Inactivation of oncogenic cAMP-specific phosphodiesterase 4D by miR-139-5p in response to p53 activation

Increasing evidence highlights the important roles of microRNAs in mediating p53’s tumor suppression functions. Here, we report miR-139-5p as another new p53 microRNA target. p53 induced the transcription of miR-139-5p, which in turn suppressed the protein levels of phosphodiesterase 4D (PDE4D), an oncogenic protein involved in multiple tumor promoting processes. Knockdown of p53 reversed these effects. Also, overexpression of miR-139-5p decreased PDE4D levels and increased cellular cAMP levels, leading to BIM-mediated cell growth arrest. Furthermore, our analysis of human colorectal tumor specimens revealed significant inverse correlation between the expression of miR-139-5p and that of PDE4D. Finally, overexpression of miR-139-5p suppressed the growth of xenograft tumors, accompanied by decrease in PDE4D and increase in BIM. These results demonstrate that p53 inactivates oncogenic PDE4D by inducing the expression of miR-139-5p. DOI: http://dx.doi.org/10.7554/eLife.15978.001

Introduction microRNAs (miRNAs) represent a class of cellular short non-coding RNAs responsible for modulating the expression of their target genes at the post-transcriptional level. Abnormal regulation of miRNAs is associated with human cancer development and staging (Lu et al., 2005). Over the past decade, increasing attention has been drawn to the role of miRNAs in p53 signaling network, and a number of miRNAs have been identified as p53 target genes (Liao et al., 2014a). These miRNAs are involved in multiple biological processes, including cell cycle arrest, apoptosis, glycolysis and so on. Also, they often connect p53 with other signaling pathways (Christoffersen et al., 2010;Sachdeva et al., 2009;Liang et al., 2013). Although miRNAs have been appreciated as important mediators of p53's tumor suppression functions, a lot still remain unexplored to better understand the fine-tuning of p53 signaling and crosstalk with other pathways by these RNAs. In this study, we identified miR-139-5p as a novel p53 target gene and demonstrated a new pathway connecting p53 and miR-139-5p with an oncogenic protein PDE4D as a new target of this miRNA and its downstream cAMP signaling.

miR-139 is a direct p53 target gene
After carefully analyzing the genomic sequence of the miR-139 gene using p53MH algorism (Hoh et al., 2002), we found a highly conserved putative p53 responsive element (RE) located at the 5' flanking region ( Figure 2A). To test if endogenous p53 binds to this p53RE sequence, we conducted a chromatin-associated immunoprecipitation (ChIP) assay after treating H460 or HCT116 p53 +/+ cells with 0.5 mM Doxorubicin for p53 induction. In both cell lines, the binding of p53 with the p53RE was dramatically increased upon Doxorubicin treatment as compared to non-treatment control, indicated by p53RE sequence pulled down with p53 specific antibody DO-1, but not nonspecific immunoglobulin G ( Figure 2B). Also, we assessed luciferase expression driven by either a wild type or a mutant-p53RE-motif-containing miR-139 sequence ( Figure 2C) in H1299 cells. GFP-p53 markedly induced luciferase activity in a dose-dependent manner when wild type miR-139 p53RE, but not the mutant p53RE, was used ( Figure 2D). These results clearly show that p53 binds to the miR-139 promoter region and thus regulates the transcription of miR-139.
p53 regulates PDE4D via miR-139-5p By using the online microRNA target prediction tool (Maragkakis et al., 2009), we searched for potential RNA targets of miR-139-5p. After screening several candidates, PDE4D turned out to be eLife digest The human body is kept mostly free from tumors by the actions of so-called tumor suppressor genes. One such gene encodes a protein called p53, which prevents tumors from growing by regulating the activity of many other genes that either inhibit cell growth or cause cells to die. For example, p53 regulates genes that encode short molecules called microRNAs, which in turn suppress the activity of other target genes.
Although a number of microRNAs have been reported as p53-regulated genes, there are still more to find. Discovering these genes would in turn help researchers to better understand exactly how p53 acts to suppress the growth of tumors, and to treat cancers caused by mutations in this tumor suppressor gene.
Cao, Wang et al. now discover a new microRNA -called miR-139-5p -as one that is activated by p53 in human cells. Colon tumors produce much lower levels of this microRNA than normal tissues, while the cancer cells with a higher level of miR-139-5p grow slower than do the cancer cells with less miR-139-5p. Further experiments showed that this is because miR-139-5p can suppress the production of a protein called PDE4D, which is often highly expressed in human cancers. The suppression of PDE4D by this microRNA results in an increase in the levels of a protein that can cause cancer cells to die. Cao, Wang et al. suggest that miR-139-5p and PDE4D form part of a signaling pathway that plays an important role in suppressing the growth of colon cancer cells. Since microRNAs often have more than one target, future studies could explore if miR-139-5p regulates the production of other cancer-related proteins as well.
In line with the above results, activation of p53 by doxorubicin in H460 and A549 cells significantly reduced the protein levels of PDE4D ( Figure 3E, left panel). Notably, the PDE4D variants with smaller molecular weight have been reported to possess stronger oncogenic activities due to the lack of functional inhibitory domains as compared to the longer forms (Lin et al., 2013). Nevertheless, miR-139-5p transfection and doxorubicin treatment led to similar expression inhibition of both the long and the short variants of PDE4D, indicating that this newly identified pathway has broader inhibition on PDE4D. In contrast, knocking down p53 elevated PDE4D expression in these two cell lines ( Figure 3E, right panel). We then assessed the effect of miR-139-5p on PDE4D in PC-3, a p53 null cell line, and found similar reduction of PDE4D by miR-139-5p mimic (Figure 3-figure supplement 2). To further validate the regulatory role of p53 on PDE4D, we also treated HCT116 p53+/+ and HCT116 p53-/-cells with 10 mM Nutlin-3 (Nut), which disrupts MDM2-p53 interaction and therefore activates p53 (Vassilev et al., 2004), and 5 nM Actinomycin D (ActD), which causes ribosomal stress-mediated p53 activation (Iapalucci-Espinoza and Franze-Fernández, 1979), and found PDE4D was reduced by both of these two drugs only in p53 positive, but not p53 negative, cells Furthermore, doxorubicin inhibited pMIR-PDE4D activity, but introduction of miR-139-5p inhibitor reversed this inhibition ( Figure 3F). Consistently, the suppression of PDE4D protein by doxorubicin was also compromised in the presence of miR-139-5p inhibitor ( Figure 3G). Collectively, these findings demonstrate that activation of p53 can induce the expression of miR-139-5p that in turn suppresses the expression of oncogenic protein PDE4D.

