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

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.

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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

Correspondence to Shivendra V. Singh.

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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.

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Kim, S., Hahm, E., 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

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Keywords

  • Notch2
  • Breast cancer
  • Cytokines
  • Withaferin A
  • Chemoprevention