title: Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells.
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.
authors.affiliation: Department of Radiation Oncology, Unit 1202, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA.
authors.forename: Bisrat G
authors.initials: BG
authors.lastname: Debeb
authors.forename: Evan N
authors.initials: EN
authors.lastname: Cohen
authors.forename: Kimberly
authors.initials: K
authors.lastname: Boley
authors.forename: Erik M
authors.initials: EM
authors.lastname: Freiter
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Fredika M
authors.initials: FM
authors.lastname: Robertson
authors.forename: James M
authors.initials: JM
authors.lastname: Reuben
authors.forename: Massimo
authors.initials: M
authors.lastname: Cristofanilli
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Buchholz
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Woodward
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors; D015703:Antigens, CD / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001552:Benzazepines / Q000494:pharmacology*; D045744:Cell Line, Tumor / Q000187:drug effects / Q000528:radiation effects; D002465:Cell Movement; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D058922:Inflammatory Breast Neoplasms / Q000188:drug therapy*; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008213:Lymphocyte Activation / Q000187:drug effects; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000528:radiation effects; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D051880:Receptors, Notch / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D018874:Spheroids, Cellular; D005466:Fluorocarbons
chemical_list: D000444:Aldehyde Dehydrogenase; D053829:Amyloid Precursor Protein Secretases; D015703:Antigens, CD; D000970:Antineoplastic Agents; D001552:Benzazepines; D018836:Inflammation Mediators; D015850:Interleukin-6; D016209:Interleukin-8; D011838:Radiation-Sensitizing Agents; D011948:Receptors, Antigen, T-Cell; D051880:Receptors, Notch; C508600:IL6 protein, human; C545185:2,2-dimethyl-N-(6-oxo-6,7-dihydro-5H-dibenzo(b,d)azepin-7-yl)-N'-(2,2,3,3,3-pentafluoropropyl)malonamide; D005466:Fluorocarbons