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

  1. Bo Cao
  2. Kebing Wang
  3. Jun-Ming Liao
  4. Xiang Zhou
  5. Peng Liao
  6. Shelya X Zeng
  7. Meifang He
  8. Lianzhou Chen
  9. Yulong He
  10. Wen Li  Is a corresponding author
  11. Hua Lu  Is a corresponding author
  1. Tulane University School of Medicine, United States
  2. The First Affiliated Hospital, Sun Yat-Sen University, China
  3. The First Affiliated Hospital, Sun Yat-sen University, China
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Cite as: eLife 2016;5:e15978 doi: 10.7554/eLife.15978

Abstract

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.

https://doi.org/10.7554/eLife.15978.001

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.

https://doi.org/10.7554/eLife.15978.002

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.

Results and discussion

Identification of miR-139 as a novel p53 target gene

In order to identify potential p53 target microRNAs, we used colon cancer cell lines, HCT116 p53+/+ and HCT116 p53-/-, the latter of which were genetically engineered to lose the expression of wild type (WT) p53 (Bunz et al., 1998). Both cells were treated with 4 μM Inauhzin-C (INZ-C), which is a p53 activating compound discovered in our lab (Zhang et al., 2012). After confirmation of p53 and its targets induction through Western blot (Figure 1A) and quantitative real-time PCR (qRT-PCR) (Figure 1B), the total RNA was extracted and sent to ArrayStar for miRNA-sequencing analysis. The results revealed that in addition to known p53 target microRNAs, such as miR-34a, miR-1246 and miR-143 (Liao et al., 2014a; Suzuki et al., 2009), miR-139 was also significantly induced in HCT116 p53+/+, but not HCT116 p53-/-, cells, suggesting miR-139 as a potential p53 target (Figure 1C). We independently confirmed this observation by detecting miR-139-5p expression after treating HCT116 p53+/+/HCT116 p53-/- and H460 (WT p53)/H1299 (p53 null) cells with DMSO or 4 μM INZ-C. miR-139-5p was significantly induced only in p53 positive, but not in p53 null, cells (Figure 1D). In contrast, p53 knockdown decreased miR-139-5p expression by more than 50% in H460 and U2OS cells (Figure 1E). These data indicate that miR-139-5p is a possible p53 target gene.

Identification of miR-139 as a novel p53-responsive gene.

Validation of induction of p53 and its targets gene. HCT116 p53+/+ and HCT116 p53-/- cells were treated with DMSO or 4uM INZ-C for 18 hr in duplicate and the protein samples and RNA samples were prepared for Western Blot (A) and qRT-PCR (B), respectively. (C). microRNA sequencing data analysis shows significantly induced microRNAs uniquely or commonly in HCT116 p53-/- and HCT116 p53+/+ cells. miR-139 was highlighted in red among other known p53 targets. (D). Induction of miR-139-5p by p53. p53 positive (H460, HCT116 p53+/+) and p53 negative (H1299, HCT116 p53-/-) cells were treated as in (A) and (B), and qRT-PCR analysis followed. (E). Decrease of miR-139-5p by knocking down p53. H460 and U2OS cells were transfected with scramble siRNA (SC) or siRNA specific to p53 (p53) and subjected to Western blot and qRT-PCR analysis.

https://doi.org/10.7554/eLife.15978.003

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 μM 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 non-specific 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.

miR-139 is a direct target of p53.

(A) Diagram shows putative p53 responsive element (p53RE) located upstream of miR-139 gene. (B) Increased binding of p53 and the endogenous p53RE-containing miR-139 promoter in response to Doxorubicin. H460 or HCT116 p53+/+ cells were treated with 0.5μM 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 β-gal expression. Western blot was also performed to confirm the expression of GFP-p53. Error bars represent standard deviation (n = 3). β-gal, β-galactosidase; Ctrl, control; Dox, Doxorubicin; IgG, immunoglobulin G; mut, mutant.

https://doi.org/10.7554/eLife.15978.004

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 an ideal target as its 3’-untranslated region (3’-UTR) contains miR-139-5p targeting sequence (Figure 3A). Overexpression of miR-139-5p mimic markedly reduced the expression of PDE4D in H460 and A549 cells (Figure 3B). The type-I insulin-like growth factor receptor (IGF-IR), a previously reported miR-139-5p target (Shen et al., 2012), was also decreased by miR-139-5p mimic, indicating the activity of miR-139-5p used here (Figure 3—figure supplement 1). PDE4D belongs to the family of phosphodiesterases and is a cyclic AMP (cAMP) specific phosphodiesterase with several splice variants (Omori and Kotera, 2007). It is an oncogenic protein that regulates cancer cell proliferation, angiogenesis and apoptosis (Lin et al., 2013; Ogawa et al., 2002; Pullamsetti et al., 2013Rahrmann et al., 2009). To further test whether the downregulation of PDE4D by miR-139-5p is through direct regulation on its mRNA, we constructed the WT or mutant predicted miR-139-5p target sites into the pMIR-Report system that contains the luciferase reporter gene subjected to regulation mimicking the microRNA target (Figure 3C). When co-transfecting H1299 cells with miR-139-5p, only the pMIR-PDE4D-WT, but not the pMIR-PDE4D-mutant, displayed suppressed expression (Figure 3D), suggesting that miR-139-5p regulates PDE4D expression by directly binding to the target sequence at 3’-UTR of PDE4D mRNA.

Figure 3 with 3 supplements see all
p53 modulates PDE4D expression via miR-139-5p.

