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Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells

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Abstract

Basal breast cancer, common among patients presenting with inflammatory breast cancer (IBC), has been shown to be resistant to radiation and enriched in cancer stem cells. The Notch pathway plays an important role in self-renewal of breast cancer stem cells and contributes to inflammatory signaling which promotes the breast cancer stem cell phenotype. Herein, we inhibited Notch signaling using a gamma secretase inhibitor, RO4929097, in an in vitro model that enriches for cancer initiating cells (3D clonogenic assay) and conventional 2D clonogenic assay to compare the effect on radiosensitization of the SUM149 and SUM190 IBC cell lines. RO4929097 downregulated the Notch target genes Hes1, Hey1, and HeyL, and showed a significant reduction in anchorage independent growth in SUM190 and SUM149. However, the putative self-renewal assay mammosphere formation efficiency was increased with the drug. To assess radiosensitization of putative cancer stem cells, cells were exposed to increasing doses of radiation with or without 1 μM RO4929097 in their standard (2D) and self-renewal enriching (3D) culture conditions. In the conventional 2D clonogenic assay, RO4929097 significantly sensitized SUM190 cells to ionizing radiation and has a modest radiosensitization effect in SUM149 cells. In the 3D clonogenic assays, however, a radioprotective effect was seen in both SUM149 and SUM190 cells at higher doses. Both cell lines express IL-6 and IL-8 cytokines known to mediate the efficacy of Notch inhibition and to promote self-renewal of stem cells. We further showed that RO429097 inhibits normal T-cell synthesis of some inflammatory cytokines, including TNF-α, a potential mediator of IL-6 and IL-8 production in the microenvironment. These data suggest that additional targeting agents may be required to selectively target IBC stem cells through Notch inhibition, and that evaluation of microenvironmental influences may shed further light on the potential effects of this inhibitor.

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Acknowledgments

The small molecule inhibitor RO4929097 was kindly provided by Dr. John Boylan of Roche Pharmaceuticals. This work was supported by grants from the National Institute of Health R01CA138239-01; the State of Texas Grant for Rare and Aggressive Cancers; the University of Texas MD Anderson Cancer Center Institutional Research Grant; the University of Texas Health Sciences Center KL2 RR024149 and Susan G. Komen Breast Cancer Foundation Grant KG081287; Assessment of Circulating Breast Cancer Stem Cells To Predict Recurrent Disease, W81XWH-09-1-0031 01, DOD.

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The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Radiation Oncology, Unit 1202, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, 77030, USA

    Bisrat G. Debeb, Li Li, Thomas A. Buchholz & Wendy A. Woodward

  2. Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Evan N. Cohen & James M. Reuben

  3. Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Kimberly Boley, Erik M. Freiter & Fredika M. Robertson

  4. Fox Chase Cancer Center, Philadelphia, PA, USA

    Massimo Cristofanilli

  5. Morgan Welch Inflammatory Breast Cancer Research Center and Clinic, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA

    Bisrat G. Debeb, Evan N. Cohen, Kimberly Boley, Erik M. Freiter, Li Li, Fredika M. Robertson, James M. Reuben, Massimo Cristofanilli, Thomas A. Buchholz & Wendy A. Woodward

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  1. Bisrat G. Debeb
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  2. Evan N. Cohen
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  3. Kimberly Boley
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  4. Erik M. Freiter
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  5. Li Li
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  8. Massimo Cristofanilli
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  9. Thomas A. Buchholz
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Correspondence to Wendy A. Woodward.

Additional information

Bisrat G. Debeb and Evan N. Cohen contributed equally to this work.

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Debeb, B.G., Cohen, E.N., Boley, K. et al. Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells. Breast Cancer Res Treat 134, 495–510 (2012). https://doi.org/10.1007/s10549-012-2075-8

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