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. Author manuscript; available in PMC: 2011 Jul 3.
Published in final edited form as: Oncogene. 2011 Feb 7;30(22):2587–2594. doi: 10.1038/onc.2010.634

Myc overexpression brings out unexpected antiapoptotic effects of miR-34a

E Sotillo 1, T Laver 2, H Mellert 3, JM Schelter 4,6, MA Cleary 4,6, S McMahon 3,5, A Thomas-Tikhonenko 1,2,3
PMCID: PMC3128883  NIHMSID: NIHMS299619  PMID: 21297663

Abstract

Downregulation of microRNA-34a by Myc is known to be essential for tumorigenesis and improve tumor-cell survival. Conversely, upregulation of miR-34a by p53 is thought to enhance its acetylation and activity and contribute to the pro-apoptotic effects of this tumor suppressor. We sought to determine whether restoration of miR-34a levels in B-lymphoid cells with Myc overexpression would aid therapeutic apoptosis. Unexpectedly, delivery of miR-34a, which doesn’t target p53 directly, severely compromised steady-state p53 levels. This effect was preceded and mediated by direct targeting of Myc, which sustained p53 protein levels via the Arf–Hdm2 pathway. As a result, in the presence of Myc, miR-34a inhibited p53-dependent bortezomib-induced apoptosis as efficiently as anti-p53 small interfering RNA. Conversely, inhibition of miR-34a using antisense RNA sensitized lymphoma cells to therapeutic apoptosis. Thus, in tumors with deregulated Myc expression, miR-34a confers drug resistance and could be considered a therapeutic target.

Keywords: microRNA, Myc, p53

Introduction

Burkitt’s lymphoma (BL) is a hematological neoplasm carrying a telltale t(8;14) cytogenetic alteration, which juxtaposes the MYC proto-oncogene and the immunoglobulin heavy chain enhancer resulting in constitutive expression of Myc protein in B-cells. The majority (70%) of BL retain wild-type p53 (Lindstrom and Wiman, 2002), although the incidence of p53 mutations in cell lines is double that found in human biopsies (Bhatia et al., 1992). This combination has profound consequences for cell homeostasis as Myc is a key transcription factor that regulates cell proliferation, growth, differentiation and p53-dependent apoptosis, primarily via the ARF/HDM2/p53 axis. Specifically, deregulated Myc activates p19ARF, which in turn interacts with and inhibits HDM2, an E3 ubiquitin ligase targeting p53 for proteasomal degradation. Arf-stabilized p53 is capable of launching an apoptotic program that includes direct transcriptional activation of death-inducing genes, such as NOXA, PUMA and BAX (reviewed in Junttila and Evan, 2009). Yet for this to occur p53 has to be acetylated on residue K382 and moreover, K382-acetylation has to be protected from SIRT-1, a NAD-dependent deacetylase (Vaziri et al., 2001). This is accomplished, at least in part, through the capacity of p53 to induce expression of several micro-RNAs, in particular miR-34a.

miRNAs are short, 18–24 nucleotides molecules of RNA that regulate the stability and translational efficiency of target mRNAs (Bartel, 2009) and have important roles in cancer. For example, miR-34a has been described as pro-apoptotic and growth-suppressive (reviewed in He et al., 2007b). Consistent with these findings, the corresponding chromosomal region (1p36) is frequently deleted in a variety of cancers, including neuroblastomas, pancreatic, breast, hepatic and colon carcinomas (reviewed in Bagchi and Mills, 2008). Also, epigenetic silencing of miR-34a has been identified in tumor-derived cell lines and in primary melanomas (Lodygin et al., 2008). Conversely, p53 robustly activates miR-34a expression under conditions of genotoxic stress (Bommer et al., 2007; Chang et al., 2007; Corney et al., 2007; Raver-Shapira et al., 2007; Tarasov et al., 2007; He et al., 2007a). Furthermore, one of the better validated targets of miR-34a is SIRT1 (Yamakuchi et al., 2008), an NAD-dependent deacetylase capable of de-modifying amino acid K382 in p53 and thus compromising p53 activity (Vaziri et al., 2001).

