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Cellular Oncology

, Volume 39, Issue 6, pp 523–536 | Cite as

NFκB activation demarcates a subset of hepatocellular carcinoma patients for targeted therapy

  • Vignesh Ramesh
  • Karthikeyan Selvarasu
  • Jaishree Pandian
  • Soundarajan Myilsamy
  • Chidambaranathan Shanmugasundaram
  • Kumaresan GanesanEmail author
Original Paper

Abstract

Background

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. It is a heterogeneous disorder and >80 % of the tumors develop in patients with liver cirrhosis, resulting from chronic inflammation and/or fibrosis. Here, we set out to identify novel targets for HCC therapy and to define a subgroup of patients that might benefit most from it.

Methods

Cellular pathway activation profiling of 45 transcription factors in a HCC-derived cell line (HEP3B), in vitro analysis of NFκB reporter activity in additional HCC-derived cell lines and pathway-focused integrative analyses of publicly available primary HCC-derived expression profiling data (GSE6764, GSE9843, E-TABM-36 and E-TABM-292) were employed to reveal a role of NFκB in HCC development. In order to identify potential targeting agents, a luciferase-based NFκB reporter screening assay was established in HEP3B cells. After screening of a drug library through this assay, a potent NFκB pathway inhibitor was identified and characterized using an array of additional in vitro assays.

Results

Using cellular pathway activation profiling, we found a high activation of NFκB-mediated signaling in HCC-derived cell lines and in primary HCC tumors. Through NFκB inhibitor screening we observed a highly efficacious NFκB pathway inhibitory potential of ornithogalum in HCC-derived HEP3B cells. Although its active component still remains to be defined, ornithogalum has been found to inhibit endoplasmic reticulum (ER) and oxidative stress responses. ER stress, oxidative stress and NFκB signaling were found to be enhanced in a subset of HCCs, as well as in (precancerous) liver cirrhosis tissues.

Conclusion

From our data we conclude that NFκB signaling is activated in precancerous cirrhosis tissues and in a subset of HCCs. We found that ornithogalum exhibits NFκB targeting and stress relieving activities. NFκB inhibitors, including the active component of ornithogalum, may serve as putative preventive and targeted therapeutic agents for at least a subset of HCCs in which the NFκB pathway is activated. These latter notions require further investigation in a translational context.

Keywords

Hepatocellular carcinoma NFkB pathway Targeted therapy Ornithogalum ER stress Oxidative stress Anti-inflammatory 

Notes

Acknowledgments

This work was supported by the Department of Atomic Energy, Government of India, through research grant No. 6/6/2008/R&D-II-230R and the Department of Biotechnology, Government of India through research grant BT/PR4500/PID/6/676/2012 to Dr. Kumaresan Ganesan, Madurai Kamaraj University. We acknowledge the award of CSIR-NET fellowship to Vignesh Ramesh. Instrumentation support of the UGC-CEGS, UGC-CAS, DBT-IPLS, DST-PURSE and UGC-NRCBS program and the central facilities of the School of Biological Sciences, Madurai Kamaraj University, are also acknowledged. The authors thank Dr. Piyush Trivedi for providing the drug screening library and Mrs. Jaishree, Publication Division, IIT-Madras, for editorial assistance.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest

