Abstract
Previous studies have demonstrated that the C-terminal of E1A binding proteins (CtBPs) influences tumorigenesis by participating in cell signal transduction in various human malignancies. However, the detailed expression patterns of CtBP isoforms in human osteosarcoma (OS) and the molecular mechanisms of CtBP involvement in tumor cell phenotypes requires further investigation. In the present study, the expression patterns of CtBP2 in OS cells and tissues were explored by immunohistochemistry. Fetal osteoblast cells were transfected with a eukaryotic expression plasmid to overexpress CtBP2, and the endogenous CtBP2 in OS cells was silenced via a short hairpin RNA. These transfections were validated and the phosphorylation levels of the JAK1/Stat3 signaling pathway were explored via western blotting. Furthermore, the malignant phenotype of OS cells was evaluated via a Cell Counting Kit-8 assay, cell colony formation assay, cell migration assay and scratch wound healing assay. The results revealed that the expression of CtBP2, but not CtBP1, was upregulated in OS tissue samples and the elevated expression level of CtBP2 was notably associated with distant metastasis. CtBP2 was demonstrated to modulate cell migration and invasion via JAK1/Stat3 signaling pathway in fetal osteoblast cells. In addition, genetic silencing of CtBP2 expression in OS cells notably reduced cell migration abilities and the phosphorylation of the JAK1/Stat3 pathway. In summary, the present studies revealed that the loss of CtBP2 constrained distant metastasis through the JAK1/Stat3 pathway in OS, suggesting that targeting CtBP2 may be a practical anti-tumor approach to prevent OS tumor progression.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Abbreviations
- CTBP:
-
C-terminus of the E1A binding protein
- IHC:
-
immunohistochemical analysis
- CCK-8:
-
cell counting kit-8
- JAK1:
-
Janus Kinase 1
- Stat3:
-
signal transducer and activator of transcription 3
- OS:
-
osteosarcoma
References
Anderson ME (2016) Update on Survival in Osteosarcoma. Orthop Clin North Am 47:283–292. https://doi.org/10.1016/j.ocl.2015.08.022
Balasubramanian P, Zhao LJ, Chinnadurai G (2003) Nicotinamide adenine dinucleotide stimulates oligomerization, interaction with adenovirus E1A and an intrinsic dehydrogenase activity of CtBP. FEBS Lett 537:157–160
Bellesis AG, Jecrois AM, Hayes JA, Schiffer CA, Royer WE Jr (2018) Assembly of human C-terminal binding protein (CtBP) into tetramers. J Biol Chem 293:9101–9112. https://doi.org/10.1074/jbc.RA118.002514
Blevins MA, Huang M, Zhao R (2017) The role of CtBP1 in oncogenic processes and its potential as a therapeutic target. Mol Cancer Ther 16:981–990. https://doi.org/10.1158/1535-7163.MCT-16-0592
Blevins MA, Zhang C, Zhang L, Li H, Li X, Norris DA, Huang M, Zhao R (2018) CPP-E1A fusion peptides inhibit CtBP-mediated transcriptional repression. Mol Oncol 12:1358–1373. https://doi.org/10.1002/1878-0261.12330
Chen S, Whetstine JR, Ghosh S, Hanover JA, Gali RR, Grosu P, Shi Y (2009) The conserved NAD(H)-dependent corepressor CTBP-1 regulates Caenorhabditis elegans life span. Proc Natl Acad Sci U S A 106:1496–1501. https://doi.org/10.1073/pnas.0802674106
Chinnadurai G (2003) CtBP family proteins: more than transcriptional corepressors. BioEssays : news and reviews in molecular, cellular and developmental biology 25:9–12. https://doi.org/10.1002/bies.10212