The existence of the positive feed-back loop wherein p53 induces miR-34a and mir-34a activates p53 by inhibiting SIRT1, suggests that increasing the amount of miR-34a in cells that retain wild-type p53 could enhance therapeutic apoptosis (Yamakuchi and Lowenstein, 2009). This is because p53 dictates apoptotic response not only to DNA-damaging agents, but also to other anticancer drugs. For example, in Myc-induced bone marrow-derived B-lymphomas (Yu and Thomas-Tikhonenko, 2002; Yu et al., 2005) we observed that tumor cell killing by proteasome inhibitors (for example, bortezomib) requires functional p53. Specifically, when the dormant p53-ER fusion protein was activated with estrogen (Amaravadi et al., 2007), previously resistant B-lymphoma cells became sensitive to bortezomib (Yu et al., 2007).

The concept of miR-34a augmentation seemed especially important in the context of Myc overexpression. This is because Myc also regulates multiple miRNAs (O’Donnell et al., 2005; Dews et al., 2006; Chang et al., 2008; Lin et al., 2009), including miR-34a. However, unlike p53, Myc represses miR-34a expression, and low miR-34a levels are a hallmark of Myc-driven lymphomas (Chang et al., 2008; Chang et al., 2009). Thus, we set out to test whether restoration of miR-34a expression in Myc-overexpressing, TP53wt-containing tumor cells could be of therapeutic use.

Results and discussion

miR-34a expression or Myc silencing induce downregulation of p53 in B-lymphoid cell lines

We first asked whether restoration of miR-34a in B-lymphoid cell lines with intact p53 results in increased p53 activity. To answer this question, we chose P493-6 cells, an established human Epstein-Barr virus EBNA1/EBNA2-positive B-cell line, in which Myc is expressed under the control of a tetracycline-regulated promoter. Thus, the genetic make-up of P493-6 cells resembles that of BL (Pajic et al., 2000; Schuhmacher et al., 2001). We transfected miR-34a mimics into P493-6 cells via electroporation, and measured steady-state levels of miR-34a by quantitative reverse transcription–PCR. Relatively modest, <10-fold overexpression of miR-34a was observed (Figure 1a). It was similar in scope to that seen in lymphomas with inactivated Myc (Chang et al., 2008) and thus not supra-physiological.

Figure 1.

Figure 1

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Ectopic expression of miR-34a or silencing of Myc downregulates p53 in B-lymphoid cells. (a) miR-34a levels in P493-6 cells transfected using Amaxa nucleofector (Amaxa, Lonza, Switzerland) with 300 nM of miR-34a mimic or control miRNA (Dharmacon, Lafayette, CO, USA) and harvested after 24 h. Total RNA was isolated following the TRIzol reagent protocol (Invitrogen, Carlsbad, CA, USA). miRNA expression was analyzed using TaqMan microRNA assay and relative abundance of miR-34a was measured by qRT–PCR and normalized to that of RNU6B. (b, c) Levels of SIRT-1 and other lysine-acetylated proteins in P493-6 cells transfected with miR-34a or control miRNA as in a. Acetylated proteins were extracted as previously described (Sykes et al., 2006) and protein levels were assessed by western blotting using the following antibodies: anti-SIRT-1(H-300) and anti-p53(FL-393) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-acetyl-p53-Lys382 (#2525) from Cell Signaling (Danvers, MA, USA), anti-acetyl-histone 4-K16 (#39167) from Active Motif (Carlsbad, CA, USA), anti-acetyl lysine (#06-933) from Upstate (Lake Placid, NY, USA) and anti-β-actin from Sigma-Aldrich (St Louis, MO, USA). Arrows denote unidentified proteins, levels of acetylation of which increased upon overexpression of miR-34a. (d) Indicated B-lymphoid cell lines were cultured and maintained in RPMI 1640 medium supplemented with 10% tetracycline-free fetal bovine serum, penicillin and streptomycin at 37 °C. Cells were transfected as in a and protein levels where analyzed by western blotting. (e) Protein levels of Myc and p53 in P493-6 cells treated with 0.1 μg/ml doxycycline (DOX) (Sigma-Aldrich). Cell lysates were prepared at the indicated time points and analyzed by western blotting using the anti-Myc (9E10) (Calbiochem, EMD Biosciences, Darmstadt, Germany) or p53 antibodies. (f) mRNA levels of p53 and Myc in P493-6 cells from panel b were analyzed by qPCR. Extracted RNAs were treated with DNAse (Turbo DNA-free KIT, Ambion, Austin, TX, USA) and then reverse-transcribed into DNA using SuperScript III First-Strand Synthesis System from Invitrogen. Real-time PCR was performed using an ABI 7900 Sequence Detection system with the SYBR Green PCR core reagent Kit (Applied Biosystems, Foster City, CA, USA). QuantiTect Primer Assays for Myc, p53 and β-Actin (control) were obtained from Qiagen (Valencia, CA, USA). (g) Protein levels of Myc and p53 in P493-6 cells treated with doxycycline in the presence of 12.5 μM Nutlin-3 (Sigma-Aldrich), as measured by Western blotting.

