Research ArticleCancer

p53 and MicroRNA-34 Are Suppressors of Canonical Wnt Signaling

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Science Signaling  01 Nov 2011:
Vol. 4, Issue 197, pp. ra71
DOI: 10.1126/scisignal.2001744

Abstract

Although loss of p53 function and activation of canonical Wnt signaling cascades are frequently coupled in cancer, the links between these two pathways remain unclear. We report that p53 transactivated microRNA-34 (miR-34), which consequently suppressed the transcriptional activity of β-catenin–T cell factor and lymphoid enhancer factor (TCF/LEF) complexes by targeting the untranslated regions (UTRs) of a set of conserved targets in a network of genes encoding elements of the Wnt pathway. Loss of p53 function increased canonical Wnt signaling by alleviating miR-34–specific interactions with target UTRs, and miR-34 depletion relieved p53-mediated Wnt repression. Gene expression signatures reflecting the status of β-catenin–TCF/LEF transcriptional activity in breast cancer and pediatric neuroblastoma patients were correlated with p53 and miR-34 functional status. Loss of p53 or miR-34 contributed to neoplastic progression by triggering the Wnt-dependent, tissue-invasive activity of colorectal cancer cells. Further, during development, miR-34 interactions with the β-catenin UTR affected Xenopus body axis polarity and the expression of Wnt-dependent patterning genes. These data provide insight into the mechanisms by which a p53–miR-34 network restrains canonical Wnt signaling cascades in developing organisms and human cancer.

Introduction

Canonical Wnt signaling controls events ranging from cell fate determination and cell cycle regulation to cell motility and metabolism (17). In each case, Wnt signaling depends on the posttranslational regulation of β-catenin levels whereby the Wnt-stabilized protein translocates to the nuclear compartment and binds to the T cell factor and lymphoid enhancer factor (TCF/LEF) family of transcriptional cofactors (17). In turn, β-catenin–TCF/LEF complexes activate transcriptional cascades that affect development (for example, the polarity of the Xenopus primary body axis), the maintenance of stem cell niches in adult tissues, and the induction of epithelial-mesenchymal transition (EMT) programs that characterize gastrulation, wound healing, and fibrosis (17). In addition to the role of the canonical Wnt pathway in normal cell function, pathologic increases in Wnt signaling are frequently implicated in neoplastic states (17). The importance of Wnt signaling in human cancer is highlighted by its coordinate control of the transcriptional programs underlying EMT, cancer stem cell generation, and cancer progression (36).

In addition to the heightened Wnt activity that characterizes many neoplastic states, the tumor suppressor p53 is also frequently inactivated in human cancers (8, 9), its transcriptional activity playing critical roles in maintaining its function as a tumor suppressor (10, 11). In Li-Fraumeni syndrome, an inherited disorder associated with TP53 mutations, the age of tumor onset—especially in breast and colon cancer—directly correlates with the decrease in p53’s transactivational activity (12, 13). The importance of p53 function further manifests itself in p53-null mouse models, where most mice appear normal at birth but develop tumors at early age (14, 15). Despite the biological and clinical consequences of p53 loss of function, the mechanistic links whereby p53 suppresses oncogenic signaling remain unclear (913). Thus, heretofore unexplored processes may exist that allow wild-type p53 to repress signaling events critical to tumorigenesis and cancer progression. Indeed, in vivo, Wnt1-overexpressing mice bred into a p53-deficient background develop mammary adenocarcinomas at an earlier age, with higher rates of tumor incidence and accelerated tumor growth relative to that of Wnt1 transgenic mice with wild-type p53 function (1618), suggesting that p53 may act upstream of Wnt to suppress its oncogenic activity.

MicroRNAs (miRNAs) are small, noncoding RNAs that interact with complementary or near-complementary target sites in the untranslated regions (UTRs) of mRNA targets, thereby repressing gene expression posttranscriptionally (19, 20). The miRNA-mRNA interactions form complex regulatory networks whose effects appear to be more pervasive than previously appreciated with vertebrate miRNAs affecting early embryonic development as well as tumorigenesis and metastasis (1927). Although recent studies have demonstrated that miR-8 in Drosophila or miR-203 in zebrafish inhibits Wnt during development, in part by targeting wntless, a gene required for Wnt secretion, and LEF1, respectively (28, 29), little is known regarding the ability of miRNAs to modulate the canonical Wnt cascade.

Transcription of the evolutionarily conserved microRNA-34 (miR-34) family falls under the direct control of p53 (3033). Further, in many types of sporadic and hereditary cancers, the miR-34 family is silenced not only by functional inactivation of p53 but also by its chromosomal deletion or epigenetic silencing (3439), raising the possibility that miR-34 constitutes an important arm of the p53 network. Here, we demonstrate a direct link from p53 activity and miR-34 regulation to the canonical Wnt signaling pathways operative in development and cancer.

Results

Tumor suppressor p53 and miR-34 inhibit Wnt activity

Supporting the hypothesis that p53 represses Wnt signaling, loss of wild-type p53 increased the endogenous β-catenin levels and potentiated TOPflash activity (a reporter construct that monitors β-catenin–TCF/LEF transcriptional activity) in p53flox/flox mouse embryonic fibroblasts (MEFs) as well as in human embryonic kidney (HEK) 293 cells (Fig. 1A and fig. S1A). Because β-catenin abundance and TCF/LEF activity are co-regulated (1), we examined the abundance of upstream Wnt genes as a function of p53 status. Stable knockdown of wild-type p53 increased not only the abundance of β-catenin but also that of WNT1 and its co-receptor, low-density lipoprotein receptor–related protein 6 (LRP6), in both A549 lung cancer cells and MCF-7 breast cancer cells (Fig. 1B). Loss of p53 function induced by (i) small interfering RNA (siRNA)–mediated knockdown, (ii) stable transduction with a human papilloma virus E6 open reading frame (ORF), or (iii) expression of mutant p53 elicited similar effects (fig. S1, B and C), suggesting that p53 represses canonical Wnt signaling at multiple levels. Multiple Wnt target genes directly downstream of β-catenin, including CD44, MMP-7, and MYCN, showed increased mRNA abundance after p53 knockdown (fig. S2A), supporting an upstream suppressor function for p53. To determine the effect of p53 loss of function on Wnt signaling in clinical samples, we analyzed a publicly available gene expression data set of 251 breast cancer samples (40). In this data set, the functional and mutational status of p53 had been analyzed in terms of its transcriptional function (see Materials and Methods for details regarding the p53 status of the patient data set). We independently validated the transcriptional activity of p53 in this breast cancer cohort by analyzing the transcript abundance of p53 target genes (fig. S2B). To assess potential associations between p53 status and Wnt activity in these patients, we examined the mRNA abundance of TCF/LEF target genes (4, 41). In an unsupervised hierarchical analysis, the TCF/LEF gene signature in breast cancer identified two distinct functional as well as mutational p53 subsets (Fig. 1C). Because the mRNA abundance of the TCF/LEF gene set associated with loss of p53 function, these data support the contention that canonical Wnt activity is linked to p53 status in primary breast cancer tissues. Because the tumor suppressor role of p53 derives primarily from its transcriptional activity (10), we used p53−/− MEFs to determine the role of p53-mediated transcription on Wnt repression. As expected, both cellular β-catenin levels and TOPflash activity fell significantly after stable transduction of wild-type p53 into p53-null MEFs (Fig. 2A). In contrast to wild-type p53, neither mutant p53 with a deleted N-terminal transcriptional activation domain (TAD) nor transcriptionally inactive p53 mutants (R175H, R273H) suppressed Wnt activity, indicating that p53’s transcriptional function is required for Wnt repression. Because the miR-34 family is a direct transcriptional target of p53 (3033), we examined miR-34 abundance in conjunction with p53 status (Fig. 2B and fig. S3A). An inverse correlation between Wnt activity and miR-34 abundance was identified, leading us to postulate that p53 represses canonical Wnt signaling through the transcriptional activation of miR-34. Indeed, TOPflash activity was suppressed in HEK293 cells by exogenous miR-34, whereas its activity was enhanced when endogenous miR-34 was depleted (Fig. 2C); similarly, p53 and miR-34 limited Wnt signaling in MEFs even in the presence of exogenous Wnt (fig. S3, B to D).

