NE-AUA 2006 Annual Meeting, September 28 - 30, 2006, The Westin Hotel & Rhode Island Convention Center Providence, Rhode Island
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Silencing of the NOX4 NADP(H) Oxidase Inhibits Tumor Growth Of VHL-Deficient Renal Cancer Cells
Jodi Maranchie, MD, Ye Zhan, MS.
University of Massachusetts, Worcester, MA,

Background: Loss of VHL occurs in greater than 60% of clear cell kidney cancer (RCC). VHL is a ubiquitin ligase for hypoxia inducible factor-α (HIF-α) and absence of VHL results in HIF-α accumulation and activation of HIF-dependent transcription of an array of genes including VEGF and TGF-α. We recently reported that expression of Nox4 is critical for expression and activation of HIF2-α even in the absence of VHL. To determine if Nox4 silencing could abrogate the tumorigenic phenotype of VHL-deficient cells, we measured the ability to form tumors in a xenograft model.
Methods: Small inhibitory RNA for Nox4 (siRNA) was cloned into pSilencer 4.1-CMV puro (Ambion) and transfected into the VHL-deficient human RCC cell lines 786-0. Single cell clones were screened for reduced production of ROS using a 2,7-dichlorofluorescin diacetate florescent assay. Non-specific (scramble) siRNA served as a negative control. One million cells were injected subcutaneously into 5 SCID-Beige mice and tumors were measured twice weekly for 14 weeks. Error bars represent standard error of the mean.
Results: Mice injected with 786-0-scramble cells developed measurable tumors at 5 weeks compared with 9 weeks for the Nox4 knockdown clones. Further, Nox4 knockdown tumors grew to approximately half the size of scramble tumors and demonstrated less ulceration and bleeding in size-matched tumors Figure 1.
Conclusions: Consistent with the Nox4 requirement for HIF2-alpha activity, Nox4 expression is critical to the full tumorigenic phenotype of 786-0, further supporting Nox4 as a candidate for molecularly targeted RCC therapy.


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