To assess the functionality of the miR-34a mimic, we first analyzed its well-known target SIRT1. As expected, under our experimental conditions, the protein levels of SIRT1 were considerably reduced by ectopic miR34a expression, causing a commensurate increase in the acetylation of known SIRT1 targets such as lysine-16 of histone 4 (H4K16) (Brachmann et al., 1995) and other lysine residues detectable using a pan-specific acetyl lysine antibody (Figure 1b). Surprisingly, the acetylation status of the SIRT1-specific residue K382 on p53 was not affected despite strongly reduced SIRT1 levels (Figure 1c). One possible explanation of this result was that miR-34a-induced acetylation of p53 was mitigated by reduced p53 steady-state levels.

To test this idea, we transfected miR-34a or a control mimic into four B-lymphoid cell lines: P493-6 (see above), LY47 (BL), GM609 (EBV-immortalized lymphoblasts) and Nalm-6 (B-cell acute lymphocytic leukemia), all of which retain wild-type p53 (Lindstrom et al., 2001; Adachi et al., 2006) and express similar levels of endogenous Myc (data not shown). In all cell lines tested, ectopic expression of miR34a induced downregulation of both SIRT1 and p53 (Figure 1d).

As p53 is not a predicted target of miR-34a and its mRNA was not affected by miR-34a in a genome-wide gain-of-function screen ((Linsley et al., 2007) (data not shown)), we considered the involvement of key positive regulators of p53, such as Myc. To determine whether Myc regulates p53 in P493-6 cells, we used treatment with doxycycline. Myc production was shut down quickly after addition of doxycycline, and as Myc levels plunged, so did levels of p53 (Figure 1e). We next sought to determine whether regulation of p53 by Myc occurs at a transcriptional or a post-transcriptional level. Although mRNA levels of Myc decreased sharply, p53 mRNA levels did not change appreciably upon addition of doxycycline (Figure 1f). This suggested that the TP53 gene is transcribed even in the absence of Myc, but its protein levels cannot be sustained unless Myc is co-expressed. We were unsuccessful in verifying Arf expression by western blotting. However, in the presence of Nutlin-3a, a direct inhibitor of HDM2 (Vassilev et al., 2004), there was no difference in p53 levels between Myc-positive and -negative cells (Figure 1g). Therefore, in P493-6 cells Myc-mediated stabilization of p53 takes place at a post-transcriptional level through the Myc -> ARF -| HDM2 axis. Thus, it was possible that the observed effects of miR-34a on p53 were mediated by Myc.

miR-34a regulates p53 in a Myc-dependent manner

Indeed, of all known positive regulators of p53, only Myc exhibited marginal downregulation at the mRNA level in the genome-wide gain-of-function screen performed ((Linsley et al., 2007) (data not shown)). In addition, others have recently shown that Myc 3′ untranslated region (UTR) contains a non-canonical seed sequence for all miR-34 family members (Kong et al., 2008; Christoffersen et al., 2010), suggesting that miR-34a could inhibit Myc protein expression and in doing so bring down p53 levels. Unfortunately, P493-6 cells are not amenable to retrovirus transduction. Thus, to validate Myc as a miR-34a target, we first used human primary fibroblasts immortalized with telomerase (BJ-hTERT) and expressing undetectable levels of endogenous Myc.

