Suppression of renal cell carcinoma growth by inhibition of Notch signaling in vitro and in vivo
Jonas Sjölund, … , Börje Ljungberg, Håkan Axelson
Jonas Sjölund, … , Börje Ljungberg, Håkan Axelson
Published December 13, 2007
Citation Information: J Clin Invest. 2008;118(1):217-228. https://doi.org/10.1172/JCI32086.
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Research Article Oncology

Suppression of renal cell carcinoma growth by inhibition of Notch signaling in vitro and in vivo

Abstract

Loss of the tumor suppressor gene von Hippel–Lindau (VHL) plays a key role in the oncogenesis of clear cell renal cell carcinoma (CCRCC). The loss leads to stabilization of the HIF transcription complex, which induces angiogenic and mitogenic pathways essential for tumor formation. Nonetheless, additional oncogenic events have been postulated to be required for the formation of CCRCC tumors. Here, we show that the Notch signaling cascade is constitutively active in human CCRCC cell lines independently of the VHL/HIF pathway. Blocking Notch signaling resulted in attenuation of proliferation and restrained anchorage-independent growth of CCRCC cell lines. Using siRNA targeting the different Notch receptors established that the growth-promoting effects of the Notch signaling pathway were attributable to Notch-1 and that Notch-1 knockdown was accompanied by elevated levels of the negative cell-cycle regulators p21Cip1 and/or p27Kip1. Treatment of nude mice with an inhibitor of Notch signaling potently inhibited growth of xenotransplanted CCRCC cells. Moreover, Notch-1 and the Notch ligand Jagged-1 were expressed at significantly higher levels in CCRCC tumors than in normal human renal tissue, and the growth of primary CCRCC cells was attenuated upon inhibition of Notch signaling. These findings indicate that the Notch cascade may represent a novel and therapeutically accessible pathway in CCRCC.

Authors

Jonas Sjölund, Martin Johansson, Sugata Manna, Carl Norin, Alexander Pietras, Siv Beckman, Elise Nilsson, Börje Ljungberg, Håkan Axelson

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Figure 5

Ablation of endogenous Notch-1 by siRNA attenuates growth of CCRCC cells and is associated with elevation of p21Cip1 and/or p27Kip1.

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Ablation of endogenous Notch-1 by siRNA attenuates growth of CCRCC cells...
(A) Inhibition of Notch-1 and Notch-2 protein expression in CCRCC cells employing siRNA. 786-O, SKRC-17, and SKRC-52 cells were transfected either with nonspecific control, Notch-1–specific (siN-1), or Notch-2–specific (siN-2) siRNAs. Cells were harvested after 24 hours of transfection, and cell lysates were analyzed for Notch-1 and Notch-2 protein expression. (B) [3H]thymidine incorporation of 786-O, SKRC-17, and SKRC-52 cells following siRNA transfection for 24 hours and incubation for 72 hours. Bars represent mean + SD of 3 independent experiments, each performed with each transfection 6 times. Notch-1–specific (siN-1) and Notch-2–specific (siN-2) siRNA-transfected cells were normalized to nonspecific control-transfected cells. *P < 0.001, statistically significant changes (siN-1 versus c-si or siN-2 versus c-si). (C) Western blotting of p21Cip1 and/or p27Kip1 in 786-O, SKRC-17, or SKRC-52 cells transfected with either nonspecific control or Notch-1–specific siRNA. The cells were transfected for 24 hours and harvested after another 24 hours. (D) Knockdown of Jagged-1 employing Jagged-1–specific (siJ-1) siRNA. 786-O, SKRC-17, and SKRC-52 cells were transfected with either nonspecific control or Jagged-1–specific siRNAs. Cells were harvested after 24 hours of transfection, and cell lysates were analyzed for Jagged-1 and actin protein expression. (E) [3H]-thymidine incorporation of CCRCC cells following control or Jagged-1 siRNA transfection for 24 hours and incubation for 72 hours. Bars represent mean + SD of 3 independent experiments, each performed with each transfection 6 times. Jagged-1–specific siRNA-transfected cells were normalized to nonspecific control-transfected cells.