Chinnadurai G (2009) The transcriptional corepressor CtBP: a foe of multiple tumor suppressors. Cancer Res 69:731–734. https://doi.org/10.1158/0008-5472.CAN-08-3349
Corda D, Colanzi A, Luini A (2006) The multiple activities of CtBP/BARS proteins: the Golgi view. Trends Cell Biol 16:167–173. https://doi.org/10.1016/j.tcb.2006.01.007
Cuilliere-Dartigues P, el-Bchiri J, Krimi A, Buhard O, Fontanges P, Fléjou JF, Hamelin R, Duval A (2006) TCF-4 isoforms absent in TCF-4 mutated MSI-H colorectal cancer cells colocalize with nuclear CtBP and repress TCF-4-mediated transcription. Oncogene 25:4441–4448. https://doi.org/10.1038/sj.onc.1209471
Dcona MM, Morris BL, Ellis KC, Grossman SR (2017) CtBP- an emerging oncogene and novel small molecule drug target: advances in the understanding of its oncogenic action and identification of therapeutic inhibitors. Cancer Biol Ther 18:379–391. https://doi.org/10.1080/15384047.2017.1323586
de Barrios O, Győrffy B, Fernández-Aceñero MJ, Sánchez-Tilló E, Sánchez-Moral L, Siles L, Esteve-Arenys A, Roué G, Casal JI, Darling DS, Castells A, Postigo A (2017) ZEB1-induced tumourigenesis requires senescence inhibition via activation of DKK1/mutant p53/Mdm2/CtBP and repression of macroH2A1. Gut 66:666–682. https://doi.org/10.1136/gutjnl-2015-310838
Deng Y, Liu J, Han G, Lu SL, Wang SY, Malkoski S, Tan AC, Deng C, Wang XJ, Zhang Q (2010) Redox-dependent Brca1 transcriptional regulation by an NADH-sensor CtBP1. Oncogene 29:6603–6608. https://doi.org/10.1038/onc.2010.406
Deng H, Liu J, Deng Y, Han G, Shellman YG, Robinson SE, Tentler JJ, Robinson WA, Norris DA, Wang XJ, Zhang Q (2013) CtBP1 is expressed in melanoma and represses the transcription of p16INK4a and Brca1. J Invest Dermatol 133:1294–1301. https://doi.org/10.1038/jid.2012.487
Eshelman MA, Shah M, Raup-Konsavage WM, Rennoll SA, Yochum GS (2017) TCF7L1 recruits CtBP and HDAC1 to repress DICKKOPF4 gene expression in human colorectal cancer cells. Biochem Biophys Res Commun 487:716–722. https://doi.org/10.1016/j.bbrc.2017.04.123
Ichikawa K, Kubota Y, Nakamura T, Weng JS, Tomida T, Saito H, Takekawa M (2015) MCRIP1, an ERK substrate, mediates ERK-induced gene silencing during epithelial-mesenchymal transition by regulating the co-repressor CtBP. Mol Cell 58:35–46. https://doi.org/10.1016/j.molcel.2015.01.023
Jin W, Scotto KW, Hait WN, Yang JM (2007) Involvement of CtBP1 in the transcriptional activation of the MDR1 gene in human multidrug resistant cancer cells. Biochem Pharmacol 74:851–859. https://doi.org/10.1016/j.bcp.2007.06.017
Kim JH, Cho EJ, Kim ST, Youn HD (2005) CtBP represses p300-mediated transcriptional activation by direct association with its bromodomain. Nat Struct Mol Biol 12:423–428. https://doi.org/10.1038/nsmb924
Kumar V, Carlson JE, Ohgi KA, Edwards TA, Rose DW, Escalante CR, Rosenfeld MG, Aggarwal AK (2002) Transcription corepressor CtBP is an NAD(+)-regulated dehydrogenase. Mol Cell 10:857–869
Li L, Liu X, He L, Yang J, Pei F, Li W, Liu S, Chen Z, Xie G, Xu B, Ting X, Zhang Z, Jin T, Liu X, Zhang W, Yuan S, Yang Z, Wu C, Zhang Y, Yang X, Yi X, Liang J, Shang Y, Sun L (2017) ZNF516 suppresses EGFR by targeting the CtBP/LSD1/CoREST complex to chromatin. Nat Commun 8:691. https://doi.org/10.1038/s41467-017-00702-5