BJ-hTERT cells were transduced with retroviral constructs expressing either myc with the full-length 3′UTR (Myc-3′wt), or myc without 3′UTR (Myc-Δ3′UTR), or myc with the 3′UTR bearing a mutation in the putative miR-34a binding site (Myc-3′mut). As shown in Figure 2a, miR-34a strongly downregulates retrovirally encoded Myc in a manner dependent on the presence of 3′UTR. This downregulation was impaired—but interestingly, not completely abolished—when the known miR-34a binding site (Kong et al., 2008) was mutated (Figure 2b). This suggested the existence of other miR-34a binding sites, and the newer target prediction algorithms such as RNA22 (Miranda et al., 2006) indeed reveal several additional sites in MYC 3′UTR (data not shown). These sites typically have 1–2 mismatches in the seed homology region, which are offset by additional base pairing at the 5′ end. Such non-canonical sites have been recently found and validated in other genes (Lal et al., 2009).

Figure 2.

Figure 2

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miR-34a downregulates p53 in a Myc-dependent manner. (a) Myc levels in human primary fibroblasts immortalized with telomerase (BJ-hTERT), infected with Myc retroviral vectors carrying full-length 3′UTR (WT) or no 3′UTR (Δ-3′UTR), and then transfected with miR34a or control mimics for 48 h using Lipofectamine 2000 (Invitrogen) following manufacturer’s instructions. (b) Levels of expression of Myc and SIRT-1 in BJ-hTERT cells infected with retroviral Myc vector with 3′UTR in either with wild type (WT) or miR-34a seed-mutated conformation (MUT). Cells were transfected with miR-34a or control mimics as in a. (c) Protein levels of Myc and p53 in P493-6 cells transfected with miR-34a or control mimic. Cell lysates were prepared at 24 and 48 h post-transfection and analyzed by western blotting. Doxycycline-treated P493-6 cells were used for comparison. (d) Protein levels of Myc and p53 in lysates from P493-6 cells transfected with miR-34a or control mimic and treated with doxycycline for 48 h or left untreated. (e) Protein levels of HDM2, Myc and p53 in P493-6 cells transfected with siRNA against p53 (si-p53) or control siRNA (Dharmacon) for 48 h. HDM2(Ab-1) antibody was from Calbiochem. (f) Protein levels of HDM2, Myc and p53 in P493-6 cells transfected with an anti-sense inhibitor of miR-34a or control inhibitor and treated with doxycycline for 24 h or left untreated. DOX, doxycycline.

To determine whether downregulation of p53 by miR-34a is Myc-mediated and Myc-dependent, we first performed a time-course experiment wherein P493-6 cells were transfected with miR-34a and harvested after 24 and 48 h. Data in Figure 2c show that 24 h after transfection, the levels of Myc were already lower than in control cells, while the levels of p53 have not yet changed. However, 48 h after transfection, both p53 and Myc protein levels were suppressed, suggesting a sequence of events where Myc downregulation precedes that of p53.

To test whether p53 downregulation by miR-34a required Myc targeting, we repeated the miR-34a mimic experiment in the absence or presence of doxycycline (that is, in the presence or absence of Myc). When miR-34a was introduced in the absence of Myc, no changes in the levels of remaining p53 were detected (Figure 2d) indicating that Myc mediates the effect of miR-34a on p53.

Furthermore, to test whether the miR-34a -| Myc -> p53 axis functions without miR-34a overexpression, we inhibited endogenous miR-34a by transfecting the anti-miR- 34a antisense inhibitor into P493-6 cells. miR-34a levels were reduced 60–80% as a result of this treatment (data not shown). As only partial reactivation of p53 was expected, we wanted to use as an additional readout Hdm2, a transcriptional target of p53 (Barak et al., 1993; Wu et al., 1993). To confirm that in P493-6 cells p53 controls Hdm2 directly and independently of Myc, we depleted p53 using transient transfection with small interfering RNA (siRNA) and demonstrated that p53 knockdown is sufficient to sharply downregulate Hdm2 (Figure 2e). Thereafter, the effects of miR-34a inhibitor were studied in DOX-treated and -untreated cells. In the absence of doxycycline, we observed upregulation of both Myc and p53 (Figure 2f, left two lanes). The upregulation of p53, while modest, was functionally significant, as it resulted in upregulation of Hdm2. However, in the presence of doxycycline Myc was not expressed and neither p53 nor Hdm2 were induced by the miR-34a inhibitor (Figure 2f, right two lanes). Thus, previous targeting of Myc by miR-34a is both necessary and sufficient for the downregulation of p53.