Supplementary material

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References

  1. 1.
    J. Ferlay, H. R. Shin, F. Bray, D. Forman, C. Mathers, D. M. Parkin, Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int. J. Cancer 127, 2893–2917 (2010)CrossRefPubMedGoogle Scholar
  2. 2.
    L. Zender, M. S. Spector, W. Xue, P. Flemming, C. Cordon-Cardo, J. Silke, S. T. Fan, J. M. Luk, M. Wigler, G. J. Hannon, D. Mu, R. Lucito, S. Powers, S. W. Lowe, Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell 125, 1253–1267 (2006)CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    N. Nishida, A. Goel, Genetic and epigenetic signatures in human hepatocellular carcinoma: a systematic review. Curr Genomics 12, 130–137 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    R. Saeki, H. Nagai, S. Kaneko, M. Unoura, N. Yamanaka, E. Okamoto, K. Kobayashi, K. Matsubara, Intratumoral genomic heterogeneity in human hepatocellular carcinoma detected by restriction landmark genomic scanning. J. Hepatol. 33, 99–105 (2000)CrossRefPubMedGoogle Scholar
  5. 5.
    L. A. D'Alessandro, R. Meyer, U. Klingmüller, Hepatocellular carcinoma: a systems biology perspective. Front. Physiol. 4, 28 (2013)CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    S. Fatima, N. P. Lee, J. M. Luk, Dickkopfs and Wnt/β-catenin signalling in liver cancer. World. J. Clin. Oncol. 2, 311–325 (2011)Google Scholar
  7. 7.
    J. M. Llovet, J. Bruix, Molecular targeted therapies in hepatocellular carcinoma. Hepatology 48, 1312–1327 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Y. Totoki, K. Tatsuno, S. Yamamoto, Y. Arai, F. Hosoda, S. Ishikawa, S. Tsutsumi, K. Sonoda, H. Totsuka, T. Shirakihara, H. Sakamoto, L. Wang, H. Ojimo, K. Shimada, T. Kosuge, T. Okusaka, K. Kato, J. Kusuda, T. Yoshida, H. Aburatani, T. Shibata, High-resolution characterization of a hepatocellular carcinoma genome. Nat. Genet. 43, 464–469 (2011)CrossRefPubMedGoogle Scholar
  9. 9.
    S. I. Grivennikov, M. Karin, Inflammatory cytokines in cancer: tumour necrosis factor and interleukin 6 take the stage. Ann. Rheum. Dis. 70, i104–i108 (2011)CrossRefPubMedGoogle Scholar
  10. 10.
    G. Fattovich, T. Stroffolini, I. Zagni, F. Donato, Hepatocellular carcinoma in cirrhosis: incidence and risk factors. Gastroenterology 127, S35–S50 (2004)CrossRefPubMedGoogle Scholar
  11. 11.
    M. Karin, A. Lin, NF-kappaB at the crossroads of life and death. Nat. Immunol. 3, 221–227 (2002)CrossRefPubMedGoogle Scholar
  12. 12.
    K. Vazquez-Santillan, J. Melendez-Zajgla, L. Jimenez-Hernandez, G. Martinez-Ruiz, V. Maldonado, NF-κB signaling in cancer stem cells: a promising therapeutic target? Cell. Oncol. 38, 327–339 (2015)CrossRefGoogle Scholar
  13. 13.
    B. B. Aggarwal, A. Bhardwaj, R. S. Aggarwal, N. P. Seeram, S. Shishodia, Y. Takada, Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res. 24, 2783–2840 (2004)PubMedGoogle Scholar
  14. 14.
    N. Chainani-Wu, Safety and anti-inflammatory activity of curcumin: a component of tumeric (Curcuma longa. J. Altern. Complement. Med. 9, 161–168 (2003)CrossRefPubMedGoogle Scholar
  15. 15.
    L. Fredriksson, B. Herpers, G. Benedetti, Q. Matadin, J. C. Puigvert, H. de Bont, S. Dragovic, N. P. Vermeulen, J. N. Commandeur, E. Danen, M. de Graauw, B. van de Water, Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis. Hepatology 53, 2027–2041 (2011)CrossRefPubMedGoogle Scholar
  16. 16.
    S. C. Miller, R. Huang, S. Sakamuru, S. J. Shukla, M. S. Attene-Ramos, P. Shinn, D. Van Leer, W. Leister, C. P. Austin, M. Xia, Identification of known drugs that act as inhibitors of NF-kappaB signaling and their mechanism of action. Biochem. Pharmacol. 79, 1272–1280 (2010)CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    S. Shishodia, D. Koul, B. B. Aggarwal, Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis. J. Immunol. 173, 2011–2022 (2004)CrossRefPubMedGoogle Scholar
  18. 18.
