Skip to main content

Advertisement

SpringerLink
Log in

Withaferin A inhibits in vivo growth of breast cancer cells accelerated by Notch2 knockdown

  • Preclinical study
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

Abstract

The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control (P < 0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Andersson ER, Sandberg R, Lendahl U (2011) Notch signaling: simplicity in design, versatility in function. Development 138(17):3593–3612

    Article  CAS  PubMed  Google Scholar 

  2. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137(2):216–233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Arnett KL, Hass M, McArthur DG, Ilagan MX, Aster JC, Kopan R, Blacklow SC (2010) Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nat Struct Mol Biol 17(11):1312–1317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Radtke F, Raj K (2003) The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat Rev Cancer 3(10):756–767

    Article  CAS  PubMed  Google Scholar 

  5. Ranganathan P, Weaver KL, Capobianco AJ (2011) Notch signalling in solid tumours: a little bit of everything but not all the time. Nat Rev Cancer 11(5):338–351

    Article  CAS  PubMed  Google Scholar 

  6. Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD, Sklar J (1991) TAN-1, the human homolog of the Drosophila Notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 66(4):649–661

    Article  CAS  PubMed  Google Scholar 

  7. Previs RA, Coleman RL, Harris AL, Sood AK (2015) Molecular pathways: translational and therapeutic implications of the Notch signaling pathway in cancer. Clin Cancer Res 21(5):955–961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Mazur PK, Grüner BM, Nakhai H, Sipos B, Zimber-Strobl U, Strobl LJ, Radtke F, Schmid RM, Siveke JT (2010) Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras G12D-induced skin carcinogenesis in vivo. PLoS One 5(10):e13578

    Article  PubMed  PubMed Central  Google Scholar 

  9. Siegel R, Ma J, Zou Z, Jemal A (2014) Cancer statistics. CA Cancer J Clin 64(1):9–29

    Article  PubMed  Google Scholar 

  10. Hu C, Diévart A, Lupien M, Calvo E, Tremblay G, Jolicoeur P (2006) Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors. Am J Pathol 168(3):973–990

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Harrison H, Farnie G, Howell SJ, Rock RE, Stylianou S, Brennan KR, Bundred NJ, Clarke RB (2010) Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res 70(2):709–718

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Reedijk M, Odorcic S, Chang L, Zhang H, Miller N, McCready DR, Lockwood G, Egan SE (2005) High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res 65(18):8530–8537

    Article  CAS  PubMed  Google Scholar 

  13. Kim SH, Sehrawat A, Singh SV (2012) Notch2 activation by benzyl isothiocyanate impedes its inhibitory effect on breast cancer cell migration. Breast Cancer Res Treat 134(3):1067–1079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sehrawat A, Sakao K, Singh SV (2014) Notch2 activation is protective against anticancer effects of zerumbone in human breast cancer cells. Breast Cancer Res Treat 146(3):543–555

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lee J, Sehrawat A, Singh SV (2012) Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells. Breast Cancer Res Treat 136(1):45–56

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Chao CH, Chang CC, Wu MJ, Ko HW, Wang D, Hung MC, Yang JY, Chang CJ (2014) MicroRNA-205 signaling regulates mammary stem cell fate and tumorigenesis. J Clin Investig 124(7):3093–3106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. O’Neill CF, Urs S, Cinelli C, Lincoln A, Nadeau RJ, León R, Toher J, Mouta-Bellum C, Friesel RE, Liaw L (2007) Notch2 signaling induces apoptosis and inhibits human MDA-MB-231 xenograft growth. Am J Pathol 171(3):1023–1036

    Article  PubMed  PubMed Central  Google Scholar 

  18. Parr C, Watkins G, Jiang WG (2004) The possible correlation of Notch-1 and Notch-2 with clinical outcome and tumour clinicopathological parameters in human breast cancer. Int J Mol Med 14(5):779–786

    CAS  PubMed  Google Scholar 

  19. Mishra LC, Singh BB, Dagenais S (2000) Scientific basis for the therapeutic use of Withania somnifera (Ashwagandha): a review. Altern Med Rev 5(4):334–346

    CAS  PubMed  Google Scholar 

  20. Hahm ER, Lee J, Kim SH, Sehrawat A, Arlotti JA, Shiva SS, Bhargava R, Singh SV (2013) Metabolic alterations in mammary cancer prevention by withaferin A in a clinically relevant mouse model. J Natl Cancer Inst 105(15):1111–1122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Stan SD, Hahm ER, Warin R, Singh SV (2008) Withaferin A causes FOXO3a- and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo. Cancer Res 68(18):7661–7669