Fig. 1

Loss of p53 function potentiates Wnt activity in cells and samples of breast cancer tissue. (A) Immunoblot analysis of β-catenin (left panels, n = 2) and TOPflash activities (right panel) after Cre recombinase–mediated deletion of wild-type (WT) p53 in p53flox/flox MEFs. Tubulin was used as the loading control. TOPflash and FOPflash activities were normalized to the activity of a cotransfected SV-40–Renilla construct; error bars mark the SD (n = 3; **P < 0.01, t test). (B) Immunoblot analysis of WNT1, LRP6, and β-catenin after shRNA-mediated knockdown of WT p53 in A549 or MCF-7 cells (n = 2). (C) Unsupervised hierarchical clustering of a 251 breast cancer cohort using a TCF/LEF gene signature to distinguish between subsets of tumors with mutant p53 function according to the DLDA classifier (red bars; P = 1.19 × 10−22, Fisher’s exact test) as well as p53 cDNA mutation status (black bars; P = 4.59 × 10−14, Fisher’s exact test). The Uppsala breast cancer specimens (GSE3494, n = 251) were clustered (columns) using genes responsive to dominant-negative TCF. In the heat map, red denotes higher relative expression, whereas green indicates lower relative expression, with degree of color saturation reflecting the magnitude of the log expression signal. The bottom row represents the median log expression value of TCF/LEF target genes.

Fig. 2

p53 represses Wnt activity via miR-34 transactivation. (A) Immunoblot analysis of β-catenin (left panels) and TCF/LEF activities of the canonical Wnt pathway (right panel) after stable transduction with empty control (Mock) or various p53-expressing lentiviruses in p53−/− MEFs. TOPflash (blue) and FOPflash (red) firefly luciferase activity were determined in MEFs. Blots are representative of two independent experiments. Relative fold changes in TOPflash activity [as defined by changes in relative light units (RLU)] in cells expressing WT p53 were statistically significant (n = 3; **P < 0.01, t test) compared to control (n.s., not significant compared to control; dTAD, deletion construct of transactivating domain of p53). (B) Pri-miR-34a abundance was determined by quantitative RT-PCR after p53 deletion by Cre recombinase in p53flox/flox fibroblasts (left panel) or stable transduction with a control or various WT or mutant p53–expressing lentiviruses in p53−/− MEFs (right panel). Relative fold change of pri-miR-34a abundance was statistically significant compared to control (n = 3; **P < 0.01, t test; n.s., not significant compared to control transfectants). (C) Wnt transcriptional activity was inhibited by miR-34a and potentiated by the inhibition of miR-34a in HEK293 cells (n = 3; **P < 0.01, t test). TOPflash (blue) and FOPflash (red) firefly luciferase expression vectors were cotransfected with negative control (N/C) miR, synthetic miR-34a precursor (miR-34a), or inhibitor (anti–miR-34a) in HEK293 cells. (D) Unsupervised hierarchical clustering of pediatric neuroblastoma samples (GSE13141) using a TCF/LEF transcriptional signature segregates a subset of tumors with chromosomal loss of miR-34a (P = 0.000033, Fisher’s exact test; see Materials and Methods for detailed clinical information and data processing). Red bars represent patient samples with 1p loss. The bottom row denotes the median log expression value of TCF/LEF target genes.

Tumor suppressive miRNA genes are frequently located at fragile sites in chromosomes (36). Because loss of chromosome 1p and 11q are common in various primary cancers, especially pediatric neuroblastoma and glioma, and these deletions encompass the coding regions for miR-34a and miR-34b/c, respectively, we hypothesized that these deletions combined with microarray data set might serve as a miRNA loss platform in human cancer tissues (42). To assess the repressor function of miR-34 on Wnt activity in clinical samples, we used a microarray gene expression data set with relevant chromosomal information from a study of pediatric neuroblastoma patients (see Materials and Methods for detailed information) (42, 43). In an unsupervised analysis, TCF/LEF gene signatures successfully identified miR-34 status on the basis of chromosome 1p and 11q deletions (Fig. 2D and fig. S3E). Thus, the expression of genes activated downstream of TCF/LEF was specifically increased in samples from patients with tumors showing 1p loss or 11q loss, consistent with the proposed roles for miR-34 family members as repressors of Wnt signaling.

Canonical Wnt genes are direct targets of miR-34

To determine whether miR-34 constitutes an inhibitory arm of the Wnt cascade, we next investigated various putative miR-34 targets and interaction sites in terms of the miBridge target class, an algorithm for specific miR-mRNA binding predictions (44). Potential binding sites for miR-34a, miR-34b*, and miR-34c-5p were identified in the UTRs of not only β-catenin, WNT1, and LRP6 but also WNT3 and LEF1 (Fig. 3A and tables S1 and S2). Furthermore, miR-34 family member sequences (table S3) and potential miR-34 mRNA binding sites (table S4) were highly conserved in vertebrates, including various mammals as well as birds (identified in Gallus) and amphibia (identified in Xenopus). To determine whether miR-34 represses Wnt pathway genes through their respective UTRs, we cloned the UTRs of human WNT1, WNT3, LRP6, β-catenin, and LEF1 downstream of luciferase reporter constructs, expressed them in A549 or MCF-7 cells, and assessed the effects of miR-34 on luciferase activity. Transduction with expression vectors encoding either miR-34 family members or synthetic miR-34a decreased the activities of the UTR reporter genes relative to that of control luciferase without a cloned UTR (Fig. 3B and fig. S4, A and B). Mutation of putative miR-binding sites in the WNT1, WNT3, or β-catenin UTRs abolished the repressor function of miR-34 (fig. S4C). Similarly, the abundance of WNT1, LRP6, and β-catenin transcripts and proteins was also decreased by miR-34a (Fig. 3C and fig. S4D). To determine whether the putative miR binding sites contributed to miR-34–mediated inhibition of Wnt target genes, we constructed Flag-tagged expression vectors of wild-type β-catenin with either no UTR or UTRs of varying length so as to include zero, two, three, or four of the predicted binding sites. Coexpression of exogenous miR-34a decreased the abundance of these β-catenin constructs in proportion to the number of putative target sites, but failed to affect the abundance of the construct lacking the UTR (Fig. 3D).