Paredes R, Schneider M, Stevens A, White DJ, Williamson AJK, Muter J, Pearson S, Kelly JR, Connors K, Wiseman DH, Chadwick JA, Löffler H, Teng HY, Lovell S, Unwin R, van de Vrugt HJ, Smith H, Kustikova O, Schambach A, Somervaille TCP, Pierce A, Whetton AD, Meyer S (2018) EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association. Nucleic Acids Res 46:7662–7674. https://doi.org/10.1093/nar/gky536
Salunke AA, Shah J, Gupta N, Pandit J (2016) Pathologic fracture in osteosarcoma: association with poorer overall survival. Eur J Surg Oncol: the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 42:889–890. https://doi.org/10.1016/j.ejso.2016.02.255
Schaeper U, Subramanian T, Lim L, Boyd JM, Chinnadurai G (1998) Interaction between a cellular protein that binds to the C-terminal region of adenovirus E1A (CtBP) and a novel cellular protein is disrupted by E1A through a conserved PLDLS motif. J Biol Chem 273:8549–8552
Senyuk V, Sinha KK, Nucifora G (2005) Corepressor CtBP1 interacts with and specifically inhibits CBP activity. Arch Biochem Biophys 441:168–173. https://doi.org/10.1016/j.abb.2005.06.024
Shen Z, Asa SL, Ezzat S (2017) Ikaros and its interacting partner CtBP target the metalloprotease ADAMTS10 to modulate pituitary cell function. Mol Cell Endocrinol 439:126–132. https://doi.org/10.1016/j.mce.2016.10.032
Turner J, Crossley M (2001) The CtBP family: enigmatic and enzymatic transcriptional co-repressors. BioEssays : news and reviews in molecular, cellular and developmental biology 23:683–690. https://doi.org/10.1002/bies.1097
Valenta T, Lukas J, Korinek V (2003) HMG box transcription factor TCF-4's interaction with CtBP1 controls the expression of the Wnt target Axin2/Conductin in human embryonic kidney cells. Nucleic Acids Res 31:2369–2380
Verger A, Quinlan KGR, Crofts LA, Spano S, Corda D, Kable EPW, Braet F, Crossley M (2006) Mechanisms directing the nuclear localization of the CtBP family proteins. Mol Cell Biol 26:4882–4894. https://doi.org/10.1128/MCB.02402-05
Wang SY, Iordanov M, Zhang Q (2006) c-Jun NH2-terminal kinase promotes apoptosis by down-regulating the transcriptional co-repressor CtBP. J Biol Chem 281:34810–34815. https://doi.org/10.1074/jbc.M607484200
Wang W, Yang J, Wang Y, Wang D, Han G, Jia J, Xu M, Bi W (2017) Survival and prognostic factors in Chinese patients with osteosarcoma: 13-year experience in 365 patients treated at a single institution. Pathol Res Pract 213:119–125. https://doi.org/10.1016/j.prp.2016.11.009
Winklmeier A, Poser I, Hoek KS, Bosserhoff AK (2009) Loss of full length CtBP1 expression enhances the invasive potential of human melanoma. BMC Cancer 9:52. https://doi.org/10.1186/1471-2407-9-52
Yang Y, Yang H, McNutt MA, Xiong F, Nie X, Li L, Zhou R (2008) LAPTM4B overexpression is an independent prognostic marker in ovarian carcinoma. Oncol Rep 20:1077–1083
Zhang Q, Wang SY, Nottke AC, Rocheleau JV, Piston DW, Goodman RH (2006) Redox sensor CtBP mediates hypoxia-induced tumor cell migration. Proc Natl Acad Sci U S A 103:9029–9033. https://doi.org/10.1073/pnas.0603269103
Zhang XL, Huang CX, Zhang J, Inoue A, Zeng SE, Xiao SJ (2013) CtBP1 is involved in epithelial-mesenchymal transition and is a potential therapeutic target for hepatocellular carcinoma. Oncol Rep 30:809–814. https://doi.org/10.3892/or.2013.2537