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Singh SV, Kim SH, Sehrawat A, Arlotti JA, Hahm ER, Sakao K, Beumer JH, Jankowitz RC, Chandra-Kuntal K, Lee J, Powolny AA, Dhir R (2012) Biomarkers of phenethyl isothiocyanate-mediated mammary cancer chemoprevention in a clinically relevant mouse model. J Natl Cancer Inst 104(16):1228–1239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Powolny AA, Bommareddy A, Hahm ER, Normolle DP, Beumer JH, Nelson JB, Singh SV (2011) Chemopreventative potential of the cruciferous vegetable constituent phenethyl isothiocyanate in a mouse model of prostate cancer. J Natl Cancer Inst 103(7):571–584

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Beaino W, Anderson CJ (2014) PET imaging of very late antigen-4 in melanoma: comparison of 68Ga- and 64Cu-labeled NODAGA and CB-TE1A1P-LLP2A conjugates. J Nucl Med 55(11):1856–1863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Moura MB, Hahm ER, Van Houten B, Singh SV (2014) The use of seahorse extracellular flux analyzer in mechanistic studies of naturally occurring cancer chemopreventive agents. In: Bode AM, Dong Z (eds) Cancer prevention: dietary factors and pharmacology, methods in pharmacology and toxicology. Springer Science + Business Media, New York, pp 173–187

    Chapter  Google Scholar 

  26. Lahat G, Zhu QS, Huang KL, Wang S, Bolshakov S, Liu J, Torres K, Langley RR, Lazar AJ, Hung MC, Lev D (2010) Vimentin is a novel anti-cancer therapeutic target; Insights from in vitro and in vivo mice xenograft studies. PLoS One 5(4):e10105

    Article  PubMed  PubMed Central  Google Scholar 

  27. Zang S, Chen F, Dai J, Guo D, Tse W, Qu X, Ma D, Ji C (2010) RNAi-mediated knockdown of Notch-1 leads to cell growth inhibition and enhanced chemosensitivity in human breast cancer. Oncol Rep 23(4):893–899

    CAS  PubMed  Google Scholar 

  28. Simmons MJ, Serra R, Hermance N, Kelliher MA (2012) NOTCH1 inhibition in vivo results in mammary tumor regression and reduced mammary tumorsphere-forming activity in vitro. Breast Cancer Res 14(5):R126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yuan X, Zhang M, Wu H, Xu H, Han N, Chu Q, Yu S, Chen Y, Wu K (2015) Expression of Notch1 correlates with breast cancer progression and prognosis. PLoS One 10(6):e0131689

    Article  PubMed  PubMed Central  Google Scholar 

  30. Hurvitz S, Mead M (2016) Triple-negative breast cancer: advancements in characterization and treatment approach. Curr Opin Obstet Gynecol 28(1):59–69

    PubMed  Google Scholar 

  31. Yadav BS, Chanana P, Jhamb S (2015) Biomarkers in triple negative breast cancer: a review. World J Clin Oncol 6(6):252–263

    Article  PubMed  PubMed Central  Google Scholar 

  32. Chavez KJ, Garimella SV, Lipkowitz S (2010) Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer. Breast Dis 32(1–2):35–48

    PubMed  PubMed Central  Google Scholar 

  33. Barnabas N, Cohen D (2013) Phenotypic and molecular characterization of MCF10DCIS and SUM breast cancer cell lines. Int J Breast Cancer 2013:872743

    Article  PubMed  PubMed Central  Google Scholar 

  34. Vyas AR, Singh SV (2014) Molecular targets and mechanisms of cancer prevention and treatment by withaferin A, a naturally occurring steroidal lactone. AAPS J 16(1):1–10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zou J, Han Z, Zhou L, Cai C, Luo H, Huang Y, Liang Y, He H, Jiang F, Wang C, Zhong W (2015) Elevated expression of IMPDH2 is associated with progression of kidney and bladder cancer. Med Oncol 32(1):373

    Article  PubMed  Google Scholar 

  36. Zhou L, Xia D, Zhu J, Chen Y, Chen G, Mo R, Zeng Y, Dai Q, He H, Liang Y, Jiang F, Zhong W (2014) Enhanced expression of IMPDH2 promotes metastasis and advanced tumor progression in patients with prostate cancer. Clin Transl Oncol 16(10):906–913

