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. 2007 Sep;171(3):1023-36.
doi: 10.2353/ajpath.2007.061029. Epub 2007 Aug 3.

Notch2 signaling induces apoptosis and inhibits human MDA-MB-231 xenograft growth

Christine F O'Neill  1 , Sumithra Urs, Christina Cinelli, Alexis Lincoln, Robert J Nadeau, Ruth León, Jessica Toher, Carla Mouta-Bellum, Robert E Friesel, Lucy Liaw
Affiliations

Notch2 signaling induces apoptosis and inhibits human MDA-MB-231 xenograft growth

Christine F O'Neill et al. Am J Pathol. 2007 Sep.

Abstract

Notch functions as an oncogene or tumor inhibitor in various cancers, and decreases in Notch2 expression are associated with increasing grade of human breast cancer. We constitutively activated Notch signaling with intracellular domain (ICD) expression in the human adenocarcinoma line MDA-MB-231. Notch2 signaling increased apoptosis, whereas Notch4ICD (int3) significantly increased cell proliferation and growth. Cells with activated Notch2 or Notch4 were injected into nu/nu mice for analysis of in vivo tumor xenograft phenotype. Tumor growth was significantly altered depending on the receptor activated. Notch2ICD potently suppressed tumor take and growth, leading to a 60% decrease in tumors and significantly smaller, necrotic tumors. Despite this, Notch2ICD tumors were highly vascularized, although the vessels were smaller and comprised a more immature network compared with Notch4ICD tumors. Notch4ICD tumors were highly aggressive and well vascularized, indicating a role for Notch4 signaling in the promotion of the malignant phenotype in addition to its transforming ability. Although both NotchICD groups expressed angiogenic factors, Notch4ICD had selective vascular endothelial growth factor-D in both tumor and host stroma, suggesting a differential regulation of cytokines that may impact vascular recruitment and autocrine tumor signaling. Our results demonstrate that Notch2 signaling is a potent inhibitory signal in human breast cancer xenografts.