Zhang X, Ruan Y, Li Y, Lin D, Quan C (2015) Tight junction protein claudin-6 inhibits growth and induces the apoptosis of cervical carcinoma cells in vitro and in vivo. Med Oncol 32:148. https://doi.org/10.1007/s12032-015-0600-4
Zhang G, Kang L, Chen J, Xue Y, Yang M, Qin B, Yang L, Zhang J, Lu H, Guan H (2016) CtBP2 regulates TGFbeta2-induced epithelial-mesenchymal transition through notch signaling pathway in Lens epithelial cells. Curr Eye Res 41:1057–1063. https://doi.org/10.3109/02713683.2015.1092554
Zhang X, Wang H, Li Q, Li T (2018) CLDN2 inhibits the metastasis of osteosarcoma cells via down-regulating the afadin/ERK signaling pathway. Cancer Cell Int 18:160. https://doi.org/10.1186/s12935-018-0662-4
Zhao LJ, Subramanian T, Vijayalingam S, Chinnadurai G (2007) PLDLS-dependent interaction of E1A with CtBP: regulation of CtBP nuclear localization and transcriptional functions. Oncogene 26:7544–7551. https://doi.org/10.1038/sj.onc.1210569
Zhao C, Shen Y, Tao X, Xu J, Lu J, Liu C, Xu Z, Tang Q, Tao T, Zhang X (2016) Silencing of CtBP1 suppresses the migration in human glioma cells. J Mol Histol 47:297–304. https://doi.org/10.1007/s10735-016-9678-z
Zhao Z, Hao D, Wang L, Li J, Meng Y, Li P, Wang Y, Zhang C, Zhou H, Gardner K, di LJ (2018) CtBP promotes metastasis of breast cancer through repressing cholesterol and activating TGF-beta signaling. Oncogene 38:2076–2091. https://doi.org/10.1038/s41388-018-0570-z
Zheng X, Song T, Dou C, Jia Y, Liu Q (2015) CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma. Oncotarget 6:3752–3769. https://doi.org/10.18632/oncotarget.2915
Zou F, Xu J, Fu H, Cao J, Mao H, Gong M, Cui G, Zhang Y, Shi W, Chen J (2013) Different functions of HIPK2 and CtBP2 in traumatic brain injury. J Mol Neurosci: MN 49:395–408. https://doi.org/10.1007/s12031-012-9906-2
Acknowledgements
We would like to thank American Journal Experts (AJE) for help with this manuscript.
Author information
Authors and Affiliations
Contributions
PW performed the experiments and analyzed the data. SZ contributed to the conception and design of the study. BY and CW revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Disclosure of potential conflicts of interest
The authors declare that they have no competing interests.
Research involving human participants and/or animals
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 9864 Helsinki declaration and its later amendments or comparable ethical standards. Ethics approval (approval no. SD02872) was approved by the Ethics Committee of Shandong University. This article does not contain any studies with animals performed by any of the authors. The informed consent for participation was obtained from all patients and their parents who participated in this study in an appropriate method.
Informed consent
Written informed consent for publication was obtained. A copy of the consent form is available for review by the Editor of this journal.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Wang, P., Yu, B., Wang, C. et al. C-terminal of E1A binding protein 2 promotes the malignancy of osteosarcoma cells via JAK1/Stat3 signaling. J. Cell Commun. Signal. 14, 67–76 (2020). https://doi.org/10.1007/s12079-019-00523-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12079-019-00523-9