer during the current worldwide shortage of bacille Calmette-Guerin. Eur Urol. 2015;67(3):359–60. doi:10.1016/j.eururo.2014.11.031.
Schellhammer PF. A worldwide view of bladder cancer. J Urol. 1997;158(2):406–7.
Beukers W, Kandimalla R, Masius RG, Vermeij M, Kranse R, van Leenders GJ, et al. Stratification based on methylation of TBX2 and TBX3 into three molecular grades predicts progression in patients with pTa-bladder cancer. Mod Pathol. 2015;28(4):515–22. doi:10.1038/modpathol.2014.145.
Li M, Cai Y, Zhao H, Xu Z, Sun Q, Luo M et al. Overexpression of HMGB3 protein promotes cell proliferation, migration and is associated with poor prognosis in urinary bladder cancer patients. Tumour Biol. 2015. doi:10.1007/s13277-015-3130-y.
Wang K, Liu T, Liu C, Meng Y, Yuan X, Liu L, et al. TERT promoter mutations and TERT mRNA but not FGFR3 mutations are urinary biomarkers in Han Chinese patients with urothelial bladder cancer. Oncologist. 2015;20(3):263–9. doi:10.1634/theoncologist.2014-0391.
Zhao F, Lin T, He W, Han J, Zhu D, Hu K, et al. Knockdown of a novel lincRNA AATBC suppresses proliferation and induces apoptosis in bladder cancer. Oncotarget. 2015;6(2):1064–78.
Lammers T, Lavi S. Role of type 2C protein phosphatases in growth regulation and in cellular stress signaling. Crit Rev Biochem Mol Biol. 2007;42(6):437–61. doi:10.1080/10409230701693342.
Sun B, Hu X, Liu G, Ma B, Xu Y, Yang T, et al. Phosphatase Wip1 negatively regulates neutrophil migration and inflammation. J Immunol. 2014;192(3):1184–95. doi:10.4049/jimmunol.1300656.
Park JH, Smith RJ, Shieh SY, Roeder RG. The GAS41-PP2Cbeta complex dephosphorylates p53 at serine 366 and regulates its stability. J Biol Chem. 2011;286(13):10911–7. doi:10.1074/jbc.C110.210211.
Sasaki M, Ohnishi M, Tashiro F, Niwa H, Suzuki A, Miyazaki J, et al. Disruption of the mouse protein Ser/Thr phosphatase 2Cbeta gene leads to early pre-implantation lethality. Mech Dev. 2007;124(6):489–99. doi:10.1016/j.mod.2007.04.001.
Drayton RM, Peter S, Myers K, Miah S, Dudziec E, Bryant HE, et al. MicroRNA-99a and 100 mediated upregulation of FOXA1 in bladder cancer. Oncotarget. 2014;5(15):6375–86.
Liao WT, Wang HM, Li MZ, Song LB, Zhang L, Mai HQ, et al. Establishment of three-dimensional culture models related to different stages of nasopharyngeal carcinogenesis. Ai Zheng. 2005;24(11):1317–21.
Sasaki Y, Sone T, Yahata K, Kishine H, Hotta J, Chesnut JD, et al. Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: eukaryotic clones containing two and three ORF multi-gene cassettes expressed from a single promoter. J Biotechnol. 2008;136(3–4):103–12. doi:10.1016/j.jbiotec.2008.06.007.
Wu J, Hu D, Yang G, Zhou J, Yang C, Gao Y, et al. Down-regulation of BMI-1 cooperates with artemisinin on growth inhibition of nasopharyngeal carcinoma cells. J Cell Biochem. 2011;112(7):1938–48. doi:10.1002/jcb.23114.
Lin H, Dai T, Xiong H, Zhao X, Chen X, Yu C, et al. Unregulated miR-96 induces cell proliferation in human breast cancer by downregulating transcriptional factor FOXO3a. PLoS One. 2010;5(12), e15797. doi:10.1371/journal.pone.0015797.
Zhang Z, Li J, Zheng H, Yu C, Chen J, Liu Z, et al. Expression and cytoplasmic localization of SAM68 is a significant and independent prognostic marker for renal cell carcinoma. Cancer Epidemiol Biomarkers Prev. 2009;18(10):2685–93. doi:10.1158/1055-9965.
LaBarbera KE, Hyldahl RD, O’Fallon KS, Clarkson PM, Witkowski S. Pericyte NF-kappaB activation enhances endothelial cell proliferation and proangiogenic cytokine secretion in vitro. Physiol Rep. 2015;3(4). doi:10.14814/phy2.12309
Gu K, Li MM, Shen J, Liu F, Cao JY, Jin S, et al. Interleukin-17-induced EMT promotes lung cancer cell migration and invasion via NF-kappaB/ZEB1 signal pathway. Am J Cancer Res. 2015;5(3):1169–79.
Mise-Omata S, Alles N, Fukazawa T, Aoki K, Ohya K, Jimi E, et al. NF-kappaB RELA-deficient bone marrow macrophages fail to support bone formation and to maintain the hematopoietic niche after lethal irradiation and stem cell transplantation. Int Immunol. 2014;26(11):607–18. doi:10.1093/intimm/dxu062.
Jiang L, Lin C, Song L, Wu J, Chen B, Ying Z, et al. MicroRNA-30e* promotes human glioma cell invasiveness in an orthotopic xenotransplantation model by disrupting the NF-kappaB/IkappaBalpha negative feedback loop. J Clin Invest. 2012;122(1):33–47. doi:10.1172/JCI58849 58849.
