Notch1 is an effector of Akt and hypoxia in melanoma development
Barbara Bedogni, … , Amato J. Giaccia, Marianne Broome Powell
Barbara Bedogni, … , Amato J. Giaccia, Marianne Broome Powell
Published October 16, 2008
Citation Information: J Clin Invest. 2008;118(11):3660-3670. https://doi.org/10.1172/JCI36157.
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Research Article Oncology

Notch1 is an effector of Akt and hypoxia in melanoma development

Abstract

Melanomas are highly aggressive neoplasms resistant to most conventional therapies. These tumors result from the interaction of altered intracellular tumor suppressors and oncogenes with the microenvironment in which these changes occur. We previously demonstrated that physiologic skin hypoxia contributes to melanomagenesis in conjunction with Akt activation. Here we show that Notch1 signaling is elevated in human melanoma samples and cell lines and is required for Akt and hypoxia to transform melanocytes in vitro. Notch1 facilitated melanoma development in a xenograft model by maintaining cell proliferation and by protecting cells from stress-induced cell death. Hyperactivated PI3K/Akt signaling led to upregulation of Notch1 through NF-κB activity, while the low oxygen content normally found in skin increased mRNA and protein levels of Notch1 via stabilization of HIF-1α. Taken together, these findings demonstrate that Notch1 is a key effector of both Akt and hypoxia in melanoma development and identify the Notch signaling pathway as a potential therapeutic target in melanoma treatment.

Authors

Barbara Bedogni, James A. Warneke, Brian J. Nickoloff, Amato J. Giaccia, Marianne Broome Powell

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Figure 7

Notch1 inhibition reduces cell proliferation and increases cell death both in vitro and in vivo.

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Notch1 inhibition reduces cell proliferation and increases cell death bo...
(A–D) Tumor sections from Akt-dependent melanomas expressing a control shGFP (A) or shNotch1 (B) were stained with anti Ki67, and were counterstained with DAPI, to label proliferating cells. (C and D) Adjacent sections stained with anti–cyclin D1 antibody. (E) Quantification of proliferating cells in A and B, shown as percent positive cells in 5 microscopic fields from 3 different tumor sections per group. (F) Melanocytes expressing either shGFP or shNotch1 were grown in culture and counted every 3–4 d. (G) Western blot analysis for Notch1-NIC, β-catenin, and cyclin D1 of cells transfected with shGFP or shNotch1. β-Actin was used as loading control. (H–K) Tumor sections from cells expressing shGFP (H) or shNotch1 (I) were stained for Notch1-NIC. (J and K) Adjacent sections were stained with anti–cleaved caspase-3. (L) Quantification of cell death under stringent hypoxia (0.5% O2), measured as positivity to annexin V/propidium iodide. (M) Western blot analysis for Notch1-NIC and cleaved caspase-3 (c-casp3) on cells grown in normoxia or stringent hypoxia for 48 h. Glut-1 was included as an indicator of the hypoxia treatment. β-Actin was used as loading control. Data in E, F, and L are mean ± SD. *P < 0.05 versus respective shGFP control, Student’s t test. Scale bars: 25 μm.