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Figures

Figure 1
Figure 1
Notch2 expression correlates with tumor aggressiveness in human breast cancer. A: Notch2 mRNA levels were analyzed using gene expression data sets from cancer gene microarray meta-analysis database that was originally described by Miller et al. Sixty-seven cases of Elston grade 1, 128 cases of Elston grade 2, and 54 cases of Elston grade 3 were included in this study. The y axis represents normalized expression units. Shaded boxes represent interquartile range making the 25th to 75th percentile; whiskers represent the 10th to 90th percent range; bars represent the median. The P value was calculated using Student’s t-test. B: Real-time PCR for Notch2 in a panel of human breast tumor cell lines.
Figure 2
Figure 2
Modulation of Notch signaling in MDA-MB-231 cells. A: MDA-MB-231 cell lysates were used for immunoblot using antibodies against the proteins indicated. B: Cells were transfected with expression constructs for hNotch2ICD or mNotch4ICD, and proteins were detected by immunoblot using antibodies against the epitope tags. C: Stable cell populations were assessed for Notch activation using CBF-1, HES1, and HRT1 luciferase reporter constructs. D: Transcript levels of HES1 and HRT1 were determined by real-time PCR. Shown are averages of three independent experiments. E: γ-Secretase inhibitor XXI was added every 2 days at the concentrations indicated, and MDA-MB-231 cells were counted up to 11 days after plating. Graphed are means ± SEM analysis of variance analysis at day 11; P = 0.0013. F: Cells were treated with shRNA to target Notch2 sequences or treated with a nontargeting control. Stable populations were generated with reduced Notch2 transcripts.
Figure 3
Figure 3
NotchICD affects cell proliferation and apoptosis in MDA-MB-231 stable transfectants. A: Cells stably expressing hNotch2ICD or mNotch4ICD were analyzed in a growth curve assay. Cells were plated in 24-well plates at 15,800 cells/cm2. At days indicated after plating, cells were trypsinized and counted. Values graphed are the average of quadruplicates. B: Cultures were pulsed with BrdU and then quantified for BrdU incorporation in each group. Graphed are means ± SEM. C and D: 7-Amino-actinomycin D incorporation was used for cell cycle analysis of stable populations. Shown are percentages of cells in S, G2, and G1 phases. E–H: Cells were used for TUNEL labeling to detect apoptotic cells by cell counting (E) or flow cytometry (F–H). The control in (H) is the nontargeting control population. FITC, fluorescein isothiocyanate.
Figure 4
Figure 4
NotchICD affects clonal and anchorage independent growth of MDA-MB-231 cells. A: Stably transfected cell populations were plated at clonal growth density at 50 cells/well in a six-well plate. After 2 weeks in culture, cells were fixed in methanol and stained with toluidine blue. Shown are representative wells from each group and the quantitation of average number of colonies/well. Graphed are means ± SEM. Notch2ICD had significantly fewer colonies, and Notch4ICD had significantly more colonies compared with vector transfected controls. B: Conditioned medium from stable cell populations was assayed by enzyme-linked immunosorbent assay for levels of the cytokines indicated. C: Cell lysates were prepared for immunoblot analysis to detect the proteins listed. hNotch2ICD has a V5 epitope tag, and mNotch4ICD has an HA epitope tag.
Figure 5
Figure 5
Activation of Notch2 decreases tumor take and tumor growth in xenografts. A: MDA-MB-231 cells stably expressing control vector, hNotch2ICD, or mNotch4ICD were grown as xenografts in athymic nu/nu mice. Shown are representative mice from each group. B: The length and width of the tumors were measured at days indicated, and volumes calculated. Shown are the quantitation of three independent experiments (total, n = 15/group) and the corresponding analysis of variance P value. Control and mNotch4ICD cells showed 100% tumor penetrance, whereas the hNotch2ICD cells led to tumors in approximately 40% of the mice. C: Cell proliferation was assessed by BrdU incorporation at the end of the experiment, and apoptosis was measured by TUNEL labeling (D). E: Similar tumor xenograft results were obtained with injection of cells into the mammary fat pad.
Figure 6
Figure 6
Histological features of NotchICD xenografts. Tumors were collected at 44 days after injection, and processed for hematoxylin and eosin staining (top two rows) or Masson’s trichrome staining (bottom row). Representative pictures are shown from the tumor periphery below the skin (top) and the tumor core (middle). Scale bar = 100 μm in all panels.
Figure 7
Figure 7
NotchICD tumors have different patterns of vascularization. MECA-32 (A, C, E, and G) and LYVE-1 (B, D, F, and H) staining was used to determine the presence of blood and lymphatic vessels within tumor sections. As shown in serial sections (A and B), these two antibodies showed a nonoverlapping pattern of staining, with MECA-32 recognizing red blood cell-filled blood vessels that are not stained with anti-LYVE-1 (white arrows). Conversely, lymphatic vessels (L) were stained prominently with anti-LYVE-1 but not MECA-32 (black arrowheads). Tumor cells were found frequently within lymphatic vessels. Sections were counterstained with hematoxylin. Scale bars: 50 μm (A and B); 100 μm (C–H).
Figure 8
Figure 8
Vascularization and angiogenic cytokine expression of NotchICD xenografts. A–C: Tumor-bearing mice were perfused with bismuth for X-ray angiography. D: MDA-MB-231 tumors stably expressing NotchICD were collected, fixed, and stained using PECAM (black bars) to detect blood vessels and LYVE-1 (white bars) to detect lymphatic vessels. The percentage of area covered with vessels in each case was quantified, and shown are means ± SEM. Both Notch2ICD and Notch4ICD tumors show an increase in tumor vascularization. E and F: RT-PCR was used to determine transcript levels of angiogenic cytokines in the tumors. Primers were designed specifically to detect tumor-derived human (E) versus host-derived mouse transcripts (F).
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References

    1. del Amo FF, Gendron-Maguire M, Swiatek PJ, Jenkins NA, Copeland NG, Gridley T. Cloning, analysis, and chromosomal localization of Notch-1, a mouse homolog of Drosophila Notch. Genomics. 1993;15:259–264. - PubMed
    1. Lardelli M, Lendahl U. Motch A and motch B: two mouse Notch homologues coexpressed in a wide variety of tissues. Exp Cell Res. 1993;204:364–372. - PubMed
    1. Uyttendaele H, Marazzi G, Wu G, Yan Q, Sassoon D, Kitajewski J. Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development. 1996;122:2251–2259. - PubMed
    1. Mumm JS, Schroeter EH, Saxena MT, Griesemer A, Tian X, Pan DJ, Ray WJ, Kopan R. A ligand-induced extracellular cleavage regulates γ-secretase-like proteolytic activation of Notch1. Mol Cell. 2000;5:197–206. - PubMed
    1. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, Cumano A, Roux P, Black RA, Israel A. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell. 2000;5:207–216. - PubMed

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