NAD(P)H quinone oxidoreductase protects TAp63gamma from proteasomal degradation and regulates TAp63gamma-dependent growth arrest

Abstract

Background: p63 is a member of the p53 transcription factor family. p63 is expressed from two promoters resulting in proteins with opposite functions: the transcriptionally active TAp63 and the dominant-negative DeltaNp63. Similar to p53, the TAp63 isoforms induce cell cycle arrest and apoptosis. The DeltaNp63 isoforms are dominant-negative variants opposing the activities of p53, TAp63 and TAp73. To avoid unnecessary cell death accompanied by proper response to stress, the expression of the p53 family members must be tightly regulated. NAD(P)H quinone oxidoreductase (NQO1) has recently been shown to interact with and inhibit the degradation of p53. Due to the structural similarities between p53 and p63, we were interested in studying the ability of wild-type and polymorphic, inactive NQO1 to interact with and stabilize p63. We focused on TAp63gamma, as it is the most potent transcription activator and it is expected to have a role in tumor suppression.

Principal findings: We show that TAp63gamma can be degraded by the 20S proteasomes. Wild-type but not polymorphic, inactive NQO1 physically interacts with TAp63gamma, stabilizes it and protects it from this degradation. NQO1-mediated TAp63gamma stabilization was especially prominent under stress. Accordingly, we found that downregulation of NQO1 inhibits TAp63gamma-dependant p21 upregulation and TAp63gamma-induced growth arrest stimulated by doxorubicin.

Conclusions/significance: Our report is the first to identify this new mechanism demonstrating a physical and functional relationship between NQO1 and the most potent p63 isoform, TAp63gamma. These findings appoint a direct role for NQO1 in the regulation of TAp63gamma expression, especially following stress and may therefore have clinical implications for tumor development and therapy.

Figure 1. TAp63γ protein is stabilized by NQO1.

(A) 293, HCT116 and HCT116^−/− cells expressing endogenous NQO1 or over-expressing wild-type FLAG-NQO1 were transfected with a plasmid expressing TAp63γ. Forty-eight hours post-transfection cell lysates were prepared and resolved by SDS-PAGE. TAp63γ, NQO1 and GAPDH levels were detected by Western blot analysis using anti-p63 (4A4), anti-FLAG and anti-GAPDH (loading control) antibodies, respectively. (B) HCT116 cells expressing endogenous NQO1 or over-expressing wild-type FLAG-NQO1 were transfected with a plasmid expressing TAp63γ. Twenty-four hours post transfection, 10µg/ml cyclohexamide (CHX) was added for 4 hours. Cell lysates were prepared and resolved by SDS-PAGE. TAp63γ, NQO1 and GAPDH levels were detected by Western blot analysis using anti-p63 (4A4), anti-FLAG and anti-GAPDH (loading control) antibodies, respectively. (C) RNA was prepared from these same cells, reverse transcribed and RT-PCR was performed using primers specific for TAp63 and for gapdh. Data is represented as relative levels of TAp63γ normalized to gapdh. (D) 293, HCT116 and HCT116^−/− cells were co-transfected with plasmids expressing TAp63γ and HA-C609T NQO1. Forty-eight hours post-transfection cell lysates were prepared and resolved by SDS-PAGE. TAp63γ, NQO1 and GAPDH levels were detected by Western blot analysis using anti-p63 (4A4), anti-HA and anti-GAPDH (loading control) antibodies, respectively. (E) 293, HCT116 and HCT116^−/− cells were transfected with scrambled oligonucleotides or siNQO1 oligonucleotides. Twenty-four hours post-transfection cell were transfected with a plasmid expressing TAp63γ. Twenty-four hours post-transfection cell lysates were prepared and resolved by SDS-PAGE. TAp63γ, NQO1 and GAPDH levels were detected by Western blot analysis using anti-p63 (4A4), anti-NQO1 and anti-GAPDH (loading control) antibodies, respectively.

Figure 2. TAp63γ expression is stabilized by NQO1 in response to genotoxic stress.

293, HCT116 and HCT116^−/− cells expressing endogenous NQO1 or over-expressing wild-type FLAG-NQO1 were transfected with a plasmid expressing TAp63γ. Twenty-four hours post-transfection cells were treated with 1µM DOX for 24h. At this point, cell lysates were prepared and resolved by SDS-PAGE. TAp63γ, NQO1 and GAPDH levels were detected by Western blot analysis using anti-p63 (4A4), anti-FLAG and anti-GAPDH (loading control) antibodies, respectively.

Figure 3. NQO1 physically associates with TAp63γ and protects it from 20S proteasomal degradation.

(A) 293 cells stably expressing HA-NQO1 or HA-C609T NQO1 were transfected with a plasmid expressing TAp63γ. Cellular extracts were prepared and subject to immunoprecipitated (IP) with anti-HA or anti-p63 antibodies. Immunoprecipitated proteins and 5% of input material were detected by Western blot using anti-p63 and anti-HA antibodies. (B) Degradation of in-vitro translated, biotin-labeled TAp63γ with 20S proteasome was carried out in the presence or absence of in-vitro translated, biotin-labeled NQO1 and 1mM NADH at 37°C for 4h. Biotin-labeled TAp63γ and NQO1 were detected with fluorescently-labeled streptavidin.

Figure 4. Downregulation of NQO1 inhibits TAp63γ-dependent growth arrest in DOX treated HCT116^−/− cells.

HCT116^−/− cells were transfected with scrambled oligonucleotides (siControl) (A–D) or siNQO1 oligonucleotides (E–H) and transfected (B, D, F, H) or not (A, C, E, G) with a plasmid expressing TAp63γ. Twenty-four hours post-transfection the cells were treated or not with 0.05µM DOX. 24h after this treatment, DNA content was analyzed by propidium-iodide staining. The percentage of cells in G1 and G2 is presented. (I) RNA was prepared from these same cells, reverse transcribed and real time PCR was performed using primers specific for p21 and 18S rRNA. Data is represented as relative levels of p21 normalized to 18S rRNA. *p<0.05, **p<0.01.