(A) Bioinformatic analysis shows the miR-139-5p-targeted 3’-untranslated region (3’UTR) sequence of PDE4D mRNA. (B) Overexpression of miR-139-5p decreases the level of PDE4D protein in cells. H460 and A549 cells were collected 48 hr after transfection with miR-139-5p mimic for Western blot analysis. (C) Schematic of the pMIR–PDE4D luciferase reporter constructs used, which contain either a wild-type or a mutant miR-139-5p target site derived from the PDE4D mRNA. (D) 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 β-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.

https://doi.org/10.7554/eLife.15978.005

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 μM 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 (Figure 3—figure supplement 3). These results suggest that this suppression of PDE4D is p53 dependent in response to various stresses.

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.

Figure 4 with 6 supplements see all
miR-139-5p induces cAMP/BIM mediated cell growth arrest.

(A). Overexpression of miR-139-5p increases cellular cAMP levels. A549 cells were transfected with miR-139-5p mimic control or miR-139-5p mimic and were subjected to cAMP measurement 48 hr after transfection. (B). Nutlin-3 increases cellular cAMP levels via miR-139-5p. A549 cells were transfected with miR-139-5p inhibitor control or miR-139-5p inhibitor for 48 hr followed by 10 μM Nutlin-3 treatment for 18 hr and subjected to cAMP measurement. (C). Overexpression of miR-139-5p induces BIM expression. H460 and A549 cells were transfected with miR-139-5p mimic and Western blot performed 48 hr after transfection. (D). Knockdown of BIM rescues miR-139-5p induced cell growth arrest. A549 cells were transfected with miR-139-5p mimic or siRNA against BIM (si-BIM) or both and subjected to MTT assay 48 hr after transfection. Cells were treated with solvent for Nutlin-3 or transfected with the same concentration of negative control oligos as miR-139-5p mimic or BIM siRNA. Error bars represent standard deviation (n = 3). *p<0.05 as compared to negative control oligos. Δ, p<0.05.

https://doi.org/10.7554/eLife.15978.009

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 μM 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 (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.

Figure 5 with 1 supplement see all
miR-139-5p is negatively correlated with PDE4D expression in human colorectal tumor samples, and represses the growth of SW480 xenograft tumors.

(A) miR-139-5p is negatively correlated with PDE4D expression in colon tumor samples. miR-139-5p (top panel) and PDE4D (middle panel) RNA expression was determined in 50 tumors (T) and paired adjacent normal tissues (ANT). The Pearson’s correlation of miR-139-5p and PDE4D RNA expression was analyzed combining tumor samples and normal tissues (bottom panel). (B) The sizes of SW480 xenograft tumors stably overexpressing pEZX-control (Vector) or pEZX-miR-139-5p (miR-139-5p) were measured every three days starting at seven days after inoculation. Mean tumor sizes were presented. Error bar, SD; *p<0.05. (C) Expression of miR-139-5p of xenograft tumors was determined by qRT-PCR. (D) Representative images of the xenograft tumors stained with hematoxylin and eosin (H&E), or immunohistochemistry analyzed with PDE4D or Ki67 antibody. (E) Xenograft tumors were subjected to Western blot analysis of BIM expression. (F) Proposed model of the p53/miR-139-5p/PDE4D pathway.

https://doi.org/10.7554/eLife.15978.016

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 Figure 5—figure supplement 1). 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).

Materials and methods

Cell lines

Human HCT116 p53+/+ (RRID: CVCL_0291) and HCT116 p53-/- (RRID: CVCL_S744) cells were generous gifts from Dr. Bert Vogelstein at the John Hopkins Medical Institutes. A549 (RRID: CVCL_0023), HepG2 (RRID: CVCL_0027), U2OS (RRID: CVCL_0042), H460 (RRID: CVCL_0459) and H1299 (RRID: CVCL_0060) cells were purchased from American Type Culture Collection (ATCC). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and PC3 (RRID: CVCL_0035) cells (also from ATCC) in RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C in 5% CO2. STR profiling was performed to ensure cell identity. No mycoplasma contamination was found.

Plasmids and antibodies

The pGL3-miR-139 luciferase reporter plasmid was constructed from the miR-139 promoter with primers as listed in Supplementary file 1. The fragment was inserted into pGL3 at the MluI and XhoI sites. The pGL3-miR-139-mut was generated by site-directed mutagenesis with primers listed in Supplementary file 1 using pGL3-miR-139 as template. The pMIR-PDE4D and pMIR-PDE4D mutant plasmids were constructed by inserting the miR-139-5p-targeted PDE4D mRNA-coding sequence or its mutant into the pMIR vector (Ambion) at the SpeI and HindIII sites. The pSIF-H1-miR-139-5p was constructed by inserting annealed oligos as listed in Supplementary file 1 into the pSIF-H1 vector at BamHI and EcoRI sites, as per manufacturer’s instruction. The anti-p21 (Thermo Fisher Scientific, Waltham, MA, RRID: AB_10986834), anti-p53 (DO-1, Santa Cruz, Dallas, TX, RRID: AB_628082), anti-PDE4D (Aviva Systems Biology, San Diego, CA, RRID: AB_10879817), and anti-BIM (Cell Signaling, Danvers, MA, RRID: AB_10692515) antibodies used here were commercially purchased. The anti-MDM2 (2A10 and 4B11) antibodies were described previously (Jin et al., 2002).

Chemicals

Inauhzin-C (INZ-C) was reported previously (Zhang et al., 2012). Doxorubicin (Dox) was purchased from Sigma-Aldrich.

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 (Sun et al., 2008). Relative gene expression was calculated using the ΔΔCT 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 μg, 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 (Sun et al., 2008).

Luciferase reporter assays

Cells were transfected with pCMV-β-galactoside and indicated plasmids (total plasmid DNA 1 μg/well) as indicated in figures. Luciferase activity was determined and normalized by a factor of β-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 × 107) 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 × length × width2 (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.