Fig. 3

The miR-34 family directly targets the UTR of genes involved in the canonical Wnt signaling pathway. (A) Potential matching target sites of the miR-34 family in the 3′UTR of WNT1, WNT3, LRP6, β-catenin, and LEF1. Predicted matching sites of miR-34a, -34b*, and -34c-5p are denoted as miR-matched sites (MMS; open arrowheads). (B) Inhibition of UTR reporter activity of Wnt genes by miR-34 family expression. Reporter constructs in which Wnt gene UTRs were cloned downstream of firefly luciferase were transfected into A549 (left panel) or MCF-7 cells (right panel) with control (mock) or miR-34 family expression vectors. Activity of the UTR reporter constructs was normalized to the activity of cotransfected SV-40–Renilla constructs after a 48-hour culture period. Relative luciferase activity in comparison to the non-UTR control reporter is shown (27); the error bar represents the SD (n = 3; *P < 0.05, t test; n.s., not significant compared to each control). (C) RT-PCR (left panels) and immunoblot analysis (n = 2, right panels) of pri-miR-34a and Wnt-pathway proteins after treatment with doxycycline (Dox, 0.5 μg/ml) for a 24-hour period in miR-34a–inducible MCF-7 cells. (D) Immunoblot analysis of HEK293 cells transfected with a Flag-tagged β-catenin cDNA expression vector without its UTR (β-catenin–CDS) or with cDNA expression vectors harboring different lengths of the β-catenin UTR [nucleotide (nt) +1 to 170, nt +1 to 480, nt +1 to 790]. The β-catenin expression vectors (100 ng each) were cotransfected with 20 nM negative control or synthetic miR-34a precursor, and the cell lysates were analyzed by immunoblotting with anti-Flag mAb after a 2-day culture period (n = 2).

Endogenous miR-34 mediates repression of canonical Wnt signaling by p53

We next examined the role of endogenous miR-34 in regulating Wnt activity in two carcinoma cells that retain wild-type p53 activity (A549 and MCF-7 cells). Functional inhibition of miR-34a, the most abundant member of the miR-34 family, with complementary RNA increased the activity of a Wnt UTR reporter in both cell lines (Fig. 4A and fig. S4E), but not that of the reporters in which the UTRs contained mutant miR-34 binding sites (Fig. 4B). Furthermore, depletion of functional miR-34a increased the abundance not only of WNT1, LRP6, and β-catenin mRNA in A549 or MCF-7 cells (Fig. 4C and fig. S4, E and F) but also that of known β-catenin target genes (Fig. 4D). Moreover, the abundance of β-catenin encoded by expression vectors with UTRs of varying length was decreased in proportion to the number of target sites in the UTR, and this was reversed when endogenous miR-34a function was inhibited (Fig. 4E).

Fig. 4

Functional activity of endogenous miR-34. (A) Inhibition of miR-34a increased activity of Wnt gene UTR reporters in A549 or MCF-7 cells. Transduction of anti–miR-34a (+) with each UTR reporter increased UTR activities relative to that of the negative control (N/C). The relative activities were measured as described in Fig. 3B; error bars depict the SD from three independent experiments. All results were statistically significant compared to the respective control; P < 0.05, t test. (B) miR-34a–dependent regulation of UTR activities was relieved by mutation of potential target sites in WNT1, WNT3, or β-catenin UTRs in MCF-7 or A549 cells. The relative WT and mutant UTR activities (not significant compared to each control t test) were compared after transduction with a negative control (−) or miR-34a. (C) Immunoblot analysis of WNT1, LRP6, or β-catenin abundance after transduction of control (−) or anti–miR-34a (+) in A549 or MCF-7 cells. Blots are representative of two independent experiments. (D) Quantitative RT-PCR analysis of β-catenin target genes after inhibition of miR-34a in MCF-7 or A549 cells. mRNA abundance of β-catenin target genes in anti–miR-34a–transfected cells relative to that in cells transfected with negative control miRNA, determined in triplicate experiments. *P > 0.05, not significant. (E) Immunoblot analysis of β-catenin expression vectors with or without UTR after inhibition of miR-34a. The Flag-tagged β-catenin expression vectors described in Fig. 3D were transfected into 293 cells after transduction of negative control miRNA (−) or depletion of miR-34a (+), and relative abundance of β-catenin was determined with an anti-Flag antibody. Blots are representative of two independent experiments. (F) UTR reporter activities of canonical Wnt genes after knockdown of WT p53 in A549 or MCF-7 cells. The UTR reporter activities were significantly increased by knockdown of WT p53 (dsRed-shp53) relative to the negative control (dsRed). n = 3; P < 0.05, t test. (G) Immunoblot (upper, n = 2) and RT-PCR (lower, n = 2) analyses of β-catenin and pri-miR-34a abundance in MCF-7 cells transduced with dsRed vector control (−) or with shRNA mediated knockdown of p53 (dsRed-shp53). Pri-miR-34a expression was induced by treatment with doxycycline (0.125, 0.25, and 0.5 μg/ml) for 24 hours in p53 knockdown cells. Abundances of p53 and β-catenin were determined by immunoblot analysis of cell lysates (1 μg). (H) Wnt UTR reporter activity after induction of miR-34a in p53-silenced MCF-7 cells (right panel, n = 3) relative to control cells (shp53-, Dox-). *P > 0.05 (not significant) compared to each negative control.

