Notch1 is an effector of Akt and hypoxia in melanoma development
Barbara Bedogni, James A. Warneke, Brian J. Nickoloff, Amato J. Giaccia, Marianne Broome Powell
Barbara Bedogni, James A. Warneke, Brian J. Nickoloff, Amato J. Giaccia, Marianne Broome Powell
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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 2

Notch1-NIC expression is a function of PI3K/Akt pathway activation and is transcriptionally regulated by Akt.

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Notch1-NIC expression is a function of PI3K/Akt pathway activation and i...
(A) We analyzed 26 low-passage melanoma cell lines for activation of the PI3K/Akt and Raf/MEK/Erk pathways with respect to normal human melanocytes in correlation with Notch1-NIC. M, melanocytes; p-, phosphorylated; t-, total. (B) Two of the cell lines analyzed in A (WM266, lane 3; K457, lane 8) were treated with 50 μM Ly294002, 10 μM U0126, or the combination (Ly/U). Effectiveness of the treatment was assessed as the inhibition of phosphorylation of Akt and Erk1/2. (C) K457 and WM266 expressing dominant-negative PI3K (Δp85) were assessed for Notch1-NIC protein. (D) Mouse melanocytes overexpressing oncogenic Akt or oncogenic BRaf were assessed for Notch1-NIC expression. The ratio of Notch1-NIC to α-tubulin was 1.3 for Babe, 4.8 for Akt, and 1.2 for BRaf. (E) Western blot for TM-Notch1 showed higher expression in Akt-expressing cells. (F) qRT-PCR for Notch1. As an internal control, 18S was used for normalization. (G) Notch1 activity, measured as induction of a HES1-dependent reporter construct. Data in F and G are mean ± SD. *P < 0.05 versus Babe control, Student’s t test. In A–E, α-tubulin and β-actin were used as indicated as loading controls.