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Decision letter

  1. Kevin Struhl
    Reviewing Editor; Harvard Medical School, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after two rounds of review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for sending your work entitled "Inactivation of Oncogenic cAMP-specific Phosphodiesterase 4D by miR-139-5p in Response to p53 Activation" for consideration at eLife. Your article has been evaluated by a Senior editor and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers found the work potentially quite interesting, but raised a large number of concerns, all of which would need to be addressed successfully to make the paper suitable, after re-review, for eLife.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

The authors performed a differential expression analysis to look at p53 regulated micro RNAs in HCT116 cells that were treated with Inauhzin-C (INZ-C) and identified miR-139-5p as a new micro RNA p53 target. Their data show quite convincingly that this is a real p53 target since it is induced by Doxorubicin and INZ-C in a p53-dependent manner, that the miR-139-5p gene contains a p53 binding site to which Dox-induced p53 binds (by ChIP analysis) and that this sequence can serve as a p53 response element in a luciferase reporter assay. The authors go further and use computational analysis to identify a target of miR-139-5p as cAMP-specific phosphodiesterase4D (PDE4D) and in Figure 3 show that a mimic of miR-139-5p leads reduces expression of PDE4D while and inhibitor of miR-139-5p increased the levels of PDE4D. PDE4D is known to increase conversion of cAMP to AMP and in Figure 4 the authors show increased levels of cAMP upon introduction of the miR-139-5p mimic, and corresponding increased levels of Bim, shown previously to be downstream of cAMP mediated cell death. Finally, they show results from a clinical colon cancer patient dataset indicating a reciprocal relationship between miR-139-5p and PDE4D levels.

Identifying novel target of p53 is an important area of study, especially when these targets provide new insights into the mechanism of action of p53. Thus, the focus of this study could be appropriate for eLife if the authors were to provide more information as outlined in the comments below. The problem with this study, however, is that the authors do not have enough information as to whether, how or when the induction of miR-139-5p by p53 actually plays a role in any p53 outcomes in cells, in animals or in human cancer. There is no evidence for this newly described process in a physiological or pathological setting in vivo. For publication in a journal, such as eLife, some demonstration of the importance of this process in vivo needs to be provided. The authors would need to produce extensive and convincing data to allay the above concerns as well as the following more specific comments:

1) Figure 3. What is the relative concentration of the mature endogenous miR-139-5p when induced by p53 compared to the concentration of the miR-139-5p mimic? This is important because it is not clear whether p53 induction of endogenous miR-139-5p is sufficient to cause enough of a drop in PDE4D to have any impact on cAMP.

2) Figure 3G shows that there is a massive increase in the levels of PDE4D when the miR-139-5p inhibitor in introduced into cells even without Dox treatment. One would therefore expect that the levels of cAMP would be reduced. But in Figure 4B this is not the case. They only see an impact of the inhibitor on the levels that are increased by Dox treatment. Why is this?

3) Figure 4D: the authors show data indicating a modest but apparently convincing drop in cell viability upon introduction of the miR-139-5p mimic into cells. If BIM is the reason for this, then one would assume that knockdown of BIM would increase cell viability. But there does not appear to be any rescue by BIM knockdown in cells treated with the mimic and so their claim that the impact of miR-139-5p mimic is attenuated by BIM is not substantiated by the data. Understanding the biological consequences of this pathway is central to establishing this microRNA as an important target for p53. Knockdown of BIM is an important experimental approach. Multiple targeting sequences should be used for this knockdown to lessen the possibility of off-target effects. The authors also need to show that knockdown of BIM blocks some cellular outcome due to p53, presumably proliferation inhibition.

4) If p53 works via miR-139-5p to increase levels of cAMP after doxorubicin as the authors propose, they need to show this. Similarly, they need to show that the increase in BIM is p53 dependent. This is central to their conclusion.

5) While the authors show in Figure 4E an inverse correlation between miR-139-5p and PDE4D, is there a positive correlation between wild-type p53 and miR-139-5p in tumors?

6) Many of the differences shown, especially, the impact of mir-139-p on cell death and the impact of BIM knockdown on this cell death (Figure 4D) are very small. This questions the importance of this newly described process.

7) It is unclear whether the authors examined the p53 status in the clinical samples. Given the focus of the manuscript on p53 regulation, one would expect that p53 loss of mutation would be associated with higher levels of PDE4D. If this is not the case, the authors should comment on this.

8) The authors also need to address whether p53 is sufficient for these effects or do they only occur with DNA damage.

9) Along these lines as well, it should be tested whether the miR-139-5p inhibitor blocks this p53-dependent cellular outcome.

10) Although the authors cite examples from the published literature, it remains to be shown what is the nature of inhibition of proliferation that is seen. Use of additional assays beyond that of MTT would be informative in this regard.

11) The biological significance of the two bands for PDE4D that are seen should be discussed.

[Editors’ note: The second decision letter after peer review is shown below.]

Thank you for resubmitting your work entitled "Inactivation of Oncogenic cAMP-specific Phosphodiesterase 4D by miR-139-5p in Response to p53 Activation" for further consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. We regret to inform you that your work will not be considered further for publication in eLife.

While the referees indicate that your revised paper is of potential interest for eLife, the remaining issues are substantial, and both referees were concerned that you did not respond adequately to the previous critiques. If you can provide convincing data to address their concerns, your paper can be reconsidered for eLife. Alternately, as more experimentation would be required, you might want to submit your study to a more specialized journal.

Major critique:

In this revised paper the authors have responded to the most serious criticism that came from Reviewers 1 and 3, who made it clear that it did not go far enough to show physiological significance of the p53 target. The authors provide xenograft data, which are for the most part convincing and strengthen the paper. However, some of the previous concerns remain. If the authors can respond to these comments and provide better data where indicated this paper would be suitable for eLife. However, it should be noted that these are not new concerns, but rather points that the authors failed to properly address.