Next, Wnt gene UTR reporter activities in A549 or MCF-7 cells were examined after knockdown of wild-type p53. After stable or transient knockdown of wild-type p53, the activities of the Wnt gene UTR reporters increased, whereas primary miR-34 (pri-miR-34) transcription and mature miR-34a abundance decreased (Fig. 4F and fig. S5). Under physiologic conditions, the E3 ubiquitin ligase activity of MDM2 (murine double minute 2) acts to limit the abundance of wild-type p53 (9, 10). Treatment of A549 or MCF-7 cells with an MDM2 inhibitor, Nutlin-3, decreased Wnt abundance and the activity of the Wnt gene UTR reporter, whereas pri-miR-34 abundance increased (fig. S6). To further verify the role of miR-34 in p53-mediated inhibition of Wnt, we constructed a miR-34a–inducible system in cells in which endogenous p53 was knocked down with a specific short hairpin RNA (shRNA). As expected, p53 knockdown decreased miR-34 abundance, resulting in increased β-catenin abundance and β-catenin UTR gene reporter activity (Fig. 4, G and H). The effects of p53 knockdown were largely relieved by induction of miR-34 with doxycycline (Fig. 4, G and H). Similarly, in a p53-null background, Wnt activity was inhibited after the expression of wild-type p53 and restored after miR-34a silencing (Fig. 5, A and B, and fig. S6C), indicating that p53-mediated inhibition of Wnt is mainly attributable to miR-34. Because p53 can directly or indirectly increase the abundance of multiple miRNAs (32), it remained possible that additional miRNAs regulated by p53 could also inhibit Wnt activity. To investigate this possibility, we tested the effects on Wnt activity of several intergenic miRNAs, including miR-27, miR-30, and miR-192, that are up-regulated by p53 (32). After transfection of each of these miRNAs in cells coexpressing Wnt UTR reporters or TOPflash, only miR-34 decreased the UTR reporter and TOPflash activities, although the other miRNAs inhibited their respective control targets (Fig. 5, C and D).

Fig. 5

Endogenous miR-34 mediates p53-dependent inhibition of TCF/LEF target genes. (A) p53 represses UTR reporter activities and Wnt transcriptional activity in a miR-34–dependent manner in p53-null MEFs. Activity of Wnt pathway reporter genes was inhibited after transduction of WT p53, and this inhibition was reversed by loss of miR-34a (n = 3; *P < 0.01, t test). (B) p53-dependent decrease in TCF/LEF reporter activity (left panel) and β-catenin abundance (right panels) were relieved by depleting miR-34a (*P < 0.01, t test). (C) UTR reporter constructs for Wnt-pathway genes were coexpressed with miR-34a, miR-27a, miR-30d, or miR-192 for 48 hours, and the relative expression of firefly luciferase was determined. The functional activity of the miRNA expression vectors was verified with positive control reporters harboring a complementary UTR target downstream of the respective luciferase construct (*P < 0.01, one-sided t test). (D) miR-34–dependent regulation of TOPflash activity in p53-null MEFs. TOP/FOPflash constructs were coexpressed with miR-27, miR-30, miR-192, or miR-98, and relative Wnt transcriptional activity was determined (*P < 0.01, t test). (E) Hierarchical clustering of breast cancer (GSE3494, left panel) or neuroblastoma (GSE13141, right panel) samples with a miR-34 signature distinguishes subsets of tumor with TCF/LEF activity and p53/1p function. The publicly available data set used in Figs. 1C and 2D was clustered (columns) using 56 genes responsive to miR-34a (32). Hierarchical clustering of clinical samples with miR-34 signature associated with a subset of tumors with high TCF/LEF activity and defects in p53 functionality in breast cancer (P = 1.20 × 10−29 in TCF/LEF activity, P = 1.60 × 10−24 in p53 function) or 1p deletion in neuroblastoma (P = 0.002 in TCF/LEF activity, P = 6.73 × 10−5 in 1p deletion), respectively. Blue bars represent patient samples with a high TCF/LEF subset in Figs. 1C and 2D, and red bars represent mutant p53 function or 1p-deleted samples. The bottom row denotes the median expression value (log2) of miR-34–responsive genes.

We further compared an expression signature responsive to miR-34 in Dicer−/− cells (32) to clinical data sets to determine whether an analysis of miR-34 gene targets could strengthen evidence for the link between p53 activity and canonical Wnt signaling. Indeed, changes in gene expression predicted to occur in response to low miR-34 activity (that is, increased expression of miR-34–responsive genes) clearly associated with sample subsets displaying high β-catenin–TCF/LEF gene signatures, mutant p53 function in the breast cancer cohort, and with chromosome 1p loss in the neuroblastoma data set, respectively (Fig. 5E). Hence, miR-34 expression functionally links p53 and Wnt signaling in affected patient populations.

miR-34 suppresses Wnt activity during Xenopus development

Although physiological and developmental functions of p53 in Xenopus have not been clearly defined (45), the aberrant duplication of the embryonic axis in early Xenopus embryos is controlled by Wnt and β-catenin signaling (2, 46). Because miR-34 and its binding sites in the β-catenin UTR are highly conserved in vertebrates, and β-catenin’s stimulation of TCF/LEF transcriptional activity largely depends on interactions between its UTR and miR-34 (Fig. 6A), we tested the hypothesis that these miR-UTR interactions play a role in limiting β-catenin activity in Xenopus development. When β-catenin mRNA lacking its UTR was injected into the Xenopus ventral blastomere, axis duplication was observed in more than 70% of the embryos, whereas β-catenin constructs containing 790 base pairs of the UTR induced axis duplication in less than 10% of the embryos (Fig. 6B). UTR-dependent repression of β-catenin function, as assessed by either TOPflash activity or axis duplication, was abolished by mutating putative miR-34 target sites (Fig. 6, C and D), indicating that miR-UTR interactions regulate β-catenin function in Xenopus development. Furthermore, injection of miR-34 into the ventral blastomeres of Xenopus embryos blocked Wnt-induced axis duplication (Fig. 6E). Because injection of anti–miR-34 into the ventral marginal zone (n = 18) or dorsal marginal zone (n = 17) of Xenopus embryos induced embryonic lethality by the end of gastrulation, whereas injection of a negative control miRNA did not (n = 20), we chose a cap explant assay with the animal pole of blastomeres to investigate the role of endogenous miR-34 in limiting signaling through the Wnt/β-catenin pathway. Indeed, in a fashion similar to that observed after the application of exogenous Wnt, depletion of endogenous miR-34 increased mRNA abundance for the Wnt–β-catenin–responsive patterning genes Siamois, Xnr3, and Chordin (Fig. 6F). In contrast, the addition of exogenous precursors of miR-34a decreased the expression of these patterning genes (Fig. 6F). Thus, as observed in human cancer tissues, miR-34 also inhibits Wnt signaling during Xenopus development (Fig. 6G).