miR-139-5p induces cellular cAMP and BIM-mediated cell growth arrest
Since PDE4D is a cAMP specific phosphodiesterase, ectopic expression of miR-139-5p in A549 cells led to significant increase of cellular cAMP levels ( Figure 4A). Also, consistent with our results shown in Figure 3F and Figure 3G, doxorubicin treatment increased cellular cAMP levels in A549 cells, which were alleviated by the miR-139-5p inhibitor (Figure 4-figure supplement 1). In addition, Nutlin-3, a more specific p53-activating reagent, showed similar effect on cAMP level, which was also reversed in the presence of miR-139-5p inhibitor ( Figure 4B). In contrast, in H1299 cells, which are p53-null and express non-detectable PDE4D, neither doxorubicin nor miR-139-5p affected cellular cAMP level (Figure 4-figure supplement 2). Notably, the rescue effect of miR-139-5p on A549 cell growth inhibition by Nutlin-3 was correlated with the change of cAMP levels ( Figure 4-figure  supplement 3). These data indicate that p53 activation could modulate cAMP levels through miR-139-5p.
Depletion of PDE4D was previously reported to induce BIM-mediated apoptosis through activation of the cAMP pathway (Lin et al., 2013;Zambon et al., 2011). We found that introduction of miR-139-5p into H460 and A549 cells dramatically increased BIM protein expression ( Figure 4C). As expected, treatment with 10 mM Nutlin-3 also induced BIM expression in HCT116 p53 +/+ , but not HCT116 p53 -/-, cells (Figure 4-figure supplement 4), supporting that BIM induction is p53-dependent. Consequently, A549 cell growth was significantly inhibited by miR-139-5p or Nutlin-3, both of which were attenuated by knocking down BIM using siRNA ( Figure 4D, Figure 4-figure supplement 5). Inversely, introduction of miR-139-5p inhibitor into A549 cells significantly alleviated cell growth inhibition by several p53 activating drugs, including INZ-C, Actinomycin D and Nutlin-3 Increased binding of p53 and the endogenous p53RE-containing miR-139 promoter in response to Doxorubicin. H460 or HCT116 p53+/+ cells were treated with 0.5mM Doxorubicin for 18 hr to stimulate the endogenous p53 before chromatin-associated immunoprecipitation assays were conducted with DO-1 p53 antibodies and the p53RE specific primers listed in Supplementary file 1 online. Western blot analysis was performed to confirm induction of p53. (C) Schematic of the pGL3 luciferase reporter constructs used. The plasmids either contain wild-type or mutant p53RE sequences of the miR-139 promoter, as underlined. (D). Enhancement of miR-139-promoter-driven luciferase activity by p53. H1299 cells were co-transfected with pGL3-139 or pGL3-139mut and increasing amount of GFP-p53 and collected 48 hr after transfection for assessment of luciferase activity, which was normalized against b-gal expression. Western blot was also performed to confirm the expression of GFP-p53. Error bars represent standard deviation (n = 3). b-gal, b-galactosidase; Ctrl, control; Dox, Doxorubicin; IgG, immunoglobulin G; mut, mutant. DOI: 10.7554/eLife.15978.004 Overexpression of miR-139-5p specifically inhibits luciferase activity from the plasmid harboring a wild-type, but not a mutant, miR-139-5p targeted sequence. H1299 cells were co-transfected with the indicated plasmids and collected 48 hr after transfection for luciferase assay. Luciferase activity was measured and normalized against b-gal expression. Even amount of oligos was achieved by adding Negative control oligos accordingly. (E) p53 modulates PDE4D expression. H460 and A549 cells were collected for Western blot analysis 18 hr after treatment with Doxorubicin (left panel) or 48 after transfection with si-SC or si-p53 (right panel). (F and G) p53 modulation of PDE4D expression is through miR-139-5p. H460 cells were co-transfected with indicated plasmids and 24 hr later were treated with Doxorubicin for 18 hr followed by measurement of luciferase activity (F) or Western blot analysis (G). 139-5p m, miR-139-5p mimic; 139-5p Inh, miR-139-5p inhibitor. Cells were treated with solvent for Dox or transfected with the same concentration of negative control oligos as miR-139-5p mimic or inhibitor. Error bars represent standard deviation (n = 3). *p<0.05. DOI: 10.7554/eLife.15978.005 The following figure supplements are available for figure 3: ( Figure 4-figure supplements 3 and 6). These results demonstrate that in response to p53 activation, increased miR-139-5p induces BIM-mediated cell growth arrest via the PDE4D/cAMP pathway.