1) Most importantly, there was a concern about the biological relevance of the findings. The authors have now included a xenograft study. While this extends observations to an in vivo setting, they still have not shown that cellular outcomes are dependent upon this pathway. They state that there is Bim-mediated cell-cycle arrest and yet data was presented to argue that the outcome may in fact be apoptosis. It is not clear how BIM would induce a cell-cycle effect and the authors have not clearly stated which outcome they think is occurring in response to miR-139-5p regulation by p53. They note that BIM siRNA causes effects on cell viability and on the percent of cells with a hypodipold DNA content and yet the model in Figure 5F shows BIM causes a cell cycle effect. This is confusing.

2) As pointed out by reviewers 1 and 3 there are some cases (for example Figure 3D and Figure 4D) where effects were very modest and even if statistically significant. It is difficult to reconcile such modest effects with meaningful physiological outcomes.

3) Responses to the reviewers' comments were in some cases not really satisfactory. In one case (their point 3) they combine comments by two referees and respond to them as if they were one referee's comment. To this melded comment they provide data for the reviewers' eyes that 2 different siRNAs vs BIM have the same effect on Doxorubicin suppression of H460 cell proliferation. First, the "rescue" by BIM knockdown was very modest in the H460 cells. Second, the cells they use to show this are not the same that were in the paper in Figure 4D, where they used A549 cells. It does not seem very relevant to present data on a different assay in another cell line to make their point.

4) In another case their response to the comment "If p53 works via miR-139-5p to increase levels of cAMP as the authors propose, they need to show this". It was noted that cAMP levels do not track with outcomes. The authors' response was one of speculation rather than providing additional insights through experimental data.

5) Similarly, they need to show that the increase in BIM is p53 dependent. What they show is not very convincing. First, in Figure 4 doxorubicin is not really appropriate to use as a demonstration of p53-specific response because this drug has other p53-independent effects on cells, so Nutlin would be a better control. Second, in Figure 4—figure supplement 2, it is not appropriate to compare two cell lung cancer lines that differ in p53 status as a way to prove that BIM is induced in a p53-specific manner. They use wild-type p53 expressing H460 cells and compare these to p53-null H1299 cells. Yet these two cell lines differ in many more ways besides p53 status. An isogenically matched set of cells is needed to make this important point that the increase in BIM needs p53 under these conditions. Also the extent of induction is really very modest in the H460 cells. Either use the HCT116 pair of cells or knockdown p53 in the H460 cells.

Reviewer #1:

This revised study by Cao et al. has identified a miR139-5p as a gene that is transactivated by p53 in Figures 1 and 2. In Figure 3 they provide evidence that PDE4D is a target of miR139-5p. They show in Figure 4 that levels of cAMP a known target of PDE4D, are up in miR139-5p mimic-containing cells or in Doxorubicin-treated cells. Also BIM, previously shown to be upregulated by cAMP, is increased by the miR-139 mimic. Finally in new data in Figure 5, they provide data from mouse xenograft studies and show that SW480 cells harboring a construct expression miR-139-5p produce smaller tumors than cells with a control vector and a representative sample shows lower PDE4D and Ki67 staining.

In this revised paper they have responded to the most serious criticisms that came from Reviewers 1 and 3 who made it clear that it did not go far enough to show physiological significance of the p53 target. The new xenograft data are for the most part convincing and strengthen the paper. However, some of the previous concerns remain. If the authors can respond to these comments and provide better data where indicated this paper would be suitable for eLife.

1) As pointed out by reviewers 1 and 3 there are some cases (for example Figure 3D and Figure 4D) where effects are very modest and even if statistically significant. It is difficult to reconcile such modest effects with meaningful physiological outcomes.

2) Their responses to the comments are in some cases not really satisfactory. In one case (their point 3) they combine comments by two referees and respond to them as if they were one referee's comment. To this melded comment they provide data for the reviewers' eyes that 2 different siRNAs vs BIM have the same effect on Doxorubicin suppression of H460 cell proliferation. First, the "rescue" by BIM knockdown is very modest in the H460 cells. Second, the cells they use to show this are not the same that were in the paper in Figure 4D where they used A549 cells. It does not seem very relevant to present data on a different assay in another cell line to make their point.

3) In another case their response to the comment "If p53 works via miR-139-5p to increase levels of cAMP as the authors propose, they need to show this. Similarly, they need to show that the increase in BIM is p53 dependent". Is not very convincing. First, in Figure 4B. Doxorubicin is not really appropriate to use as a demonstration of p53-specific response because this drug has other p53-independent effects on cells, so Nutlin would be a better control. Second, in Figure 4—figure supplement 2 it is not appropriate to compare two cell lung cancer lines that differ in p53 status as a way to prove that BIM is induced in a p53-specific manner. Also the extent of induction is really very modest in the H460 cells. Either use the HCT116 pair of cells or knockdown p53 in the H460 cells.

Reviewer #2:

This is a revision of a previous submitted manuscript which attempts to argue that miR-139-5p is a novel p53 target and its mechanism of action is to increase cAMP levels via downregulation of phosphodiesterase 4 (PDE4D) and resulting Bim-mediated cell growth arrest. While the authors have addressed many of the concerns of the previous review, there remain several notable issues. If these can be addressed, the manuscript would be acceptable for publication. However, it should be noted that these are not new concerns, but rather points that the authors failed to properly address.

First, and most importantly, there was a concern about the biological relevance of the findings. The authors have now included a xenograft study. While this extends observations to an in vivo setting, they still have not shown that cellular outcomes are dependent upon this pathway. They state that there is Bim-mediated cell cycle arrest and yet data is presented to argue that the outcome may in fact be apoptosis. It is not clear how BIM would induce a cell cycle effect and the authors have not clearly stated which outcome they think is occurring in response to miR-139-5p regulation by p53. They note that BIM siRNA causes effects on cell viability and on the percent of cells with a hypodipold DNA content and yet the model in Figure 5F shows BIM causes a cell cycle effect. This is confusing.