Fig. 6

Specific interactions between miR-34 and the β-catenin UTR regulate the Wnt pathway during Xenopus development. (A) TOPflash activities of wild-type β-catenin expression vectors harboring variable UTR lengths with negative control (N/C) or synthetic miR-34a inhibitor (anti–miR-34a) in HEK293 cells. The relative Wnt transcriptional activity of TOPflash was determined and normalized with cotransfected SV-40–Renilla activity. Relative fold increase of β-catenin expression vectors to empty vector is shown (*P < 0.01 compared to control, t test). (B) Axis duplication by β-catenin during Xenopus development is blocked by miR-UTR interaction. One of the ventral vegetal blastomeres of four- to eight-cell–stage Xenopus embryos was injected with 30 pg of β-catenin mRNA either without a UTR or with variable-sized UTRs. Embryos were scored in three categories: complete secondary axis, partial secondary axis (small posterior protrusion or pigmented spot/line), and normal single axis. *P < 0.05 between groups. (C) TOPflash activities of β-catenin expression vectors with WT or mutant UTR relative to β-catenin expression vectors without UTR (CDS) in 293 cells. Depletion of miR-34a (red bars) abolished UTR activities regardless of mutational status of putative miR-34 target sites in the UTR of β-catenin (*P < 0.01 compared to control, t test). (D) Inhibition of axis duplication by β-catenin depends on miR-34–UTR interactions during Xenopus development. Xenopus embryos were injected with 50 pg of β-catenin mRNA without UTR (CDS), with UTR (1 to 170), or with UTR mutants and scored as described above (*P < 0.01; n.s., not significant, binomial t test). (E) miR-34 suppresses Wnt8-induced axis duplication in Xenopus embryos. Wnt8 mRNAs were co-injected with control miR (N/C) or miR-34 into one of the ventral vegetal blastomeres of four- to eight-cell–stage embryos and scored as described above. Results shown are statistically significant at P < 0.01 by binomial t test. (F) Induction or repression of Wnt/β-catenin target patterning genes in X. laevis animal cap explants by respectively depleting miR-34a levels (left panels) or supplementing miR-34a levels (right panels). Fertilized two-cell–stage embryos were injected with control miR, miR-34a, anti–miR-34a, or Wnt8 mRNA as indicated. Animal caps were prepared at the blastula stage (Xenopus developmental stages 8.5 to 9.0) and cultured until the gastrula stage (10.5). Expression of Wnt/β-catenin target genes was assessed by RT-PCR. Xenopus histone H4 was used as a loading control. Lanes: WE, whole embryo; UI, uninjected. (G) Schematic diagram of p53-mediated inhibition of canonical Wnt signaling.

Hyperactivation of the canonical Wnt signaling pathway after loss of p53–miR-34 function contributes to cancer progression

Mutations in APC (adenomatous polyposis coli) or β-catenin that lead to constitutive activation of the canonical Wnt signaling pathway are key steps in initiating colorectal tumorigenesis (47). In the multistep progression of colorectal cancer, loss of wild-type p53 function tends to occur relatively late in the disease process, during the transition from adenoma to invasive colorectal carcinoma (48). To investigate the gatekeeper role of the p53–miR-34 axis in regulating constitutive Wnt signaling in cancer, we used LoVo and SNU-81 colon carcinoma cells, which express mutant APC, but retain wild-type p53 function. In these cells, either p53 knockdown or miR-34 silencing increased Wnt signaling without affecting the abundance of mutant APC (Fig. 7A and fig. S7A). In SW480 and SW620 colon carcinoma cells, which carry mutations in both APC and p53, transduction of miR-34a decreased β-catenin abundance and inhibited Wnt UTR activities (Fig. 7B). TOPflash activity, which was inhibited by wild-type APC or miR-34 alone, was further inhibited when wild-type APC and miR-34 were coexpressed (Fig. 7B). By contrast, the p53–miR-34 axis remained functional in HCT116 and SNU-407 colon carcinoma cells that express mutant β-catenin but retain wild-type p53 function (fig. S7B). To further assess the role of miR-34 in colorectal cancer progression, we performed invasion assays with the live chick chorioallantoic membrane (CAM), which provides an in vivo system for monitoring the ability of cancer cells to traverse an intact basement membrane and infiltrate underlying interstitial tissues (5, 6, 49). As predicted, the invasive activity of p53-mutant SW480 and SW620 cells was largely blocked and the CAM basement membrane was preserved when miR-34a abundance was increased in vivo (Fig. 7C). Furthermore, the anti-invasive effects of miR-34a depended on its interaction with the intact β-catenin UTR (fig. S8). Together, these data support the hypothesis that miR-34 counterbalances the effects of APC or β-catenin mutations in the early stages of colorectal tumorigenesis (48, 50) and that the hyperactive Wnt signaling program that accompanies malignant progression likely results as a consequence of the combined loss of p53 and miR-34 function.

Fig. 7

Loss of p53 and miR-34 function augments canonical Wnt activity and triggers a tissue-invasive phenotype in colorectal cancer cells. (A) Loss of WT p53 function induces Wnt transcriptional activity in LoVo or SNU-81 colorectal cancer cells. p53 knockdown increased β-catenin abundance (relative abundance reported below the blot; blots are representative of two independent experiments), leaving truncated APC abundance unaltered (left panels). Wnt UTR (middle panel; *P < 0.01 compared to control, t test) and TOPflash (right panel; *P < 0.01, t test) activities were increased significantly by knockdown of p53 in APC-mutant colorectal cancer cells with WT p53 function. (B) Restoration of miR-34 decreases Wnt activity in APC-mutant–p53-mutant SW480 or SW620 cells. Transduction of mature miR-34a decreased β-catenin abundance (left panels) and Wnt UTR activities (middle panel; *P < 0.01 compared to control, t test). Cotransfection with an APC expression vector and miR-34a additively, and independently, repressed TOPflash activities in colorectal cancer cells (right panel; *P < 0.01 compared to control, t test). Blots are representative of two independent experiments. (C) Negative control (−) or miR-34a (+)–transduced SW480 or SW620 cells were cultured atop the embryonic chick CAM (upper CAM surface demarcated by red arrows) and the invasive activity of cancer cells (labeled with green fluorescent beads) was monitored by fluorescence microscopy. Yellow arrowheads indicate invading cancer cells. White arrows and blue arrowheads denote the basement membrane of the upper CAM and blood vessels in mesenchymal CAM tissues, respectively. Scale bars, 100 μm.

Discussion

Here, we have demonstrated that the miR-34 family links p53 activity with the canonical Wnt pathway through miR-34–specific interactions with the UTRs of multiple Wnt pathway genes and thereby to the inhibition of β-catenin–TCF/LEF–dependent transcriptional activity. The UTRs of proto-oncogenes, including β-catenin, are often shortened in cancer cells as a consequence of alternative cleavage and polyadenylation, resulting in both enhanced mRNA stability and heightened transforming activity (51, 52). Our findings, in conjunction with these and other studies, suggest that the loss of normal regulatory interactions between tumor suppressor miR-34 family members and their target UTRs in genes associated with the canonical Wnt pathway can lead to a sustained amplification of oncogenic signaling (21). In addition, the activation of Wnt signaling that occurs as a consequence of the loss of p53 and miR-34 function may provide a genetic basis for the developmental defects and cancer-prone status of p53-mutant mice (14, 15, 53). Because p53–miR-34 targets such as LRP6 have also been implicated in G protein–coupled receptor signaling (54, 55), the regulatory function of the p53–miR-34 axis likely extends beyond development and neoplasia alone.

