Prolyl Hydroxylase-3 Is Down-regulated in Colorectal Cancer Cells and Inhibits IKKβ Independent of Hydroxylase Activity

Abstract

Prolyl hydroxylase (PHD) hydroxylates hypoxia inducible factor (HIF) alpha, leading to HIFalpha degradation. The PHD family comprises PHD1, PHD2, and PHD3. The enzymatic activity of PHDs is O(2)-dependent, so PHDs are believed to be oxygen sensors as well as tumor suppressors. However, the expression pattern of PHDs in colorectal cancer and the correlation between their expression level and tumorigenesis is unclear. We determined the expression of PHDs in 60 human primary colorectal carcinoma tissues, paired with normal colorectal tissues. PHD3 expression levels were knocked down using small interfering RNA (siRNA); cells were analyzed by immunoblotting, immunoprecipitation, and histochemical analyses. In vivo tumor growth was analyzed in nu/nu mice. Expression of PHD3 is decreased in colorectal cancer tissues. Decreased expression of PHD3 is associated with higher tumor grade and metastasis. PHD3 inhibits phosphorylation of inhibitor of kappaB (IkappaB) kinase (IKK) beta and activation of (NF) kappaB, independent of its hydroxylase activity. PHD3 associates with IKKbeta but does not target it for destruction; instead, PHD3 blocks the interaction between IKKbeta and Hsp90 that is required for phosphorylation of IKKbeta. Knockdown of PHD3 increased resistance of colorectal cancer cells to the effects of tumor necrosis factor-alpha and tumorigenesis. PHD3 appears to be a tumor suppressor in colorectal cancer cells that inhibits IKKbeta/NF-kappaB signaling, independent of its hydroxylase activity. Activation of NF-kappaB has been observed in colon cancer. Determination of PHD3 status could aid targeted therapy selection for patients with colorectal tumors that have increased NF-kappaB activity.