Second, it was noted that cAMP levels do not track with outcomes. The authors' response is one of speculation rather than providing additional insights through experimental data.

Third, the p53-dependence of the BIM increase remains an issue. They use wild-type p53 expressing H460 cells and compare these to p53-null H1299 cells. Yet these two cell lines differ in many more ways besides p53 status. An isogenically matched set of cells is needed to make this important point that the increase in BIM needs p53 under these conditions.

https://doi.org/10.7554/eLife.15978.024

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

[…] Identifying novel target of p53 is an important area of study, especially when these targets provide new insights into the mechanism of action of p53. Thus, the focus of this study could be appropriate for eLife if the authors were to provide more information as outlined in the comments below. The problem with this study, however, is that the authors do not have enough information as to whether, how or when the induction of miR-139-5p by p53 actually plays a role in any p53 outcomes in cells, in animals or in human cancer. There is no evidence for this newly described process in a physiological or pathological setting in vivo. For publication in a journal, such as eLife, some demonstration of the importance of this process in vivo needs to be provided.

We thank the editor for the suggestion and agree that in vivo demonstration is critical for our finding. Therefore, we performed xenograft study using SW480 cells stably overexpressing miR-139-5p or scramble control. The data are now included in Figure 5 and interpreted in the manuscript accordingly.

The authors would need to produce extensive and convincing data to allay the above concerns as well as the following more specific comments:

1) Figure 3. What is the relative concentration of the mature endogenous miR-139-5p when induced by p53 compared to the concentration of the miR-139-5p mimic? This is important because it is not clear whether p53 induction of endogenous miR-139-5p is sufficient to cause enough of a drop in PDE4D to have any impact on cAMP.

We agree with the reviewer that the relative level of miR-139-5p is an important measure for sufficiently transducing p53-mediated effect. As shown in Author response image 1 in this letter, induction of p53 by 4μM INZ-C or 0.5 μM Doxorubicin led to 6 to 8-fold increase of miR-139-5p in H460 cells (Author response image 1A). Transient transfection of pSIF-H1-miR-139-5p and miR-139-5p mimic in A549 cells resulted in ~4-fold and ~7800-fold increase of miR-139-5p, respectively (Author response image 1B and C). However, we were able to detect changes in PDE4D protein at comparable levels among cells treated with 4μM INZ-C (Image 1D), 0.5 μM Doxorubicin (Figure 3E), or transfected with pSIF-miR-139-5p (Author response image 1E) or miR-139-5p mimic (Figure 3B). Consistently, 0.5 μM Doxorubicin treatment and miR-139-5p mimic transfection also led to similar induction of cAMP (Figure 4A and B). These observations suggest that although the relative levels of miR-139-5p could differ significantly when cells were treated with different means, several folds of increase achieved by p53 induction is sufficient to suppress PDE4D protein expression and subsequently cAMP elevation, while the excess amount of miR-139-5p mimic does not cause further impact.

Author response image 1
Downregulation of PDE4D by p53 and miR-139-5p.

(A) H460 cells were treated with DMSO, 2 μM INZ-C or 0.5 μM Dox for 18 hr followed by qRT-PCR analysis of miR-139-5p. (B) and (C) H460 cells were transfected with pSIF-H1-Scramble control (SC) or pSIF-H1-miR-139-5p (pSIF-139) (B), or negative control oligos (Ctrl) or miR-139-5p mimic (Mimic), followed by qRT-PCR analysis of miR-139-5p at 48 hr after transfection. Samples from (A) and (B) were also analyzed by Western blot as shown in (D) and (E), respectively.

https://doi.org/10.7554/eLife.15978.019

2) Figure 3G shows that there is a massive increase in the levels of PDE4D when the miR-139-5p inhibitor in introduced into cells even without Dox treatment. One would therefore expect that the levels of cAMP would be reduced. But in Figure 4B this is not the case. They only see an impact of the inhibitor on the levels that are increased by Dox treatment. Why is this?

We thank the reviewer for the comment. One possible scenario is that the basal level of PDE4D in cells is sufficient to keep cAMP at a certain level, while increase in PDE4D protein would not further reduce it.

3) Figure 4D: the authors show data indicating a modest but apparently convincing drop in cell viability upon introduction of the miR-139-5p mimic into cells. If BIM is the reason for this, then one would assume that knockdown of BIM would increase cell viability. But there does not appear to be any rescue by BIM knockdown in cells treated with the mimic and so their claim that the impact of miR-139-5p mimic is attenuated by BIM is not substantiated by the data. Understanding the biological consequences of this pathway is central to establishing this microRNA as an important target for p53. Knockdown of BIM is an important experimental approach. Multiple targeting sequences should be used for this knockdown to lessen the possibility of off-target effects. The authors also need to show that knockdown of BIM blocks some cellular outcome due to p53, presumably proliferation inhibition.

We agree with the reviewer that BIM knockdown did not increase cell viability in our experiments. This is consistent with the reports that BIM knockdown itself has minor effect on cell apoptosis, while BIM is more responsible for upstream signal stimulation (Lin, Xu et al., 2013, Zambon, Wilderman et al., 2011). In Figure 4D, we showed that in the presence of BIM siRNA, introduction of miR-139-5p mimic lost the ability to significantly decrease cell viability, indicating that BIM is responsible, at least in part, for miR-139-5p effect.

To address the reviewer’s comment on the off-target effects of BIM siRNA, we also tested another BIM siRNA in addition to the original one, and found that knocking down BIM with both of these two siRNAs reversed doxorubicin suppression of H460 cell growth determined by using the sulforhodamine B (SRB) assay as described (Vichai & Kirtikara, 2006) (Author response image 2), confirming that the effect of BIM knockdown is less likely due to off-target, and also indicating BIM is a downstream effector of p53 pathway as we have proposed in this study.