Although our findings establish a direct link between the p53–miR-34 axis and WNT1, LRP6, β-catenin, and LEF1 expression, additional Wnt-related target genes of the p53–miR-34 axis are probably affected. For example, increases in the abundance of WNT1 and AXIN2, two miR-34 targets (44, 56), stabilize the nuclear abundance of the transcriptional repressor, Snail1, thereby triggering a tissue-invasive phenotype in cancer cells by promoting EMT (6, 49, 57). Although we were unable to identify Wnt repressor functions among the intergenic miRNAs miR-27, miR-30, or miR-192 (32), other p53-regulated miRNAs may work in tandem with miR-34. Indeed, recent reports have linked p53 to the regulation of miR-200 family members (58). In human cancers, the miR-200 family has also been characterized as an EMT suppressor as a result of its targeting of the E-cadherin repressors, zinc finger E-box binding homeobox 1 (ZEB1) and ZEB2 (24, 59). miR-200 also targets the UTR of β-catenin (60), although the functional outcomes of this regulation are unknown. We speculate that miR-34 and miR-200 family members coordinately regulate Wnt signaling in response to changes in p53 activity.

The tumor suppressor function of p53 in bona fide oncogenic signaling events, such as those linked to MYC, RAS, and E1A, has long been noted (8, 61), but direct molecular links between the transcriptional activity of p53 and oncogenic transformation have remained largely undefined. MYC orthologs are downstream target genes of canonical Wnt signaling cascades and have been identified as miR-34 targets (62, 63). Further, activation of Wnt–β-catenin and Myc pathways is correlated with aggressive behavior in human cancers in terms of triggering EMT-like changes in cancer cells or promoting the adoption of cancer stem cell–like features (4, 64). Given studies implicating miR-34 and p53 in the self-renewal of cancer stem cells (25, 64), both oncogenic signaling and stem cell renewal may fall within the purview of the p53–miR-34 axis described herein.

MiR-34a and miR-34b/c are located on chromosomes 1p36 and 11q23, respectively, regions in which deletions are associated with early-stage cancers and with the more bleak prognoses associated with sporadic and familial cancers (34, 35, 38, 65). Furthermore, chromosomal losses in these regions are commonly found in familial malignant melanoma–dysplastic nevus syndrome, familial gliomas, and pediatric neuroblastomas (34, 35, 65). Although various genes map to these sites, conventional tumor suppressors other than miR-34a and miR-34b/c have not been identified in these regions (33, 36). Loss of 1p and p53 mutation is mutually exclusive in gliomas, supporting a specific role for miR-34 in tumorigenesis (66, 67). Notably, epigenetic silencing of miR-34 by aberrant CpG methylation also occurs in various types of human cancer (37, 39). Therefore, sustained activation of Wnt signaling may arise independently of p53 mutations as a consequence of chromosomal loss or epigenetic silencing of miR-34. Thus, we propose that miR-34–dependent perturbations in the interconnected p53 and canonical Wnt pathways endow cancer cells with the ability to subvert p53-dependent tumor suppressor programs while activating the Wnt-dependent machinery that supports clonal expansion and the acquisition of the aggressive phenotypes associated with cancer progression.

Materials and Methods

Cell lines and chemicals

MCF-7, A549, and 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (5, 6). SW480 and SW620 cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM with 10% FBS. Colorectal cancer cells LoVo and SNU-81 were obtained from the Korean Cell Line Bank. p53-null MEFs were obtained from ATCC (CRL-2821) and p53 floxed mice were provided by J.-L. Guan (University of Michigan). Primary dermal fibroblasts were obtained from mouse skin. The functional status of p53 in various cancer cell lines was confirmed through the TP53 database (http://www-p53.iarc.fr/). The MDM2 inhibitor Nutlin-3 (Sigma) was dissolved in dimethyl sulfoxide and used at a final concentration of 10 μM. Conditioned medium for exogenous Wnt treatment was prepared from the conditioned media of Wnt3a-producing L cells (5, 6).

Constructs and viral transduction

A retroviral expression vector of HPV-E6 was constructed by cloning of HPV-E6 ORF (68) into a pQCXIP retroviral expression vector (Clontech). Lentiviral shRNA-dsRed-VSVG (vesicular stomatitis virus glycoprotein) construct for p53 (69), control vector with packaging constructs, and adenovirus expressing Cre recombinase were obtained from the University of Michigan Vector Core. TOPflash (pGL3-OT), FOPflash (pGL3-OF), reporter constructs for p21 and PUMA, wild-type p53 (plasmid 16434), and mutant p53 (plasmid 16346 for R175H and plasmid 16439 for R273H) expression vectors were obtained from Addgene. An expression vector for the p53 transactivation domain (TAD) deletion mutant was generated with polymerase chain reaction (PCR)–based methods. Wild-type and mutant p53 were subcloned into pLentilox–IRES (internal ribosomal entry site)–GFP (green fluorescent protein) for lentiviral transduction. Flag-tagged wild-type β-catenin, pCMV-APC, and pMSCV–miR-34a and -34b/c expression vectors were provided by E. R. Fearon (University of Michigan). Adenovirus expressing GFP (ad-GFP) and GFP-p53 (ad-GFP-p53) were purchased from Vector Biolabs. p53-null MEFs were transduced with adenoviral expression vectors by directly applying the diluted viruses to the culture medium at 100 multiplicity of infection. Transduction efficiency was determined by direct visualization using fluorescence microscopy of GFP-expressing cells. All expression and reporter vectors used in this study were verified by sequencing.

miR-34 family target screening and prediction

The miR-34 family and other miRNA sequences of various species were obtained from miRBase (http://www.mirbase.org/index.shtml). The initial miBridge prediction set was used to identify potential miRNAs targeting more than two Wnt signaling genes (44), one of which being either WNT1, β-catenin (CTNNB1), or LRP6. The parameters of this prediction algorithm include seven or more consecutive matches of Watson-Crick hybridization with an energy threshold at −13 kcal/mol by means of RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). The miBridge algorithm was then modified including the energy threshold for the 5′UTR interaction at −14 kcal/mol and the 3′UTR interaction at −15 kcal/mol to predict additional targets among the initially screened miRNAs. Additional targets were predicted through the miRcore service (http://mircore.org). The identified 3′UTR interaction sites were compared with TargetScan and Pictar targets and checked for conservation by means of the UCSC Genome Browser (http://genome.ucsc.edu/).