    Article  CAS  PubMed  Google Scholar 

  37. Ramos FS, Serino LT, Carvalho CM, Lima RS, Urban CA, Cavalli IJ, Ribeiro EM (2015) PDIA3 and PDIA6 gene expression as an aggressiveness marker in primary ductal breast cancer. Genet Mol Res 14(2):6960–6967

    Article  CAS  PubMed  Google Scholar 

  38. Dethlefsen C, Højfeldt G, Hojman P (2013) The role of intratumoral and systemic IL-6 in breast cancer. Breast Cancer Res Treat 138(3):657–664

    Article  CAS  PubMed  Google Scholar 

  39. Todorović-Raković N, Milovanović J (2013) Interleukin-8 in breast cancer progression. J Interferon Cytokine Res 33(10):563–570

    Article  PubMed  PubMed Central  Google Scholar 

  40. Divella R, Daniele A, Savino E, Palma F, Bellizzi A, Giotta F, Simone G, Lioce M, Quaranta M, Paradiso A, Mazzocca A (2013) Circulating levels of transforming growth factor-βeta (TGF-β) and chemokine (C-X-C motif) ligand-1 (CXCL1) as predictors of distant seeding of circulating tumor cells in patients with metastatic breast cancer. Anticancer Res 33(4):1491–1497

    CAS  PubMed  Google Scholar 

  41. Lin S, Gan Z, Han K, Yao Y, Min D (2015) Interleukin-6 as a prognostic marker for breast cancer: a meta-analysis. Tumori 101(5):535–541

    Article  PubMed  Google Scholar 

  42. Hartman ZC, Poage GM, den Hollander P, Tsimelzon A, Hill J, Panupinthu N, Zhang Y, Mazumdar A, Hilsenbeck SG, Mills GB, Brown PH (2013) Growth of triple-negative breast cancer cells relies upon coordinate autocrine expression of the proinflammatory cytokines IL-6 and IL-8. Cancer Res 73(11):3470–3480

    Article  CAS  PubMed  Google Scholar 

  43. Freund A, Chauveau C, Brouillet JP, Lucas A, Lacroix M, Licznar A, Vignon F, Lazennec G (2003) IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells. Oncogene 22(2):256–265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This investigation was supported by the National Cancer Institute, National Institutes of Health grant award RO1 CA142604-06 (to SVS). This research project used Animal Facility, In Vivo Imaging Facility, Tissue and Research Pathology Facility, and Proteomics Facility supported in part by a grant from the National Cancer Institute at the National Institutes of Health (P30 CA047904).

Author information

Author notes

    Authors and Affiliations

    1. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

      Su-Hyeong Kim, Eun-Ryeong Hahm, Suman K. Samanta, Michelle B. Moura & Shivendra V. Singh

    2. 2.32A Hillman Cancer Center Research Pavilion, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA

      Julie A. Arlotti, Stephen H. Thorne, Yongli Shuai, Carolyn J. Anderson, Alexander G. White, Anna Lokshin & Shivendra V. Singh

    3. Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

      Stephen H. Thorne

    4. Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA, USA

      Yongli Shuai

    5. Department of Radiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

      Carolyn J. Anderson & Alexander G. White

    6. Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

      Anna Lokshin

    7. Department of Food and Nutrition, Chosun University, Gwangju, Korea

      Joomin Lee

    Authors
    1. Su-Hyeong Kim
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Eun-Ryeong Hahm
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Julie A. Arlotti
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Suman K. Samanta
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Michelle B. Moura
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Stephen H. Thorne
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Yongli Shuai
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Carolyn J. Anderson
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Alexander G. White
      View author publications

      You can also search for this author in PubMed Google Scholar

    10. Anna Lokshin
      View author publications

      You can also search for this author in PubMed Google Scholar

    11. Joomin Lee
      View author publications

      You can also search for this author in PubMed Google Scholar

    12. Shivendra V. Singh
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Shivendra V. Singh.

    Ethics declarations

    Conflict of interest

    The authors do not have any conflict of interest.

    Ethical standards

    Use of mice and their care was in accordance by the Institutional Animal Care and Use Committee.

    Additional information

    Su-Hyeong Kim and Eun-Ryeong Hahm have contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Kim, SH., Hahm, ER., Arlotti, J.A. et al. Withaferin A inhibits in vivo growth of breast cancer cells accelerated by Notch2 knockdown. Breast Cancer Res Treat 157, 41–54 (2016). https://doi.org/10.1007/s10549-016-3795-y

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s10549-016-3795-y

    Keywords

    Search

    Navigation