Inhibition of janus kinase 2 by compound AG490 suppresses the proliferation of MDA-MB-231 cells via up-regulating SARI (suppressor of AP-1, regulated by IFN)

Document Type : Original Article

Authors

1 Department of Radiation Oncology, Shandong Cancer Hospital, Shandong University, Jinan, Shandong, China

2 Department of Radiation Oncology, Linyi People’s Hospital, Linyi, Shandong, China

3 Department of Traumatology, Linyi People’s Hospital, Linyi, Shandong, China

Abstract

Objective(s): The Janus kinase-signal transducers and activators of transcription signaling pathway (JAK/STAT pathway) play an important role in proliferation of breast cancer cells. Previous data showed that inhibition of STAT3 suppresses the growth of breast cancer cells, but the associated mechanisms are not well understood. This study aims to investigate the effect and associated mechanisms of JAK/STAT pathway inhibitor AG490 on proliferation and suppression of breast cancer cells.
Materials and Methods: CCK-8 assay and trypan blue exclusion assay were used to investigate the cytotoxicity of AG490 to MDA-MB-231 cells. Real-time PCR was used to detect the mRNA level of SARI (suppressor of AP-1, regulated by IFN). Western blot was used to analyze the protein levels of SARI, phospho-STAT3 and total STAT3. Luciferase reporter assay was adopted to explore the mechanism of SARI mRNA upregulation.
Results: AG490 suppressed the proliferation of MDA-MB-231 cells in a dose-dependent manner. AG490 significantly up-regulated the mRNA and protein levels of SARI in MDA-MB-231 cells. Knockdown of SARI obviously attenuated AG490-induced growth suppression effect in MDA-MB-231 cells. Furthermore, AG490 dramatically enhanced the transcription activity of SARI promoter. But the transcription activity of truncated SARI promoter, which does not contain STAT3 binding site, cannot be activated by AG490 treatment.
Conclusion: We demonstrate in this study that AG490 suppresses the proliferation of MDA-MB-231 cells through transcriptional activation of SARI.

Keywords


1. Cyr AE, Margenthaler JA. Molecular profiling of breast cancer. Surg Oncol Clin N Am 2014; 23:451-462.
2. Teshome M, Hunt KK. Neoadjuvant Therapy in the Treatment of Breast Cancer. Surg Oncol Clin N Am 2014; 23:505-523.
3. Manuel ER, Blache CA, Paquette R, Kaltcheva TI, Ishizaki H, Ellenhorn JD, et al. Enhancement of cancer vaccine therapy by systemic delivery of a tumor-targeting Salmonella-based STAT3 shRNA suppresses the growth of established melanoma tumors. Cancer Res 2011; 71:4183-4191.
4. Jing N, Tweardy DJ. Targeting Stat3 in cancer therapy. Anti-cancer Drugs 2005; 16:601-607.
5. Teng Y, Ross JL, Cowell JK. The involvement of JAK-STAT3 in cell motility, invasion, and metastasis. Jak-Stat 2014; 3:e28086.
6. Haftchenary S, Avadisian M, Gunning PT. Inhibiting aberrant Stat3 function with molecular therapeutics: a progress report. Anti-cancer Drugs 2011; 22:115-127.
7. Levitzki A, Mishani E. Tyrphostins and other tyrosine kinase inhibitors. Annu Rev Biochem 2006; 75:93-109.
8. Luo C, Laaja P. Inhibitors of JAKs/STATs and the kinases: a possible new cluster of drugs. Drug Discov Today 2004; 9:268-275.
9. Toyonaga T, Nakano K, Nagano M, Zhao G, Yamaguchi K, Kuroki S, et al. Blockade of constitutively activated Janus kinase/signal transducer and activator of transcription-3 pathway inhibits growth of human pancreatic cancer. Cancer Lett 2003; 201:107-116.
10. Rahaman SO, Harbor PC, Chernova O, Barnett GH, Vogelbaum MA, Haque SJ. Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells. Oncogene 2002; 21:8404-8413.
11. Burke WM, Jin X, Lin HJ, Huang M, Liu R, Reynolds RK, et al. Inhibition of constitutively active Stat3 suppresses growth of human ovarian and breast cancer cells. Oncogene 2001; 20:7925-7934.
12. Caceres-Cortes JR. A potent anti-carcinoma and anti-acute myeloblastic leukemia agent, AG490. Anticancer Agents Med Chem 2008; 8:717-722.
13. Chen HF, Deng HY, Fan WK. Effect of Janus kinase inhibitor AG490 on invasion and metastasis of human breast cancer cells. J Sichuan Univ Med Sci 2008; 39:63-65.
14. Gariboldi MB, Ravizza R, Molteni R, Osella D, Gabano E, Monti E. Inhibition of Stat3 increases doxorubicin sensitivity in a human metastatic breast cancer cell line. Cancer Lett 2007; 258:181-188.
15. Lin KL, Su JC, Chien CM, Chuang PW, Chang LS, Lin SR. Down-regulation of the JAK2/PI3K-mediated signaling activation is involved in Taiwan cobra cardiotoxin III-induced apoptosis of human breast MDA-MB-231 cancer cells. Toxicon 2010; 55:1263-1273.
16. Taylor SC, Posch A. The design of a quantitative western blot experiment. Biomed Res Int 2014; 2014:361590.
17. Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, et al. Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 1996; 379:645-648.
18. Catlett-Falcone R, Dalton WS, Jove R. STAT proteins as novel targets for cancer therapy. Curr Opin Oncol 1999; 11:490-496.
19. Akira S, Nishio Y, Inoue M, Wang XJ, Wei S, Matsusaka T, et al. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell 1994; 77:63-71.
20. Buettner R, Mora LB, Jove R. Activated STAT signaling in human tumors provides novel molecular
targets for therapeutic intervention. Clin Cancer Res 2002; 8:945-954.
21. Carpenter RL, Lo HW. STAT3 Target Genes Relevant to Human Cancers. Cancers 2014; 6:897-925.
22. Su ZZ, Lee SG, Emdad L, Lebdeva IV, Gupta P, Valerie K, et al. Cloning and characterization of SARI (suppressor of AP-1, regulated by IFN). Proc Natl Acad Sci USA 2008; 105:20906-20911.
23. Dash R, Su ZZ, Lee SG, Azab B, Boukerche H, Sarkar D, et al. Inhibition of AP-1 by SARI negatively regulates transformation progression mediated by CCN1. Oncogene 2010; 29:4412-4423.
24. Wang C, Su Y, Zhang L, Wang M, You J, Zhao X, et al. The function of SARI in modulating epithelial-mesenchymal transition and lung adenocarcinoma metastasis. PLoS One 2012; 7:e38046.
25. Dash R, Bhoopathi P, Das SK, Sarkar S, Emdad L, Dasgupta S, et al. Novel mechanism of MDA-7/IL-24 cancer-specific apoptosis through SARI induction. Cancer Res 2014; 74:563-574.