Gastroenterology

Gastroenterology

Volume 138, Issue 2, February 2010, Pages 606-615
Gastroenterology

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

https://doi.org/10.1053/j.gastro.2009.09.049Get rights and content

Background & Aims

Prolyl hydroxylase (PHD) hydroxylates hypoxia inducible factor (HIF) α, leading to HIFα degradation. The PHD family comprises PHD1, PHD2, and PHD3. The enzymatic activity of PHDs is O2-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.

Methods

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.

Results

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 κB (IκB) kinase (IKK) β and activation of (NF) κB, independent of its hydroxylase activity. PHD3 associates with IKKβ but does not target it for destruction; instead, PHD3 blocks the interaction between IKKβ and Hsp90 that is required for phosphorylation of IKKβ. Knockdown of PHD3 increased resistance of colorectal cancer cells to the effects of tumor necrosis factor-α and tumorigenesis.

Conclusions

PHD3 appears to be a tumor suppressor in colorectal cancer cells that inhibits IKKβ/NF-κB signaling, independent of its hydroxylase activity. Activation of NF-κB has been observed in colon cancer. Determination of PHD3 status could aid targeted therapy selection for patients with colorectal tumors that have increased NF-κB activity.

Section snippets

Cell Culture and Reagents

Human colon cancer HCT116 and SW480 cells and human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% serum. PHD3 antibody was from Novus Biologicals (Littleton, CO). IKKβ and phosphorylated IKKβ antibodies were from Cell Signaling (Beverly, MA). HA, myc, and Hsp90 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). β-Actin antibody and cycloheximide were from Sigma (St. Louis, MO). Recombinant human TNF-α was from R&D Systems

Expression of PHD3 Is Decreased in Human Colorectal Cancer

Sixty pairs of human colorectal cancer samples, including primary colorectal carcinoma tissues and paired normal colorectal tissues, were tested. Decreased expression of PHD3 occurs in 37 of 60 (61.7%) of colorectal tumors compared with the paired normal colorectal tissues (Figure 1A). Univariate analysis indicates that messenger RNA (mRNA) levels of PHD3 are significantly different between paired normal colorectal tissues and tumors (P < .001). The decreased expression of PHD3 is associated

Discussion

Hydroxylase activity of PHD is modulated by oxygen availability. The hydroxylase activity of PHD is inhibited when under hypoxia or in the presence of DMOG. Our results show that both PHD3 and the hydroxylase-defective PHD3(H196A) inhibit hypoxia- and DMOG-induced activation of NF-κB (Figure 3C). Moreover, PHD3(H196A) also inhibits TNF-α-induced phosphorylation of IKKβ and activation of NF-κB (Figure 3). These results suggest that PHD3 inhibits IKKβ/NF-κB signaling independent of its

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by National Natural Science Foundation of China (30870524), Chinese Academy of Sciences (KSCX2-YW-R-114), Chinese Ministry of Science and Technology (2007CB947100), and Shanghai Institute for Biological Sciences (2008KIP402).

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