miR-139-5p is negatively correlated with PDE4D in human and xenograft colorectal tumors
In order to determine the clinical relevance of miR-139-5p regulation of PDE4D, we obtained 50 paired human colon tumor specimens and their adjacent normal tissues and conducted qRT-PCR analysis on these specimens. miR-139-5p expression was significantly lower while PDE4D was higher in these tumor specimens than that in their normal tissues ( Figure 5A). Pearson's correlation analysis of the expression results from the tumor specimens and normal tissues revealed that miR-139-5p is inversely correlated with PDE4D expression ( Figure 5A, bottom panel). These results provide clinical evidence supporting miR-139-5p as a negative regulator of PDE4D, consistent with our above results.
To validate this clinical correlation, we established a xenograft model using SW480 cells stably expressing either scramble oligos (Control) or miR-139-5p. As expected, miR-139-5p expressing tumors grew significantly slowly as compared to control tumors starting at day 16 after inoculation ( Figure 5B). The difference of miR-139-5p expression in these two groups of tumors was comparable to that observed in human specimens ( Figure 5C vs Figure 5A). Immunohistochemistry analysis revealed that in the miR-139-5p overexpressing tumors, PDE4D expression was markedly repressed, while tumor cell proliferation was significantly inhibited as reflected by Ki67 staining (Figure 5D and  . Consistent with our in vitro observation, BIM expression was also elevated in miR-139-5p tumors ( Figure 5E). These findings suggest that the tumor suppressor role of miR-139-5p is likely attributed to its regulation of the PDE4D/BIM pathway.
In summary, this study for the first time demonstrates that p53 can induce the expression of miR-139-5p (Figure 1 and Figure 2), which in turn suppresses the expression of an oncogenic protein PDE4D ( Figure 3) and leads to cAMP/BIM-mediated cell growth arrest (Figure 4). Significantly, miR-139-5p expression is negatively correlated with PDE4D in human colorectal tumor and normal tissues, and overexpression of miR-139-5p is associated with slower tumor growth in the xenograft model, which is accompanied with PDE4D suppression, BIM induction and cell proliferation inhibition ( Figure 5). As a potential tumor suppressor, miR-139 was previously shown to be downregulated in human hepatocellular carcinoma and colorectal cancer with characterized targets including Rho-kinase 2, IGF-IR and RAP1B (Guo et al., 2012;Shen et al., 2012;Wong et al., 2011). Here, we identified PDE4D, an oncogenic protein that is upregulated in various human cancers (Lin et al., 2013), as another novel target of this miRNA. Inhibition or depletion of PDE4D significantly induces apoptosis and inhibits proliferation of cancer cells (Lin et al., 2013;Ogawa et al., 2002;Rahrmann et al., 2009). Notably, the oncogenic property of PDE4D involves the cAMP/BIM pathway (Lin et al., 2013;Zambon et al., 2011). cAMP is an important secondary messenger mediating diverse cellular processes with protein kinase A as its main effector (Taskén and Aandahl, 2004). In particular, lower cAMP levels favor cancer cell survival and proliferation, which can be reversed by inhibition of PDE4D, the cAMP specific phopsphodiesterase (Goldhoff et al., 2008;Lin et al., 2013;Murata et al., 2000;Ogawa et al., 2002). The tumor suppressor role of p53 has been extensively documented over the last two decades, and is highly attributable to its regulation of target genes involved in cell cycle arrest, apoptosis and senescence (Bieging et al., 2014;Levine, 2011). More recent discoveries revealed that p53 is also a critical mediator of metabolic pathways that are important for tumor survival (Bieging et al., 2014;Jiang et al., 2015;Wang and Gu, 2014). Based on our findings, we propose a p53-miR-139-5p-PDE4D-cAMP-BIM pathway as a novel pathway that can mediate p53's tumor suppression function to modulate cellular cAMP levels by inhibiting PDE4D expression via miR-139-5p, and deregulation of this pathway would be highly associated with tumorigenesis ( Figure 5F).