    X. Yao, J. Huang, H. Zhong, N. Shen, R. Faggioni, M. Fung, Y. Yao, Targeting interleukin-6 in inflammatory autoimmune diseases and cancers. Pharmacol. Ther. 141, 125–139 (2014)CrossRefPubMedGoogle Scholar
  19. 19.
    S. W. Tas, M. J. Vervoordeldonk, P. P. Tak, Gene therapy targeting nuclear factor-κB: towards clinical application in inflammatory diseases and cancer. Curr Gene Ther 9, 160–170 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    J. R. Nevins, Pathway-based classification of lung cancer: a strategy to guide therapeutic selection. Proc. Am. Thorac. Soc. 8, 180–182 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    P. N. Verma, I. Vaid, Encyclopaedia of Homeopathic Pharmacopoeia, Vol. IB (Jain Publishers, New Delhi, India, 2002), p. 1864Google Scholar
  22. 22.
    B. W. Dyer, F. A. Ferrer, D. K. Klinedinst, R. Rodriguez, A noncommercial dual luciferase enzyme assay system for reporter gene analysis. Anal. Biochem. 282, 158–161 (2000)CrossRefPubMedGoogle Scholar
  23. 23.
    T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63 (1983)CrossRefPubMedGoogle Scholar
  24. 24.
    A. Subramanian, P. Tamayo, V. K. Mootha, S. Mukherjee, B. L. Ebert, M. A. Gillette, A. Paulovich, S. L. Pomeroy, T. R. Golub, E. S. Lander, J. P. Mesirov, Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U. S. A. 102, 15545–15550 (2005)CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    J. Lamb, E. D. Crawford, D. Peck, J. W. Modell, I. C. Blat, M. J. Wrobel, J. Lerner, J. P. Brunet, A. Subramanian, K. N. Ross, M. Reich, H. Hieronymus, G. Wei, S. A. Armstrong, S. J. Haggarty, P. A. Clemons, R. Wei, S. A. Carr, E. S. Lander, T. R. Golub, The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929–1935 (2006)CrossRefPubMedGoogle Scholar
  26. 26.
    C. Li, W. H. Wong, Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc. Natl. Acad. Sci. U. S. A. 98, 31–36 (2001)CrossRefPubMedGoogle Scholar
  27. 27.
    J. Boelens, S. Lust, F. Offner, M. E. Bracke, B. W. Vanhoecke, The endoplasmic reticulum: a target for new anticancer drugs. In Vivo 21, 215–226 (2007)PubMedGoogle Scholar
  28. 28.
    A. Shibata, T. Nagaya, T. Imai, H. Funahashi, A. Nakao, H. Seo, Inhibition of NF-κB activity decreases the VEGF mRNA expression in MDA-MB-231 breast cancer cells. Breast Cancer Res. Treat. 73, 237–243 (2002)CrossRefPubMedGoogle Scholar
  29. 29.
    D. Hanahan, R. A. Weinberg, The hallmarks of cancer. Cell 100, 57–70 (2000)CrossRefPubMedGoogle Scholar
  30. 30.
    A. Geurts van Kessel, The cancer genome: from structure to function. Cell. Oncol. 37, 155–165 (2014)CrossRefGoogle Scholar
  31. 31.
    F. Colotta, P. Allavena, A. Sica, C. Garlanda, A. Mantovani, Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability. Carcinogenesis 30, 1073–1081 (2009)CrossRefPubMedGoogle Scholar
  32. 32.
    S. Lüth, J. Schrader, S. Zander, A. Carambia, J. Buchkremer, S. Huber, K. Reifenberg, K. Yamamura, P. Schirmacher, A. W. Lohse, J. Herkel, Chronic inflammatory IFN-γ signaling suppresses hepatocarcinogenesis in mice by sensitizing hepatocytes for apoptosis. Cancer Res. 71, 3763–3771 (2011)CrossRefPubMedGoogle Scholar
  33. 33.
    J. F. Rossi, S. Negrier, N. D. James, I. Kocak, R. Hawkins, H. Davis, U. Prabhakar, X. Qin, P. Mulders, B. Berns, A phase I/II study of siltuximab (CNTO 328), an anti-interleukin-6 monoclonal antibody, in metastatic renal cell cancer. Br. J. Cancer 103, 1154–1162 (2010)CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    F. Wang, P. Arun, J. Friedman, Z. Chen, C. Van Waes, Current and potential inflammation targeted therapies in head and neck cancer. Curr. Opin. Pharmacol. 9, 389–395 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    S. Maeda, H. Kamata, J. L. Luo, H. Leffert, M. Karin, IKKβ couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 121, 977–990 (2005)CrossRefPubMedGoogle Scholar
  36. 36.
    P. J. Wysocki, Targeted therapy of hepatocellular cancer. Expert Opin Investig Drugs 19, 265–274 (2010)CrossRefPubMedGoogle Scholar
  37. 37.