Author response image 2
Knockdown of BIM rescue doxorubicin suppression of H460 cell proliferation.

H460 cells were transfected with two siRNAs against BIM, and 48 hr after transfection, cells were treated with 0.5 μM Dox for indicated duration and cell proliferation was determined by using the sulforhodamine B (SRB) assay. * p<0.05. Error bars, SD.

https://doi.org/10.7554/eLife.15978.020

4) If p53 works via miR-139-5p to increase levels of cAMP after doxorubicin as the authors propose, they need to show this. Similarly, they need to show that the increase in BIM is p53 dependent. This is central to their conclusion.

We thank the reviewer for the advice. In Figure 4B, we showed that Doxorubicin increased cAMP to a level comparable to that induced by miR-139-5p mimic (Figure. 4A), while in the presence of miR-139-5p inhibitor, this induction no longer existed, indicating that Doxorubicin/p53 effect on cAMP is through miR-139-5p.

In order to address the dependence of BIM increase on p53, we treated H460 and H1299 cells, which are p53-positive and p53-negative cell lines, respectively, with 2 μM INZ-C or 10 μM mycophenolic acid (MPA, activator of p53 (Sun, Dai et al., 2008)), and found that BIM is induced by these two compounds only in H460 cells, but not H1299 cells (Figure 4—figure supplement 2), suggesting that the increase in BIM is p53 dependent.

5) While the authors show in Figure 4E an inverse correlation between miR-139-5p and PDE4D, is there a positive correlation between wild-type p53 and miR-139-5p in tumors?

We agree with the reviewer that p53 mutation status in the clinical samples should be associated with miR-139-5p/PDE4D levels. Unfortunately, we don’t have the first hand data on p53 status in this set of tumor samples. However, it is well accepted that p53 mutation rate in colon cancer is 50~60% according to the literature (Iacopetta, 2003, Kandoth, McLellan et al., 2013). In addition, even though p53 is wild type in some tumors, its activity should remain quite low due to other tumor favorable pathways. Therefore, it is difficult to elucidate the correlation between p53 mutation status and miR-139-5p/PDE4D levels, but we, as well as many other p53 researchers, believe that the overall p53 activity in the tumors is very low based on a large amount of literature information and the “killing” nature of p53 activity.

6) Many of the differences shown, especially, the impact of mir-139-p on cell death and the impact of BIM knockdown on this cell death (Figure 4D) are very small. This questions the importance of this newly described process.

We agree with the reviewer that miR-139-5p did not show very dramatic impact on cell death in our in vitro data. However, the difference between control and miR-139-5p transfected cells is statistically significant. Also, previous reports showed that miR-139-5p is a potential tumor suppressor against various types of cancer (Guo, Hu et al., 2012, Wong, Wong et al., 2011), which are consistent with our findings. In addition, our in vivo data in the revised manuscript further confirmed the tumor suppressor role of miR-139-5p, and tumors overexpressing miR-139-5p have elevated expression of BIM (Figure 5), suggesting the correlation between miR-139-5p and BIM as we have proposed.

7) It is unclear whether the authors examined the p53 status in the clinical samples. Given the focus of the manuscript on p53 regulation, one would expect that p53 loss of mutation would be associated with higher levels of PDE4D. If this is not the case, the authors should comment on this.

See response to point #5.

8) The authors also need to address whether p53 is sufficient for these effects or do they only occur with DNA damage.

We thank the reviewer for the advice. To address this, we treated HCT116 p53+/+ and HCT116 p53-/- cells with 10 μM Nutlin-3 (Nut), which disrupts MDM2-p53 interaction (Vassilev, Vu et al., 2004), or 5 nM Actinomycin D (ActD) (Iapalucci-Espinoza & Franze-Fernández, 1979), which leads to ribosomal stress-mediated p53 activation at low concentration, and found that these two drugs only reduced PDE4D in p53 positive, but not p53 negative, cells (Figure 3—figure supplement 3), indicating this reduction is p53 dependent, and not limited to DNA damage caused by Doxorubicin.

9) Along these lines as well, it should be tested whether the miR-139-5p inhibitor blocks this p53-dependent cellular outcome.

We thank the reviewer for the advice. As shown in Figure 4—figure supplement 3, introduction of miR-139-5p inhibitor significantly alleviated A549 cell growth inhibition by several p53 activating drugs, including INZ-C, Actinomycin D and Nutlin-3. The rescue effect was not dramatic, indicating that miR-139-5p is only partially responsible for p53-mediated downstream events.

10) Although the authors cite examples from the published literature, it remains to be shown what is the nature of inhibition of proliferation that is seen. Use of additional assays beyond that of MTT would be informative in this regard.

We thank the reviewer for the advice and performed flow cytometry analysis on H460 cells transfected with either pSIF-H1-scramble control or pSIF-H1-miR-139-5p. The result in Author response image 3 showed that miR-139-5p transfection caused significant increase in cell population of sub G1, indicating that miR-139-5p could induce apoptosis as well.

Author response image 3
miR-139-5p induces apoptosis.

H460 cells were transfected with pSIF-H1-Scramble control (Control) or pSIF-H1-miR-139-5p (139-5p), and 48 hr after transfection, the cells were subjected to flow cytometry analysis. *p<0.05. Error bars, SD.

https://doi.org/10.7554/eLife.15978.021

11) The biological significance of the two bands for PDE4D that are seen should be discussed.

We thank the reviewer for the comment and now include the discussion in the revised version.

[Editors’ note: the author responses to the second round of peer review follow.]