Bortezomib induces apoptosis in Myc-transformed cells in a p53-dependent manner

We had previously reported that the p53 status determines the response of B-cell lymphomas to bortezomib (Yu et al., 2007). To confirm that P493-6 cells die by apoptosis in response to treatment with bortezomib, we measured cell death using several flow-cytometry-based assays, as recommended in ‘Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes’ (Galluzzi et al., 2009). First, after treating P493-6 cells with 1 nM bortezomib we added tetramethylrhodamine ethyl ester perchlorate (TMRE) to the culture plates. TMRE localizes in the mitochondria of healthy cells and is released upon depolarization of the mitochondrial membrane. We also measured binding of Annexin V to phosphatidylserine-positive membrane surfaces, which are exposed on apoptotic cells. In the third assay, we determined the integrity of the nuclear membranes by adding to the culture plates Yo-Pro, a monomeric cyanine nucleic-acid stain that binds double-stranded DNA. All three flow-cytometric analyses consistently demonstrated the presence of 27–42% of apoptotic cells at 5 h 30 min after addition of bortezomib (Figure 3a).

Figure 3.

Figure 3

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Bortezomib induces cell death in a p53-dependent manner. (a) P493-6 cells were treated with 1 nM Bortezomib for 5 h 30min or left untreated, and the percentage of apoptotic cells was measured by flow cytometry. Yo-Pro or TMRE (Molecular Probes, Eugene, OR, USA) were added to cultures for 1 h and 30min before harvesting the cells, respectively. Annexin A5-binding analysis was performed using the Annexin V-EGFP Apoptosis Detection Kit (Biovision, Mountain View, CA, USA) following manufacturer’s instructions. SSC density dot plots are shown and the percentage of apoptotic cells is indicated. (b) P493-6 cells transfected with siRNA against p53 were treated with increasing concentrations of bortezomib for 5 h 30 min and the percentages of apoptotic cells were measured as in a. (c) P493-6 cells were transfected with siRNA targeting p53 followed by treatment with 1 nM bortezomib for 5 h 30min. Full-length and cleaved forms of PARP (arrows) were analyzed by western blotting using anti-PARP (#215–228) antibody (Calbiochem, EMD Biosciences). (d) P493-6 cells transfected with siRNA against p53 were treated with indicated concentrations of bortezomib, and caspase 3/7 activity was measured in 96-well clusters (5×104 cells/well) using Caspase-Glo 3/7 Assay (Promega, Madison, WI, USA). Luminescent signals were captured and analyzed in a Synergy 2 plate reader (BioTek Instruments, Winooski, VT, USA). Each concentration was assayed in triplicates and two-way Anova statistical tests were performed with a levels of significance P<0.01. Error bars represent s.ds.

To confirm that botezomib-induced cell death is dependent on p53, control and p53-depleted cultures were exposed to various concentrations of bortezomib and the percentage of apoptotic cells was measured by incorporation of TMRE and Yo-Pro and by binding of Annexin V. Data in Figure 3b show that, irrespective of the method used for cell-death detection, depletion of p53 interferes with bortezomib-induced apoptosis. Furthermore, western blot analysis of the cleavage of poly-ADP ribose polymerase (PARP) demonstrated that while control cells cleaved PARP as early as 6 h after treatment with the drug, in p53-depleted cells no PARP cleavage was detected (Figure 3c). Finally, we repeated this experiment using as death marker the activation of effector caspases 3 and 7, which were measured in a quantitative luminescence-based assay. As shown in Figure 3d, caspase 3/7 activity increased as P493-6 cells were treated with escalating concentrations of bortezomib (‘Control’ plot). This activity is reduced ~2.5-fold when p53 is knocked down with siRNA. Thus, bortezomib-induced cell death in P493-6 cells is indeed p53-dependent apoptosis, and the latter two assays (PARP cleavage and caspase 3/7 activation) were used to monitor it in subsequent experiments.

