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Link to original content: http://pubmed.ncbi.nlm.nih.gov/23812431/
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. 2014 May 29;33(22):2918-27.
doi: 10.1038/onc.2013.246. Epub 2013 Jul 1.

PPM1A is a RelA phosphatase with tumor suppressor-like activity

X Lu  1 , H An  2 , R Jin  3 , M Zou  4 , Y Guo  1 , P-F Su  5 , D Liu  4 , Y Shyr  5 , W G Yarbrough  6
Affiliations

PPM1A is a RelA phosphatase with tumor suppressor-like activity

X Lu et al. Oncogene. .

Abstract

Nuclear factor-κB (NF-κB) signaling contributes to human disease processes, notably inflammatory diseases and cancer. NF-κB has a role in tumorigenesis and tumor growth, as well as promotion of metastases. Mechanisms responsible for abnormal NF-κB activation are not fully elucidated; however, RelA phosphorylation, particularly at serine residues S536 and S276, is critical for RelA function. Kinases that phosphorylate RelA promote oncogenic behaviors, suggesting that phosphatases targeting RelA could have tumor-inhibiting activities; however, few RelA phosphatases have been identified. Here, we identified tumor inhibitory and RelA phosphatase activities of the protein phosphatase 2C (PP2C) phosphatase family member, PPM1A. We show that PPM1A directly dephosphorylated RelA at residues S536 and S276 and selectively inhibited NF-κB transcriptional activity, resulting in decreased expression of monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 and interleukin-6, cytokines implicated in cancer metastasis. PPM1A depletion enhanced NF-κB-dependent cell invasion, whereas PPM1A expression inhibited invasion. Analyses of human expression data revealed that metastatic prostate cancer deposits had lower PPM1A expression compared with primary tumors without distant metastases. A hematogenous metastasis mouse model revealed that PPM1A expression inhibited bony metastases of prostate cancer cells after vascular injection. In summary, our findings suggest that PPM1A is a RelA phosphatase that regulates NF-κB activity and that PPM1A has tumor suppressor-like activity. Our analyses also suggest that PPM1A inhibits prostate cancer metastases and as neither gene deletions nor inactivating mutations of PPM1A have been described, increasing PPM1A activity in tumors represents a potential therapeutic strategy to inhibit NF-κB signaling or bony metastases in human cancer.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PPM1A is a RelA phosphatase. (a) U2OS and HeLa cells were transfected with indicated siRNAs. PPM1A as well as endogenous phospho- and total RelA were visualized by immunoblotting. GAPDH served as loading control. (b) U2OS cells were transfected with indicated plasmids and proteins visualized by immunoblotting with GAPDH serving as loading control. (c) Wild-type (wt) mouse embryonic fibroblasts (MEFs) and IKKα−/−IKKβ −/− double-null MEFs were transfected with indicated plasmids. Endogenous IKKα and IKKβ and transfected phospho- and total RelA, as well as PPM1A, were visualized by immunoblotting. (d) Immunoprecipitated (IP) full-length Flag-RelA was used as substrate for in vitro phosphatase assay. Phospho- and total RelA were visualized by immunoblotting. Exclusion of PPM1A or magnesium in the phosphatase buffer served as negative controls. (e and f) S536- and S276-specific phosphorylated RelA peptides were incubated with equal amounts of glutathione S-transferase (GST)-PPM1A or His-Wip1 and free phosphate measured by malachite green assay. OD, optical density; PPM1A-PD, phosphatase dead PPM1A.
Figure 2
Figure 2
PPM1A inhibits NF-κB transcription activity, NF-κB-dependent cell invasion and sensitizes cells to TNFα. (a) U2OS cells were co-transfected with indicated plasmids and Renilla luciferase and the NF-κB-responsive reporter 3κB-ConA-LUC firefly luciferase. In this and subsequent luciferase assays, firefly luciferase activity was normalized to Renilla luciferase and normalized firefly luciferase activity from cells transfected with control plasmids was assigned a value of 1. Error bars, s.d. derived from three analyses. **P<0.01. (b) NF-κB-dependent luciferase activity was determined as described in a in cells expressing wild-type (wt) or phosphatase dead PPM1A and either ectopic expression of RelA or 4 h treatment with TNFα or IL-1β; **P<0.01. (c) Luciferase activity was determined following transfection of U2OS with plasmids encoding wt or mutant RelA (S536D or S276D) with or without PPM1A as