Quantitative real-time PCR (qRT-PCR)
qRT-PCR for mature microRNAs was carried out by using the methods as described previously (Tang et al., 2006). qRT-PCR for other genes were conducted using the protocol as described before . Relative gene expression was calculated using the DDCT method. All reactions were carried out in triplicate.

Transient transfection and immunoblot
As described previously (Jin et al., 2008), briefly, cells were transfected with plasmids as indicated in each figure by using TurboFect (Thermo Scientific, Waltham, MA) and following the company's manuals. Cells were harvested and lysed in lysis buffer 48 hr post transfection. The total protein concentrations were determined using a BioRad protein assay kit and equal amounts of total proteins (50 mg, otherwise indicated specifically) were then subjected to SDS-PAGE, followed by WB with antibodies as indicated in each figure.
Knockdown of the endogenous miRNA, p53 and BIM Hsa-mir-139-5p mimic and Negative control were purchased from Gene Pharma (Shanghai, China). Anti-miR miRNA Inhibitor and Anti-miR miRNA Inhibitors-Negative Control were purchased from Ambion. p53 siRNA was purchased from Santa Cruz. Two BIM siRNA (siBIM-1, ID: 19,474 and siBIM-2, ID: 195012) were purchased from Ambion. Transfection of miRNA inhibitors was performed using the same method as that for normal siRNA as described previously .

Luciferase reporter assays
Cells were transfected with pCMV-b-galactoside and indicated plasmids (total plasmid DNA 1 mg/ well) as indicated in figures. Luciferase activity was determined and normalized by a factor of b-gal activity in the same assay as described previously (Jin et al., 2006).

ChIP-PCR
ChIP analysis was performed as described previously using p53 (DO-1) antibodies for endogenous p53 (Liao and Lu, 2013;Liao et al., 2014b). Immunoprecipitated DNA fragments were analyzed by quantitative real-time PCR (qRT-PCR) amplification using primers for miR-139 gene. The primers are listed in Supplementary file 1 online.

Cellular cAMP measurement
cAMP-GloTM Assay (Promega, Madison, WI) was performed to measure the cellular cAMP concentration as per manufacturer's instructions.

Tissue specimens
Fifty paired colorectal tumors and adjacent non-tumor tissues were collected and histopathologically diagnosed at the Departments of Gastrointestinal Surgery and Pathology, the First Affiliated Hospital, Sun Yat-sen University. Patient consent and Institutional Research Ethics Committee approval were obtained prior to the use of these clinical materials for research purposes.
Xenograft SW480 cells (1 Â 10 7 ) stably expressing pEZX-scramble control sequence (Vector) or pEZX-miR-139-5p were inoculated subcutaneously into the right flank of female BALB/c nude mouse (four weeks old, n = 6 per group). The tumor volume was measured every three days and calculated as 0.524 Â length Â width 2 (Gleave et al., 1992). At the conclusion of the experiments, tumors were removed and fixed in 10% formalin for paraffin embedding and histological analysis, or flash-frozen in liquid nitrogen for Western blot and qRT-PCR analysis. All experimental procedures were approved by the Medical Ethical Committee of the First Affiliated Hospital, Sun Yat-sen University (Guangzhou, China). H & E staining and immunohistochemistry were described previously (Cao et al., 2013;Zhang et al., 2013). Quantitative analysis of IHC staining was achieved by categorizing the staining intensity to low, medium and high as determined by ImageJ software (NIH).

Statistical analysis
The Student's two-tailed t test was used to compare the mean differences between treatment and control groups, unless otherwise indicated. Data are presented as Mean ± SD (standard deviation). p<0.05 was determined as statistically significant.