miR-34a is antiapoptotic in Myc-transformed cells treated with bortezomib

We next asked whether downregulation by miR-34a of Myc and eventually p53 would compromise responses to bortezomib. As anticipated, no PARP cleavage was observed in miR-34a-treated cells while control mimic-transfected cells exhibited evidence of cell death (Figure 4a). Using the caspase 3/7 assay, we determined that in untreated cells miR-34a and anti-p53 siRNA have no effects on cell viability (‘0 nM’ in panel 4b). However, in the presence of bortezomib, miR-34a inhibited cell death as efficiently as did anti-p53 siRNA (Figure 4b). To determine if the reverse is also true, we transfected P493-6 cells with the anti-sense miR-34a inhibitor (see Figure 2f) before bortezomib treatment. As shown in Figures 4c and d, miR-34a knockdown resulted in more PARP cleavage and higher caspase 3/7 activity. This increase in bortezomib sensitivity correlated well with elevated levels of both Myc and p53 (compare lanes 2 and 4 in panel 4d).

Figure 4.

Figure 4

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miR-34a renders P493-6 cells more resistant to bortezomib-induced apoptosis. (a) P493-6 cells transfected with miR-34a mimic were treated with 1 nM bortezomib for 5 h 30 min. Full-length and cleaved forms of PARP (arrows) were analyzed by western blotting using anti-PARP antibody. (b) P493-6 cells transfected with siRNA against p53 or miR-34a mimic were treated with indicated concentrations of bortezomib for 3.5 h. Caspase 3/7 activity was measured as in Figure 3d. (c, d) P493-6 cells transfected with anti-miR-34a or control hairpin inhibitors were treated with bortezomib for 24 h and analyzed as in panels a and b, respectively. In panels b and d, each concentration was assayed in triplicates and two-way Anova statistical tests were performed with a levels of significance P<0.01. Error bars represent s.ds. (e) A model depicting complex interplay between miR-34a, Myc and p53 in Myc-driven tumors.

The identification of microRNA homology sequences in the 3′UTRs of various tumor-associated genes opened a new chapter in cancer genetics and genomics and demonstrated that classically defined oncogenes and tumor suppressor genes need not encode proteins and that chromosomal abnormalities affecting micro-RNA genes can have profound effects on cancer initiation and progression (Calin and Croce, 2007). A paradigm-establishing discovery of tumor suppressive microRNAs was the demonstration that the DLEU2 locus frequently deleted in chronic lymphocytic leukemia does not contain an open reading frame and instead serves as a host gene for the miR-15a/16 microRNA cluster (Calin et al., 2002). The causative role of miR-15a/16 in chronic lymphocytic leukemia pathogenesis was recently validated in the mouse model (Klein et al., 2010). On the oncogenic side, miR-17~92 remains the prime example of a miRNA-encoding gene amplified in human cancers (Ota et al., 2004) and strongly contributing to B-lymphomagenesis in mouse models (He et al., 2005; Ventura et al., 2008; Xiao et al., 2008; Mu et al., 2009; Olive et al., 2009). Interestingly, both miR-15a/16 and miR-17~92 are regulated by Myc in a manner consistent with its oncogenic function (repression and activation, respectively).

However, other miRNAs deregulated in cancer may not fit the strict definition of cancer genes and their binary classification into oncogenes and tumor suppressor genes might be misleading. Part of the problem is that microRNAs function in certain cellular and genetic contexts and any given miRNA that promotes neoplastic growth in one cell type could inhibit it in another, depending on the repertoire of its target genes. Another important consideration is that a seemingly ‘tumor suppressive’ microRNAs can inhibit early stages of tumor pathogenesis (that is, function ‘host-friendly’) but at the same time render existing neoplasms resistant to chemo or radiation therapy (that is, function ‘tumor-friendly’). The same duality potentially applies to ‘oncogenic’ miRNAs—or protein-coding oncogenes such as Myc itself (Sala et al., 2009).

