Summary
Transcription factor Specificity protein 1 (Sp1) and its downstream target survivin (inhibitor of apoptosis protein), play major roles in the pathogenesis of various cancers. Ewing Sarcoma (ES) is a common soft tissue/bone tumor in adolescent and young adults. Overexpression of survivin is also linked to the aggressiveness and poor prognosis of ES. Small molecule Tolfenamic acid (TA) inhibits Sp1 and survivin in cancer cells. In this investigation, we demonstrate a strategy to target Sp1 and survivin using TA and positive control Mithramycin A (Mit) to inhibit ES cell growth. Knock down of Sp1 using small interfering RNA (siRNA) resulted in significant (p < 0.05) inhibition of CHLA-9 and TC-32 cell growth as assessed by CellTiter-Glo assay kit. TA or Mit treatment caused dose/time-dependent inhibition of cell viability, and this inhibition was correlated with a decrease in Sp1 and survivin protein levels in ES cells. Quantitative PCR results showed that Mit treatment decreased the mRNA expression of both survivin and Sp1, whereas TA diminished only survivin but not Sp1. Proteasome inhibitor restored TA-induced inhibition of Sp1 protein expression suggesting that TA might cause proteasome-dependent degradation. Gel shift assay using ES cell nuclear extract and biotinylated Sp1 consensus oligonucleotides confirmed that both TA and Mit decreased DNA-binding activity of Sp1. These results demonstrate that both Mit and TA reduce expression of Sp1 and survivin, disrupt Sp1 DNA-binding and inhibit ES cell proliferation. This investigation suggests that targeting Sp1 and survivin could be an effective strategy for inhibiting ES cell growth.
Similar content being viewed by others
References
Jo VY, Fletcher CD (2014) Who classification of soft tissue tumours: an update based on the 2013 (4th) edition. Pathology 46(2):95–104
Jo VY, Doyle LA (2016) Refinements in sarcoma classification in the current 2013 world health organization classification of tumours of soft tissue and bone. Surg Oncol Clin N Am 25(4):621–643
Ushigome S, Machinami R, Sorensen PH (2002) World health organization classification of tumours: Pathology and genetics of tumours of soft tissue and bone. In: IARCPress, 69008 Lyon, France, pp 297–300
Burchill SA (2003) Ewing’s sarcoma: diagnostic, prognostic, and therapeutic implications of molecular abnormalities. J Clin Pathol 56(2):96–102
Zucman J, Melot T, Desmaze C, Ghysdael J, Plougastel B, Peter M, Zucker JM, Triche TJ, Sheer D, Turc-Carel C et al (1993) Combinatorial generation of variable fusion proteins in the Ewing family of tumours. EMBO J 12(12):4481–4487
Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Peter M, Kovar H, Joubert I, de Jong P, Rouleau G et al (1992) Gene fusion with an ets DNA-binding domain caused by chromosome translocation in human tumours. Nature 359(6391):162–165
Darnell JE Jr (2002) Transcription factors as targets for cancer therapy. Nat Rev Cancer 2(10):740–749
Wang YT, Yang WB, Chang WC, Hung JJ (2011) Interplay of posttranslational modifications in sp1 mediates sp1 stability during cell cycle progression. J Mol Biol 414(1):1–14
Beishline K, Azizkhan-Clifford J (2015) Sp1 and the ‘hallmarks of cancer. FEBS J 282(2):224–258
Liu L, Ji P, Qu N, Pu WL, Jiang DW, Liu WY, Li YQ, Shi RL (2016) The impact of high co-expression of sp1 and hif1alpha on prognosis of patients with hepatocellular cancer. Oncol Lett 12(1):504–512
Hu J, Hu H, Hang JJ, Yang HY, Wang ZY, Wang L, Chen DH, Wang LW (2016) Simultaneous high expression of pld1 and sp1 predicts a poor prognosis for pancreatic ductal adenocarcinoma patients. Oncotarget
Hingorani P, Dickman P, Garcia-Filion P, White-Collins A, Kolb EA, Azorsa DO (2013) Birc5 expression is a poor prognostic marker in Ewing sarcoma. Pediatr Blood Cancer 60(1):35–40
Greve B, Sheikh-Mounessi F, Kemper B, Ernst I, Gotte M, Eich HT (2012) Survivin, a target to modulate the radiosensitivity of ewing’s sarcoma. Strahlenther Onkol 188(11):1038–1047
Khan Z, Khan AA, Prasad GB, Khan N, Tiwari RP, Bisen PS (2016) Growth inhibition and chemo-radiosensitization of head and neck squamous cell carcinoma (hnscc) by survivin-sirna lentivirus. Radiother Oncol 118(2):359–368
Habib R, Akhtar J, Taqi M, Yu C, Zhang C (2015) Lentiviral vector-mediated survivin shrna delivery in gastric cancer cell lines significantly inhibits cell proliferation and tumor growth. Oncol Rep 34(2):859–867
Liu S, Huang W, Jin MJ, Fan B, Xia GM, Gao ZG (2016) Inhibition of murine breast cancer growth and metastasis by survivin-targeted sirna using disulfide cross-linked linear pei. Eur J Pharm Sci 82:171–182
Blume SW, Snyder RC, Ray R, Thomas S, Koller CA, Miller DM (1991) Mithramycin inhibits sp1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. J Clin Invest 88(5):1613–1621