UTR reporter assays and miRNA constructs

A luciferase expression construct with multiple cloning sites for UTRs was used as described previously (44). The 3′UTRs of WNT1 (NM_005430; +1 to +920), WNT3 (NM_030753; +1 to +151), β-catenin (NM_001904; +1 to +1157), and LEF1 (NM_016269; +6 to +712) were amplified from genomic DNA of MCF-7 cells and subcloned into the Bam HI and Not I sites downstream of luciferase. For the UTR of LRP6 (NM_002336), the UTR reporters of LRP6-1 (+1 to +230) and of LRP6-2 (+1036 to +2401) were generated using the same method. The miRNA expression vectors for miR-27a (−296 to +235), miR-30a (−358 to +197), miR-98 (−354 to +199), and miR-192 (−321 to +213) were constructed from PCR-amplified products obtained from the genomic DNA of MCF-7 cells followed by subcloning into the pLPCX vector (Clontech). As a positive control reporter for miRNAs, synthetic oligonucleotides of complementary sequences for each miRNA were hybridized and inserted into Not I and Xba I sites downstream of luciferase. β-Catenin expression vectors in which the UTR was serially deleted or retained were constructed by substituting Flag-tagged β-catenin complementary DNA (cDNA) for firefly luciferase in a control luciferase vector (β-catenin–CDS) or a β-catenin UTR reporter construct after serial deletion of the UTR by PCR [β-catenin–CDS–UTR–nucleotide (nt) +1 to +170, –nt 1 to 480, –nt −1 to 790]. Mutant UTR reporter constructs were constructed by deletion of the seed-matched portion of the potential target sites of Wnt1, Wnt3, and β-catenin (1 to 170). Cells were cotransfected with each of the UTR reporter constructs (2 to 100 ng) and miR-34 family expression vectors (200 to 500 ng) or synthetic miRNA (Ambion) as indicated. As a transfection control, 1 ng of SV-40–Renilla construct (Promega) was cotransfected with the reporter vectors. For functional analyses of miRNA precursors, 20 nM miR-34 (PM11030) or a negative control (AM17110) was transfected with Lipofectamine 2000 (Invitrogen). For functional inhibition of endogenous miRNA, cells were treated with anti–miR-34a inhibitor (AM11030) or a negative control (AM17010) a total of two times at 2-day intervals. Cells were lysed 48 hours after transfection, and the relative ratio of Renilla to firefly luciferase was determined by dual-luciferase assay (Promega). All reporter assays were performed in triplicate.

Microarray data analysis of clinical samples

Publicly available gene expression data from the Uppsala breast cancer cohort (GSE3494, raw data) and a primary neuroblastoma series (GSE13141, processed by GCOS 1.1.1 and normalized using GeneSpring GX) using HG-U133A Plus 2.0 arrays were downloaded from the Gene Expression Omnibus (40, 43). The functional status of p53 DLDA (diagonal linear discriminant analysis) classifier, as well as mutational status of p53 cDNA for 251 patients with breast cancer, was also obtained from GSE3494. The breast cancer data set consists of 193 cases expressing wild-type p53 and 58 cases harboring p53 mutations, representing 179 cases of wild-type DLDA function and 72 cases of mutant p53 function (40). The data set for 30 cases of pediatric neuroblastoma samples is composed of 12 cases of chromosome 1p deletion and 6 cases of 11q deletion. Genes regulated by TCF/LEF and miR-34a were obtained from independently published results (32, 41) and matched to the corresponding Affymetrix probes. The TCF/LEF signature consisted of 132 probes (U133A Affymetrix chip) of 74 genes that were responsive to dominant-negative TCF4 (twofold cutoff) in colon cancer cells (41) and a miR-34a signature consisting of 56 probes (U133A Affymetrix chip) that were down-regulated by miR-34a expression in HCT116 DicerEX5 cells (GSE7864) (32). The probe ID and gene list are listed in table S5. The raw breast cancer data were normalized with the Robust Multichip Average (RMA) as implemented in the “rma” function in the R Bioconductor “affy” package. For an unsupervised hierarchical cluster analysis of TCF/LEF or miR-34–responsive genes, Ward linkage method was used together with the Pearson distance (breast cancer samples) or the Euclidean distance (neuroblastoma samples) for both sample and gene clustering. The statistical significance of the association between the hierarchical clusters of TCF/LEF genes and p53 groups of breast cancer, or between the clusters and 1p/11q deletion of neuroblastoma, was determined by two-tailed Fisher’s exact test. Patient subgroups with high and low TCF/LEF activity, as determined by the hierarchical clustering, were used to assess the statistical association between hierarchical clusters of miR-34 genes and TCF/LEF subgroups, as well as the association between the clusters and p53/1p status. To validate the functional status of p53 in breast cancer samples, we compared the transcript expression levels of known p53 target genes relative to the p53 status and determined the differences in transcript levels by one-sided t test. The list of p53 downstream target genes was obtained from previously published results (11).

Xenopus axis duplication and animal cap assay

Xenopus laevis eggs were fertilized, de-jellied, and incubated in 0.1× modified Barth’s solution (MBS) [1× MBS: 88 mM NaCl, 1 mM KCl, 0.7 mM CaCl2, 1 mM MgSO4, 5 mM Hepes (pH 7.8), and 2.5 mM NaHCO3]. For the axis duplication assay, β-catenin mRNA (5 pg/nl) without its UTR, with various sized UTRs, or with mutant UTRs synthesized with the Riboprobe system (Promega) was injected into one of the ventral vegetal blastomeres of four- to eight-cell–stage embryos. The embryos were then allowed to develop at room temperature in 0.1× MBS, and axis duplication was scored at the tadpole stage. For axis duplication with Wnt8, 30 pg of Wnt8 mRNAs was co-injected with 0.4 pmol of control miRNA or miR-34 into one of the ventral vegetal blastomeres of four- to eight-cell–stage Xenopus embryos. The statistical significance of axis duplication was determined by binomial t test for single-axis embryos. For the animal cap assay, each blastomere of the X. laevis two-cell–stage embryo was injected with 5 nl of either control miRNA (40 μM), miR-34a (40 μM), anti–miR-34a (20 μM), or 50 pg of Wnt8 mRNA in the animal pole region and cultured at 16°C in 0.1× MBS buffer. Animal caps of embryos were dissected from blastula-stage embryos (stages 8.5 to 9) and cultured in 0.5× MBS buffer for 2 to 4 hours until gastrula stage (stage 10.5). Reverse transcription–PCR (RT-PCR) was performed on total RNA isolated from the animal caps with TRIzol (Invitrogen). Oligonucleotide sequences used for the RT-PCR analyses in this study are shown in table S6.