Our present study underscores both complexities. On the one hand, miR-34a is known to function down-stream of p53 and mediate pro-apoptotic and anti-proliferative effects of this tumor suppressor in non-Myc-amplified cell lines such as HTC116, H1299 and U2OS (Bommer et al., 2007; Chang et al., 2007; Corney et al., 2007; Raver-Shapira et al., 2007; Tarasov et al., 2007; He et al., 2007a). Furthermore, there are many cell lines where miR-34a has apparent growth suppressive effects. Examples include but are not limited to neuroblastoma (Welch et al., 2007; Cole et al., 2008), glioma and medulloblastoma (Guessous et al., 2010), ovarian carcinoma (Corney et al., 2010) and megakaryocytic leukemia (Navarro et al., 2009), although in other studies intrinsic growth-inhibitory effects of miR-34a were documented (Dalgard et al., 2009; Luan et al., 2010). Yet all these studies were conducted using cell lines where Myc is not known to be genetically deregulated and cell accumulation generally was not measured under treatment with chemotherapeutic drugs.

In this study we demonstrate that in Myc-driven tumors, miR-34a improves cell survival under treatment with bortezomib, based on its ability to reduce p53 levels. This surprising finding was fully attributable to Myc overexpression as in the absence of Myc (doxocyclin- treated P493-6 cells) miR-34a had no effect of p53 levels and function. Furthermore, in the absence of bortezomib, we have not observed any effects of miR-34a on intrinsic apoptosis suggesting that the regulation of p53 by miR-34a only matters in the context of chemotherapy, where miR-34a switches from being ‘host-neutral’ to ‘tumor friendly’.

Furthermore, our findings reinforce the emerging idea that Myc might be a key target of miR-34a. Although regulation of Myc 3′UTR by miR-34 family members had been observed in luciferase sensor and miRNA pull-out assays (Kong et al., 2008; Christoffersen et al., 2010), only very recently an effect of miR-34a on a Myc-driven cellular phenotype (DNA replication) has been reported (Cannell et al., 2010). Our finding that miR-34a expression compromises the Myc -> Arf -| HDM2 -| p53 axis in B-cells and overrides possible SIRT1-dependent effects on p53 (Figure 3f) is likely to have broad implications not only for B-lymphoid malignancies, but also for other tumors with Myc rearrangements.

Acknowledgments

We thank Drs Joshua Mendell and Tsung-Cheng Chang (Johns Hopkins University) for sharing unpublished data on miR-34a function in B-cells. Current and past members of our laboratories (in particular Drs Duonan Yu, James Psathas, Michael Dews and Elaine Chung) are acknowledged for many stimulating discussions. We are grateful to the Rosetta Gene Expression Laboratory for performing microarray hybridization experiments andMiho Kibukawa (Merck & Co., Inc.)—for technical support. We thank Dr Dirk Eick (GSF Research Centre, Munich) for P493-6 cells, Dr Carlo Croce (Ohio State University, Columbus) for GM607 cells and Dr Joelle Wiels (Institut Gustave Roussy, Villejuif, France) for Ly47cells. This work was supported by US National Institutes of Health grant CA 122334 to ATT and the Institutional Development Fund of the Children’s Hospital of Philadelphia (ATT), as well as NIH grants R01CA098172-07, R21CA152786-01 and R01CA090465-08 to SBM.

Footnotes

Conflict of interest

Michele Cleary is an employee of Merck Inc. The authors declare no further competing financial interests.