    W. Yeo, T. S. Mok, B. Zee, T. W. Leung, P. B. Lai, W. Y. Lau, J. Koh, F. K. Mo, S. C. Yu, A. T. Chan, P. Hui, B. Ma, K. C. Lam, W. M. Ho, H. T. Wong, A. Tang, P. J. Johnson, A randomized phase III study of doxorubicin versus cisplatin/interferon α-2b/doxorubicin/fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma. J. Natl. Cancer Inst. 97, 1532–1538 (2005)CrossRefPubMedGoogle Scholar
  38. 38.
    A. R. Baudy, N. Saxena, H. Gordish, E. P. Hoffman, K. Nagaraju, A robust in vitro screening assay to identify NF-κB inhibitors for inflammatory muscle diseases. Int. Immunopharmacol. 9, 1209–1214 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Y. Zhou, C. Garcia-Prieto, D. A. Carney, R. H. Xu, H. Pelicano, Y. Kang, W. Yu, C. Lou, S. Kondo, J. Liu, D. M. Harris, Z. Estrov, M. J. Keating, Z. Jin, P. Huang, OSW-1: a natural compound with potent anticancer activity and a novel mechanism of action. J. Natl. Cancer Inst. 97, 1781–1785 (2005)CrossRefPubMedGoogle Scholar
  40. 40.
    V. Yadav, S. Sultana, J. Yadav, N. Saini, Gatifloxacin induces S and G2-phase cell cycle arrest in pancreatic cancer cells via p21/p27/p53. PLoS One 7, e47796 (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    D. Lavelle, J. DeSimone, M. Hankewych, T. Kousnetzova, Y. H. Chen, Decitabine induces cell cycle arrest at the G1 phase via p21(WAF1) and the G2/M phase via the p38 MAP kinase pathway. Leuk. Res. 27, 999–1007 (2003)CrossRefPubMedGoogle Scholar
  42. 42.
    T. Fujimoto, M. Onda, H. Nagai, T. Nagahata, K. Ogawa, M. Emi, Upregulation and overexpression of human X-box binding protein 1 (hXBP-1) gene in primary breast cancers. Breast Cancer 10, 301–306 (2003)CrossRefPubMedGoogle Scholar
  43. 43.
    C. Jamora, G. Dennert, A. S. Lee, Inhibition of tumor progression by suppression of stress protein GRP78/BiP induction in fibrosarcoma B/C10ME. Proc. Natl. Acad. Sci. U. S. A. 93, 7690–7694 (1996)CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    M. Shuda, N. Kondoh, N. Imazeki, K. Tanaka, T. Okada, K. Mori, A. Hada, M. Arai, T. Wakatsuki, O. Matsubara, N. Yamamoto, M. Yamamoto, Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis. J. Hepatol. 38, 605–614 (2003)CrossRefPubMedGoogle Scholar
  45. 45.
    G. S. Hotamisligil, Endoplasmic reticulum stress and the inflammatory basis of me4tabolic disease. Cell 140, 900–917 (2010)CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    C. Garcia-Prieto, K. B. R. Ahmed, Z. Chen, Y. Zhou, N. Hammoudi, Y. Kang, C. Lou, Y. Mei, Z. Jin, P. Huang, Effective killing of leukemia cells by the natural product OSW-1 through disruption of cellular calcium homeostasis. J. Biol. Chem. 288, 3240–3250 (2013)CrossRefPubMedGoogle Scholar
  47. 47.
    J. Jin, X. Jin, C. Qian, Y. Ruan, H. Jiang, Signaling network of OSW-1-induced apoptosis and necroptosis in hepatocellular carcinoma. Mol. Med. Rep. 7, 1646–1650 (2013)Google Scholar
  48. 48.
    Y. Inami, S. Waguri, A. Sakamoto, T. Kouno, K. Nakada, O. Hino, S. Watanabe, J. Ando, M. Iwadate, M. Yamamoto, M. S. Lee, K. Tanaka, M. Komatsu, Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J. Cell Biol. 193, 275–284 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    J. M. Luk, C. T. Lam, A. F. Siu, B. Y. Lam, I. O. Ng, M. Y. Hu, C. M. Che, S. T. Fan, Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values. Proteomics 6, 1049–1057 (2006)CrossRefPubMedGoogle Scholar
  50. 50.
    S. Derks, B. Diosdado, Personalized cancer medicine: next steps in the genomic era. Cell. Oncol. 38, 1–2 (2015)CrossRefGoogle Scholar

Copyright information

© International Society for Cellular Oncology 2016

Authors and Affiliations

  • Vignesh Ramesh
    • 1
  • Karthikeyan Selvarasu
    • 1
  • Jaishree Pandian
    • 1
  • Soundarajan Myilsamy
    • 1
  • Chidambaranathan Shanmugasundaram
    • 1
  • Kumaresan Ganesan
    • 1
    Email author
  1. 1.Unit of Excellence in Cancer Genetics, Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological SciencesMadurai Kamaraj UniversityMaduraiIndia

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