Esteve PO, Chin HG, Pradhan S (2007) Molecular mechanisms of transactivation and doxorubicin-mediated repression of survivin gene in cancer cells. J Biol Chem 282(4):2615–2625
Eslin D, Lee C, Sankpal UT, Maliakal P, Sutphin RM, Abraham L, Basha R (2013) Anticancer activity of tolfenamic acid in medulloblastoma: a preclinical study. Tumour Biol 34(5):2781–2789
Eslin D, Sankpal UT, Lee C, Sutphin RM, Maliakal P, Currier E, Sholler G, Khan M, Basha R (2013) Tolfenamic acid inhibits neuroblastoma cell proliferation and induces apoptosis: a novel therapeutic agent for neuroblastoma. Mol Carcinog 52(5):377–386
Abdelrahim M, Baker CH, Abbruzzese JL, Safe S (2006) Tolfenamic acid and pancreatic cancer growth, angiogenesis, and sp protein degradation. J Natl Cancer Inst 98(12):855–868
Papineni S, Chintharlapalli S, Abdelrahim M, Lee SO, Burghardt R, Abudayyeh A, Baker C, Herrera L, Safe S (2009) Tolfenamic acid inhibits esophageal cancer through repression of specificity proteins and c-met. Carcinogenesis 30(7):1193–1201
Fuchs B, Inwards CY, Janknecht R (2004) Vascular endothelial growth factor expression is up-regulated by ews-ets oncoproteins and sp1 and may represent an independent predictor of survival in ewing’s sarcoma. Clin Cancer Res 10(4):1344–1353
Giorgi C, Boro A, Rechfeld F, Lopez-Garcia LA, Gierisch ME, Schafer BW, Niggli FK (2015) Pi3k/akt signaling modulates transcriptional expression of ews/fli1 through specificity protein 1. Oncotarget
Fukuda S, Pelus LM (2006) Survivin, a cancer target with an emerging role in normal adult tissues. Mol Cancer Ther 5(5):1087–1098
Yan S, Li Z, Thiele CJ (2013) Inhibition of stat3 with orally active jak inhibitor, azd1480, decreases tumor growth in neuroblastoma and pediatric sarcomas in vitro and in vivo. Oncotarget 4(3):433–445
Giorgi C, Boro A, Rechfeld F, Lopez-Garcia LA, Gierisch ME, Schafer BW, Niggli FK (2015) Pi3k/akt signaling modulates transcriptional expression of ews/fli1 through specificity protein 1. Oncotarget 6(30):28895–28910
Shin JA, Jung JY, Ryu MH, Safe S, Cho SD (2013) Mithramycin a inhibits myeloid cell leukemia-1 to induce apoptosis in oral squamous cell carcinomas and tumor xenograft through activation of bax and oligomerization. Mol Pharmacol 83(1):33–41
Wang L, Guan X, Zhang J, Jia Z, Wei D, Li Q, Yao J, Xie K (2008) Targeted inhibition of sp1-mediated transcription for antiangiogenic therapy of metastatic human gastric cancer in orthotopic nude mouse models. Int J Oncol 33(1):161–167
Choi ES, Jung JY, Lee JS, Park JH, Cho NP, Cho SD (2013) Myeloid cell leukemia-1 is a key molecular target for mithramycin a-induced apoptosis in androgen-independent prostate cancer cells and a tumor xenograft animal model. Cancer Lett 328(1):65–72
Konduri S, Colon J, Baker CH, Safe S, Abbruzzese JL, Abudayyeh A, Basha MR, Abdelrahim M (2009) Tolfenamic acid enhances pancreatic cancer cell and tumor response to radiation therapy by inhibiting survivin protein expression. Mol Cancer Ther 8(3):533–542
Grohar PJ, Woldemichael GM, Griffin LB, Mendoza A, Chen QR, Yeung C, Currier DG, Davis S, Khanna C, Khan J, McMahon JB et al (2011) Identification of an inhibitor of the ews-fli1 oncogenic transcription factor by high-throughput screening. J Natl Cancer Inst 103(12):962–978
Gately S, Li WW (2004) Multiple roles of cox-2 in tumor angiogenesis: a target for antiangiogenic therapy. Semin Oncol 31(2 Suppl 7):2–11
Jacoby RF, Seibert K, Cole CE, Kelloff G, Lubet RA (2000) The cyclooxygenase-2 inhibitor celecoxib is a potent preventive and therapeutic agent in the min mouse model of adenomatous polyposis. Cancer Res 60(18):5040–5044
Tarnawski AS, Jones MK (2003) Inhibition of angiogenesis by nsaids: molecular mechanisms and clinical implications. Journal of molecular medicine (Berlin, Germany) 81(10):627–636
Juni P, Reichenbach S, Egger M (2005) Cox 2 inhibitors, traditional nsaids, and the heart. BMJ 330(7504):1342–1343
Koki AT, Masferrer JL (2002) Celecoxib: a specific cox-2 inhibitor with anticancer properties. Cancer Control 9(2 Suppl):28–35
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
All authors (SS, UTS, WPW, MW, AR and RB) of this manuscript declare no conflict of interest.
Funding
This work is partially supported by the Institute for Cancer Research of UNT Health Science Center to RB and the Young Investigator research award from ‘Hyundai Hope on Wheels’ awarded to AR.
Ethical approval
Research presented in this manuscript does not contain studies with human participants or animals.
Rights and permissions
About this article
Cite this article
Shelake, S., Sankpal, U.T., Paul Bowman, W. et al. Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth. Invest New Drugs 35, 158–165 (2017). https://doi.org/10.1007/s10637-016-0417-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10637-016-0417-9