Reviewer #1:

This revised study by Cao et al. has identified a miR139-5p as a gene that is transactivated by p53 in Figures 1 and 2. In Figure 3 they provide evidence that PDE4D is a target of miR139-5p. They show in Figure 4 that levels of cAMP a known target of PDE4D, are up in miR139-5p mimic-containing cells or in Doxorubicin-treated cells. Also BIM, previously shown to be upregulated by cAMP, is increased by the miR-139 mimic. Finally in new data in Figure 5, they provide data from mouse xenograft studies and show that SW480 cells harboring a construct expression miR-139-5p produce smaller tumors than cells with a control vector and a representative sample shows lower PDE4D and Ki67 staining. In this revised paper they have responded to the most serious criticisms that came from Reviewers 1 and 3 who made it clear that it did not go far enough to show physiological significance of the p53 target. The new xenograft data are for the most part convincing and strengthen the paper. However, some of the previous concerns remain. If the authors can respond to these comments and provide better data where indicated this paper would be suitable for eLife.

We thank the reviewer for his/her positive view of our manuscript. In the revised manuscript, we are providing point-by-point responses to specific concerns.

1) As pointed out by reviewers 1 and 3 there are some cases (for example Figure 3D and Figure 4D) where effects are very modest and even if statistically significant. It is difficult to reconcile such modest effects with meaningful physiological outcomes.

We agree with the reviewer that in some cases, our in vitro data did not show very dramatic effects. Specifically, for Figure 3D, since the pMIR-reporter assay is an ectopic luciferase system as a very sensitive and reliable method, we believe that statistically significant changes (20-35% inhibition) are meaningful. For Figure 4D, we realized that in A549 cells, miR-139-5p is relatively less effective in inhibiting cell growth, so in our first revision, we included another cell line H460 and performed flow cytometry analysis, which showed quite dramatic induction of sub-G1 cell population (3, first rebuttal letter).

Additionally, our xenograft study further confirmed the correlation between miR-139-5p and PDE4D, BIM and tumor growth (Figure 5D, E and Figure 5—figure supplement 1). Collectively, our study demonstrates the tumor suppressor role of miR-139-5p in both in vitroand in vivosystems.

2) Their responses to the comments are in some cases not really satisfactory. In one case (their point 3) they combine comments by two referees and respond to them as if they were one referee's comment. To this melded comment they provide data for the reviewers' eyes that 2 different siRNAs vs BIM have the same effect on Doxorubicin suppression of H460 cell proliferation. First, the "rescue" by BIM knockdown is very modest in the H460 cells. Second, the cells they use to show this are not the same that were in the paper in Figure 4D where they used A549 cells. It does not seem very relevant to present data on a different assay in another cell line to make their point.

We thank the reviewer for the comment. The original decision letter was provided to us as a melded summary, so we did not have separate comments from different reviewers. That was why we tried to address point 3 as one comment. In Author response image 2 in the first rebuttal letter, we showed that BIM knockdown rescued H460 cell inhibition by Doxorubicin from 58% to 42% (siBIM-1) and 35% (siBIM-2) at 48 h, and from 67% to 47% (siBIM-1) and 37% (siBIM-2) at 72 h after treatment, respectively, which we believe is quite evident. To be consistent with the data in Figure 4D as the reviewer suggested, we also performed cell proliferation assay in A549 cells to test the rescue effect of BIM knockdown in response to Nutlin-3 treatment. As shown in Figure 4—figure supplement 5, the inhibitory effect of Nutlin-3 on cell proliferation was significantly reversed by BIM siRNA. Although the rescue effect was not 100%, we believe it is reasonably anticipated, because as one of hundreds of target genes of p53, miR-139 also has multiple target genes other than PDE4D/cAMP/BIM pathway (Guo, Hu et al., 2012, Shen, Liang et al., 2012, Wong, Wong et al., 2011). Therefore, we propose that induction of miR-139-5p mediated PDE4D/cAMP/BIM pathway contributes to p53 tumor suppressor function.

3) In another case their response to the comment "If p53 works via miR-139-5p to increase levels of cAMP as the authors propose, they need to show this. Similarly, they need to show that the increase in BIM is p53 dependent". Is not very convincing. First, in Figure 4B. Doxorubicin is not really appropriate to use as a demonstration of p53-specific response because this drug has other p53-independent effects on cells, so Nutlin would be a better control. Second, in Figure 4—figure supplement 2 it is not appropriate to compare two cell lung cancer lines that differ in p53 status as a way to prove that BIM is induced in a p53-specific manner. Also the extent of induction is really very modest in the H460 cells. Either use the HCT116 pair of cells or knockdown p53 in the H460 cells.

We agree with the reviewer that Nutlin-3 is a more appropriate approach to show p53 dependence. We now have replaced Figure 4B with new data showing that Nutlin-3 treatment also increased cAMP level in A549 cells, but not in the presence of miR-139-5p inhibitor.

We agree with the reviewer that comparing BIM induction in H460 and H1299 cells is not appropriate to show p53 dependence and therefore performed Western blot analysis in HCT116 p53+/+ and HCT116 p53-/- cells, and replaced Figure 4—figure supplement 2 with new data (now Figure 4—figure supplement 4) showing that Nutlin-3 induced BIM expression only in HCT116 p53+/+, but not HCT116 p53-/-, cells.

Reviewer #2:

This is a revision of a previous submitted manuscript which attempts to argue that miR-139-5p is a novel p53 target and its mechanism of action is to increase cAMP levels via downregulation of phosphodiesterase 4 (PDE4D) and resulting Bim-mediated cell growth arrest. While the authors have addressed many of the concerns of the previous review, there remain several notable issues. If these can be addressed, the manuscript would be acceptable for publication. However, it should be noted that these are not new concerns, but rather points that the authors failed to properly address.

We thank the reviewer for his/her positive view of our manuscript, which is potentially acceptable upon satisfactory responses to specific concerns.