Generation of stable transfectant cells and Tet-inducible miR-34a cells

pQCXIP–HPV-E6 expression or control vector was used to generate retroviral stocks in 293 cells for subsequent infection of MCF-7 or A549 cells. Stable HPV-E6 transfectants were obtained after selection with puromycin (1.0 μg/ml). For stable knockdown of p53, each cell line was transduced with lentiviral shRNA for p53 or control vector. Stable knockdown of p53 by HPV-E6 or shRNA was confirmed by Western blot analysis. A primary transcript sequence from pMSCV–miR-34a vector was subcloned into episomal doxycycline-inducible expression vector (6). MCF-7 cells were then transfected with episomal miR-34a expression vector and selected with hygromycin (200 μg/ml). The Tet-inducible cells were maintained in Tet-free condition, and doxycycline was directly added to Tet-free culture medium at the indicated concentrations for miR-34a induction.

RT-PCR and immunoblot analysis

Conventional reverse transcriptase with random hexamers was used for synthesis of cDNA for primary transcripts of human and mouse miR-34a and miR-34b/c. Primer3 software was used to design the primers to amplify the miR-34 family pri-miRNA and the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCRs were performed for miR-34a (30 cycles) and miR-34b/c (34 cycles) and annealed at 57°C. The amplified product was visualized by an ultraviolet illuminator and then cloned into TA vector to verify the sequence of the PCR products. Real-time quantitative PCR (qPCR) analysis for pri-miR-34a and proven β-catenin target genes was performed with an ABI-7300 instrument under standard conditions and SBGR mix (n = 3). The list of 13 direct target genes of β-catenin was obtained from the Nusse home page (http://www.stanford.edu/~rnusse/wntwindow.html). The expression of ΔCt value from each sample was calculated by normalizing with GAPDH. Primer specificity and PCR were verified by dissociation curve after PCR. For quantitative analysis of mature miR-34a levels, human TaqMan miRNA assay kits (Applied Biosystems, assay ID 000426 for miR-34a, ID 001001 for RNU24, and ID001973 for U6 snRNA) were used for reverse transcription with specific primers, and qPCR was performed with corresponding probes (n = 3). The expression of mature miR-34a ΔCt values from each sample was calculated by normalizing with RNU24 or U6 expression values. Conservation of UTR and miR-34 target sites in β-catenin mRNA in HCT116 and SNU-407 cells was confirmed by RT-PCR and sequence analysis. siRNA oligo duplexes for negative control and p53 were purchased from Santa Cruz. The specificity of antibodies for WNT1 and LRP6 detection was determined with hemagglutinin-tagged expression vectors. Truncated APC in colon cancer cells was detected by Western blot and the corresponding molecular weight. To detect endogenous protein expression, we subjected total cell lysates in Triton X-100 buffer (20 μg for LRP6, WNT1, and APC; 1 to 2 μg for p53 and β-catenin) to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Antibodies directed against LRP6 (C5C7; Cell Signaling Technology), WNT1 (Abcam), β-catenin (BD Biosciences), APC (H-290, Santa Cruz), M2 monoclonal antibody (mAb) Flag tag (Sigma), p53 (DO-1 or pAb240, Santa Cruz), and tubulin (LabFrontier) were obtained from the commercial vendors.

CAM invasion assay

Noninvasive, dsRed-expressing SW480 or SW620 cells transfected with negative control or miR-34a were labeled with Fluoresbrite carboxylate nanospheres (Polysciences Inc.). Colon cancer cells were cultured on the CAM of 11-day-old chick embryos for 3 days as described previously (5, 6, 49). Type IV collagen staining of frozen CAM sections was performed with a chick-specific mAb (IIB12) (5) and Alexa Fluor–labeled secondary antibodies. Invasion was monitored in cross sections of the fixed CAM by fluorescence microscopy as described (5, 6, 49). DAPI (4′,6-diamidino-2-phenylindole) (Molecular Probes) was used to stain cell nuclei.

Statistical analysis

Statistical significance of reporter assays and qPCR was determined by the Student’s t test. Differences were considered significant when the P value was less than 0.05 or 0.01 as indicated in the text.

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/4/197/ra71/DC1

Fig. S1. Loss of p53 function increases Wnt activity.

Fig. S2. Loss of p53 function increases TCF/LEF target gene expression.

Fig. S3. miR-34 transactivated by p53 suppresses Wnt activity.

Fig. S4. miR-34 targets UTRs of Wnt genes.

Fig. S5. Loss of p53 function potentiates Wnt UTR activity.

Fig. S6. Gain of p53 function attenuates Wnt activity.

Fig. S7. Loss of p53/miR-34 potentiates Wnt activity in colorectal cancer cells.

Fig. S8. Interactions between miR-34 and β-catenin UTR are functional during in vivo invasion.

Table S1. hsa–miR-34a match sites on Wnt genes.

Table S2. hsa–miR-34b* and hsa–miR-34c-5p match sites on Wnt genes.

Table S3. Highly conserved miR-34a sequences in vertebrates.

Table S4. Conserved miR-34a match sites in vertebrate Wnt genes.

Table S5. List of TCF/LEF and miR-34 signature genes using hierarchical clustering analysis.

Table S6. Primer oligonucleotide sequences for qPCR analysis.

References and Notes

  1. Acknowledgments: We thank Y. S. Lee for critical reading of the manuscript, E. Tunkle and Y. H. Cha for preparation of the manuscript, and Y.-M. Huh for supervising microarray data analysis. E. R. Fearon and G. T. Bommer provided constructs and cell lines along with helpful discussion. Funding: This study was supported by grants from the National R&D Program for Cancer Control (0720270 and 1020110), Korea Health Technology (A080916 and A084699), Ministry of Health and Welfare, National Research Foundation of Korea funded by the Korean government (Ministry of Education, Science and Technology) (2011-0000358, 2011-0002620, 2010-0029703, R15-2004-024-00000-0), Korea Research Institute of Chemical Technology (SI-1105), and GIST Systems Biology Infrastructure Establishment Grant through Ewha Research Center for Systems Biology. This work is also supported by NIH grants R37 GM037432 (B.M.G.) and CA116516 (S.J.W.) and the Breast Cancer Research Foundation (S.J.W.). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions: H.S.K., X.-Y.L., K.K., S.J.W., and J.I.Y. conceived and designed the project. N.H.K., H.S.K., S.E.K., S.Y.C., J.K.R., and J.M.N. performed experiments. N.-G.K. performed the Xenopus experiments. I.L., H.-S.C., C.P., and S.L. analyzed bioinformatics data. H.S.K., B.M.G., J.I.Y., and S.J.W. handled project planning and wrote the paper. Competing interests: I.L. is the founder of the nonprofit organization miRcore; there is a patent pending on the miBridge target prediction method, which is owned by the University of Michigan and exclusively licensed to miRcore. The other authors declare that they have no competing financial interests.
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