Yu J, Wang Y, Yan F, Zhang P, Li H, Zhao H, et al. Noncanonical NF-kappaB activation mediates STAT3-stimulated IDO upregulation in myeloid-derived suppressor cells in breast cancer. J Immunol. 2014;193(5):2574–86. doi:10.4049/jimmunol.1400833.
Nair VS, Gevaert O, Davidzon G, Plevritis SK, West R. NF-kappaB protein expression associates with (18)F-FDG PET tumor uptake in non-small cell lung cancer: a radiogenomics validation study to understand tumor metabolism. Lung Cancer. 2014;83(2):189–96. doi:10.1016/j.lungcan.2013.11.001.
Ma J, Liu J, Wang Z, Gu X, Fan Y, Zhang W, et al. NF-kappaB-dependent microRNA-425 upregulation promotes gastric cancer cell growth by targeting PTEN upon IL-1beta induction. Mol Cancer. 2014;13:40. doi:10.1186/1476-4598-13-40.
Guan Z, Ding C, Du Y, Zhang K, Zhu JN, Zhang T, et al. HAF drives the switch of HIF-1alpha to HIF-2alpha by activating the NF-kappaB pathway, leading to malignant behavior of T24 bladder cancer cells. Int J Oncol. 2014;44(2):393–402. doi:10.3892/ijo.2013.2210.
Park SL, Won SY, Song JH, Kim WJ, Moon SK. EPO gene expression induces the proliferation, migration and invasion of bladder cancer cells through the p21WAF1mediated ERK1/2/NF-kappaB/MMP-9 pathway. Oncol Rep. 2014;32(5):2207–14. doi:10.3892/or.2014.3428.
Park SL, Kim WJ, Moon SK. p21WAF1 mediates the IL-15-induced migration and invasion of human bladder cancer 5637 cells via the ERK1/2/NF-kappaB/MMP-9 pathway. Int Immunopharmacol. 2014;22(1):59–65. doi:10.1016/j.intimp.2014.06.008.
Lee EJ, Lee SJ, Kim S, Cho SC, Choi YH, Kim WJ, et al. Interleukin-5 enhances the migration and invasion of bladder cancer cells via ERK1/2-mediated MMP-9/NF-kappaB/AP-1 pathway: involvement of the p21WAF1 expression. Cell Signal. 2013;25(10):2025–38. doi:10.1016/j.cellsig.2013.06.004.
Kim JK, Kim KD, Lee E, Lim JS, Cho HJ, Yoon HK, et al. Up-regulation of Bfl-1/A1 via NF-kappaB activation in cisplatin-resistant human bladder cancer cell line. Cancer Lett. 2004;212(1):61–70. doi:10.1016/j.canlet.2004.02.021.
Yao F, Long LY, Deng YZ, Feng YY, Ying GY, Bao WD, et al. RACK1 modulates NF-kappaB activation by interfering with the interaction between TRAF2 and the IKK complex. Cell Res. 2014;24(3):359–71. doi:10.1038/cr.2013.162.
Yang J, Park Y, Zhang H, Xu X, Laine GA, Dellsperger KC, et al. Feed-forward signaling of TNF-alpha and NF-kappaB via IKK-beta pathway contributes to insulin resistance and coronary arteriolar dysfunction in type 2 diabetic mice. Am J Physiol Heart Circ Physiol. 2009;296(6):H1850-8. doi:10.1152/ajpheart.01199.2008.
Sun W, Yu Y, Dotti G, Shen T, Tan X, Savoldo B, et al. PPM1A and PPM1B act as IKKbeta phosphatases to terminate TNFalpha-induced IKKbeta-NF-kappaB activation. Cell Signal. 2009;21(1):95–102. doi:10.1016/j.cellsig.2008.09.012.
Duan S, Wang Y, Wang H, Wang S, Ji L, Dai D, et al. A novel PCR-based approach to discover miRNA target genes. Int J Med Sci. 2014;11(12):1270–4. doi:10.7150/ijms.9343.
Zhang X, Wan G, Mlotshwa S, Vance V, Berger FG, Chen H, et al. Oncogenic Wip1 phosphatase is inhibited by miR-16 in the DNA damage signaling pathway. Cancer Res. 2010;70(18):7176–86. doi:10.1158/0008-5472.CAN-10-0697.
Tonevitsky AG, Maltseva DV, Abbasi A, Samatov TR, Sakharov DA, Shkurnikov MU, et al. Dynamically regulated miRNA-mRNA networks revealed by exercise. BMC Physiol. 2013;13:9. doi:10.1186/1472-6793-13-9.
Jurmeister S, Baumann M, Balwierz A, Keklikoglou I, Ward A, Uhlmann S, et al. MicroRNA-200c represses migration and invasion of breast cancer cells by targeting actin-regulatory proteins FHOD1 and PPM1F. Mol Cell Biol. 2012;32(3):633–51. doi:10.1128/MCB.06212-11.
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This work was supported by the Science and Technology Program of Guangzhou Medical and Health System (20141A011094).
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Yang, J., Yuan, D., Li, J. et al. miR-186 downregulates protein phosphatase PPM1B in bladder cancer and mediates G1-S phase transition. Tumor Biol. 37, 4331–4341 (2016). https://doi.org/10.1007/s13277-015-4117-4
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DOI: https://doi.org/10.1007/s13277-015-4117-4