First, and most importantly, there was a concern about the biological relevance of the findings. The authors have now included a xenograft study. While this extends observations to an in vivo setting, they still have not shown that cellular outcomes are dependent upon this pathway. They state that there is Bim-mediated cell cycle arrest and yet data is presented to argue that the outcome may in fact be apoptosis. It is not clear how BIM would induce a cell cycle effect and the authors have not clearly stated which outcome they think is occurring in response to miR-139-5p regulation by p53. They note that BIM siRNA causes effects on cell viability and on the percent of cells with a hypodipold DNA content and yet the model in Figure 5F shows BIM causes a cell cycle effect. This is confusing.

We thank the reviewer for the comments. In addition to Figure 3—figure supplement 3 and Figure 4—figure supplement 3 (now Figure 4—figure supplement 6), in which we showed that Nutlin-3 treatment led to p53 specific suppression of PDE4D and miR-139-5p inhibitor significantly rescued Nutlin-3 inhibition (in addition to INZ-C and Actinomycin D) of cell proliferation, we have now included more data (Figure 4B, Figure 4—figure supplement 3, Figure 4—figure supplement 4 and Figure 4—figure supplement 5), showing that specific p53 activation by Nutlin-3 induced cAMP dependent on miR-139- 5p, and cell growth inhibition by Nutlin-3 is at least partially dependent on miR-139-5p and BIM. Combined with our in vivo observation, we believe that the tumor suppressor function of p53 is at least partially through our proposed pathway.

We agree with the reviewer that BIM is a mediator of apoptosis. Indeed, in Image 3 in the first rebuttal letter, we used sub G1 population as an indicator of apoptosis (Zhou, Hao et al., 2015). In fact, in our proposed model in Figure 5F, we showed that BIM actually caused cell growth arrest as a general term of cellular outcome, instead of a cell cycle effect.

Second, it was noted that cAMP levels do not track with outcomes. The authors' response is one of speculation rather than providing additional insights through experimental data.

We thank the reviewer for the comment. We now have included Figure 4—figure supplement 3 to show that the rescue effect of miR-139-5p on A549 cell growth inhibition by Nutlin-3 is correlated with the change of cAMP levels (Figure 4B).

Third, the p53-dependence of the BIM increase remains an issue. They use wild-type p53 expressing H460 cells and compare these to p53-null H1299 cells. Yet these two cell lines differ in many more ways besides p53 status. An isogenically matched set of cells is needed to make this important point that the increase in BIM needs p53 under these conditions.

We agree with the reviewer that H460 and H1299 cells are not an appropriate pair of cells for comparing p53 dependent BIM induction. Therefore, we performed Western blot analysis in HCT116 p53+/+ and HCT116 p53-/- cells, and replaced Figure 4—figure supplement 2 with new data (now Figure 4—figure supplement 4) showing that Nutlin-3 induced BIM expression only in HCT116 p53+/+, but not in HCT116 p53-/- cells.

https://doi.org/10.7554/eLife.15978.025

Article and author information

Author details

  1. Bo Cao

    Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, United States
    Contribution
    BC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Kebing Wang
    Competing interests
    The authors declare that no competing interests exist.
  2. Kebing Wang

    Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
    Contribution
    KW, Conception and design, Acquisition of data, Analysis and interpretation of data
    Contributed equally with
    Bo Cao
    Competing interests
    The authors declare that no competing interests exist.
  3. Jun-Ming Liao

    Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, United States
    Contribution
    J-ML, Conception and design, Acquisition of data
    Competing interests
    The authors declare that no competing interests exist.
  4. Xiang Zhou

    Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, United States
    Contribution
    XZ, Conception and design, Acquisition of data
    Competing interests
    The authors declare that no competing interests exist.
  5. Peng Liao

    Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, United States
    Contribution
    PL, Conception and design, Acquisition of data
    Competing interests
    The authors declare that no competing interests exist.
  6. Shelya X Zeng

    Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, United States
    Contribution
    SXZ, Conception and design, Acquisition of data
    Competing interests
    The authors declare that no competing interests exist.
  7. Meifang He

    Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
    Contribution
    MH, Acquisition of data
    Competing interests
    The authors declare that no competing interests exist.
  8. Lianzhou Chen

    Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
    Contribution
    LC, Acquisition of data
    Competing interests
    The authors declare that no competing interests exist.
  9. Yulong He

    Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
    Contribution
    YH, Acquisition of data
    Competing interests
    The authors declare that no competing interests exist.
  10. Wen Li

    Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
    Contribution
    WL, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    wenli28@163.com
    Competing interests
    The authors declare that no competing interests exist.
  11. Hua Lu

    Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, United States
    Contribution
    HL, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    hlu2@tulane.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-9285-7209

Funding

National Natural Science Foundation of China (81172337)

  • Wen Li

National Natural Science Foundation of China (30973395)

  • Wen Li

NIH Office of the Director (R01CA095441)

  • Hua Lu

NIH Office of the Director (R01CA172468)

  • Hua Lu

NIH Office of the Director (R01CA127724)

  • Hua Lu

NIH Office of the Director (R21 CA190775)

  • Hua Lu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

Hua Lu was supported in part by NIH-NCI grants R01CA095441, R01CA172468, R01CA127724, and R21 CA190775 as well as the Reynolds and Ryan Families Chair fund. Wen Li was supported by the National Natural Science Foundation of China (No. 81172337, 30973395).

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#4257R) of Tulane University. All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.

Reviewing Editor

  1. Kevin Struhl, Reviewing Editor, Harvard Medical School, United States

Publication history

  1. Received: March 11, 2016
  2. Accepted: June 29, 2016
  3. Accepted Manuscript published: July 7, 2016 (version 1)
  4. Version of Record published: July 25, 2016 (version 2)

Copyright

© 2016, Cao et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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