indicated; **P<0.01. (d) Quantitative real-time PCR (q-RT-PCR) analyses of MCP-1 expression in HeLa cells selected after infection of PPM1A short hairpin RNA (shRNA) encoding lentivirus. Error bars = s.d.; *P<0.05. PPM1A, pS536RelA and RelA levels were confirmed by immunoblotting. (e) ChIP assays of the binding of RelA to MCP-1 proximal promoter. Samples from HeLa cells as described in d were prepared and analyzed using antibodies specific for RelA or immunoglobulin G (IgG) as control. Immunoprecipitated (IP) DNA fragments and input DNA were analyzed by real-time PCR. Values were normalized to input DNA in each group. Error bars = s.d.; *P<0.05. (f) HeLa cells infected with indicated viruses were plated on transwells with matrigel and invasion measured by direct counting of trespassed cells. Representative photomicrographs are shown (scale bars, 100 µm). Quantification of cell invasion in results represents cell counts from 10 randomly selected low-powered fields (× 200); **P<0.01. PPM1A and kBα-super-repressor (IκBα-SR) expression levels were confirmed by immunoblotting (Supplementary Figure S2D). (g) HT1080 cells were infected as indicated and treated with indicated doses of TNFα for 24 h. Cells were stained with Trypan blue and live cells were counted. Error bars = s.d.; *P<0.05.
Figure 3
Figure 3
PPM1A does not interfere with nuclear translocation or DNA binding of RelA. (a) U2OS cells transfected with indicated plasmids were lysed and total or p-RelA visualized by immuno-blotting from total lysate or subcellular fractions. Nucleolin and β-actin served as nuclear and cytosol controls, respectively. Amount of protein loaded is indicated. (b) Following TNFα stimulation, nuclear extracts were prepared from HeLa cells stably expressing PPM1A, IκBα-super-repressor (IκBα-SR) or control. RelA DNA-binding activity was measured using TransAM NF-κB p65 transcription factor kit. Error bars = s.d. Wt, wild type.
Figure 4
Figure 4
PPM1A inhibits NF-κB and prostate cancer cell invasion. (a) Endogenous total and p-RelA (S536) and PPM1A were visualized in LNCaP and PC3 cells by immunoblotting. GAPDH serves as loading control. (b) LNCaP cells were transfected with indicated siRNA and indicated phospho- or total proteins visualized by immunoblotting. (c) MCP-1 expression was measured by quantitative real-time PCR (q-RT–PCR) in LNCaP cells transfected as indicated. Error bars = s.d.; *P<0.05; (d) Transwell invasion of LNCaP cells following transfection with indicated constructs were quantified as described in Figure 2f; **P<0.001. (e) PC3-LUC cells were infected with indicated retrovirus, selected using puromycin and expression of indicated phospho- or total proteins visualized by immunoblotting. (f) PC3-LUC cells were infected with indicated retroviruses and expression of MCP-1 mRNA or protein, determined by q-RT–PCR or enzyme-linked immunosorbent assay, Error bars = s.d.; *P<0.05. (g) PC3-LUC cells were infected with indicated retroviruses and transwell invasion determined as in d; **P<0.001.
Figure 5
Figure 5
PPM1A expression is lower in metastatic human prostate cancer and inhibits a model of prostate cancer cells metastasis. (a) A Gene Expression Omnibus search indicated that PPM1A expression is lower in metastatic deposits of prostate cancer compared to: primary prostate cancers without distant metastases (P= 2.43E — 9, fold change 1.82); normal prostate tissue adjacent to cancer (P= 9.77E — −14, fold change 2.00); or normal prostate (P=2.17E –7, fold change 2.06). (b) Percentage of mice developed metastases. On top is the number of animals that develop metastases/the number of animals successfully injected; *P<0.05. (c) The number of metastases in each mouse; *P<0.05. (d) Xenogen bioluminescent images of representative mice 4 weeks after intracardiac injection of PC3-LUC cells infected with control retrovirus or retrovirus driving expression of PPM1A. (e) X-ray images of representative mice in b. Lytic bone lesions are indicated by white arrows. (f) Representative hematoxylin/eosin staining of bone and adjacent tissue samples obtained from mice in b are shown. Magnification: top, × 40; bottom, × 200. (g) Sections of bony metastases described in b were immunostained with cleaved-caspase-3. Percentage of positive staining cells in each group was measured from at least three representative tumors and in each tumor at least five randomly chose fields (× 200). Error bars, s.d.; **P<0.001.
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