References

  1. Adachi N, So S, Iiizumi S, Nomura Y, Murai K, Yamakawa C, et al. The human pre-B cell line Nalm-6 is highly proficient in gene targeting by homologous recombination. DNA Cell Biol. 2006;25:19–24. doi: 10.1089/dna.2006.25.19. [DOI] [PubMed] [Google Scholar]
  2. Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, et al. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 2007;117:326–336. doi: 10.1172/JCI28833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bagchi A, Mills AA. The quest for the 1p36 tumor suppressor. Cancer Res. 2008;68:2551–2556. doi: 10.1158/0008-5472.CAN-07-2095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barak Y, Juven T, Haffner R, Oren M. mdm2 expression is induced by wild type p53 activity. EMBO J. 1993;12:461–468. doi: 10.1002/j.1460-2075.1993.tb05678.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–233. doi: 10.1016/j.cell.2009.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bhatia KG, Gutierrez MI, Huppi K, Siwarski D, Magrath IT. The pattern of p53 mutations in Burkitt’s lymphoma differs from that of solid tumors. Cancer Res. 1992;52:4273–4276. [PubMed] [Google Scholar]
  7. Bommer GT, Gerin I, Feng Y, Kaczorowski AJ, Kuick R, Love RE, et al. p53-mediated activation of miRNA34 candidate tumor-suppressor genes. Curr Biol. 2007;17:1298–1307. doi: 10.1016/j.cub.2007.06.068. [DOI] [PubMed] [Google Scholar]
  8. Brachmann CB, Sherman JM, Devine SE, Cameron EE, Pillus L, Boeke JD. The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev. 1995;9:2888–2902. doi: 10.1101/gad.9.23.2888. [DOI] [PubMed] [Google Scholar]
  9. Calin GA, Croce CM. Chromosomal rearrangements and microRNAs: a new cancer link with clinical implications. J Clin Invest. 2007;117:2059–2066. doi: 10.1172/JCI32577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA. 2002;99:15524–15529. doi: 10.1073/pnas.242606799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cannell IG, Kong YW, Johnston SJ, Chen ML, Collins HM, Dobbyn HC, et al. p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication. Proc Natl Acad Sci USA. 2010;107:5375–5380. doi: 10.1073/pnas.0910015107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH, et al. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell. 2007;26:745–752. doi: 10.1016/j.molcel.2007.05.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chang TC, Yu D, Lee YS, Wentzel EA, Arking DE, West KM, et al. Widespread microRNA repression by Myc contributes to tumorigenesis. Nat Genet. 2008;40:43–50. doi: 10.1038/ng.2007.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chang TC, Zeitels LR, Hwang HW, Chivukula RR, Wentzel EA, Dews M, et al. Lin-28B transactivation is necessary for Myc-mediated let-7 repression and proliferation. Proc Natl Acad Sci USA. 2009;106:3384–3389. doi: 10.1073/pnas.0808300106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Christoffersen NR, Shalgi R, Frankel LB, Leucci E, Lees M, Klausen M, et al. p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC. Cell Death Differ. 2010;17:236–245. doi: 10.1038/cdd.2009.109. [DOI] [PubMed] [Google Scholar]
  16. Cole KA, Attiyeh EF, Mosse YP, Laquaglia MJ, Diskin SJ, Brodeur GM, et al. A functional screen identifies miR-34a as a candidate neuroblastoma tumor suppressor gene. Mol Cancer Res. 2008;6:735–742. doi: 10.1158/1541-7786.MCR-07-2102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Corney DC, Flesken-Nikitin A, Godwin AK, Wang W, Nikitin AY. MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. Cancer Res. 2007;67:8433–8438. doi: 10.1158/0008-5472.CAN-07-1585. [DOI] [PubMed] [Google Scholar]
  18. Corney DC, Hwang CI, Matoso A, Vogt M, Flesken-Nikitin A, Godwin AK, et al. Frequent downregulation of miR-34 family in human ovarian cancers. Clin Cancer Res. 2010;16:1119–1128. doi: 10.1158/1078-0432.CCR-09-2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dalgard CL, Gonzalez M, deNiro JE, O’Brien JM. Differential microRNA-34a expression and tumor suppressor function in retinoblastoma cells. Invest Ophthalmol Vis Sci. 2009;50:4542–4551. doi: 10.1167/iovs.09-3520. [DOI] [PubMed] [Google Scholar]
  20. Dews M, Homayouni A, Yu D, Murphy D, Sevignani C, Wentzel E, et al. Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster. Nat Genet. 2006;38:1060–1065. doi: 10.1038/ng1855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ. 2009;16:1093–1107. doi: 10.1038/cdd.2009.44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Guessous F, Zhang Y, Kofman A, Catania A, Li Y, Schiff D, et al. microRNA-34a is tumor suppressive in brain tumors and glioma stem cells. Cell Cycle. 2010;9:1031–1036. doi: 10.4161/cc.9.6.10987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. He L, He X, Lim LP, de SE, Xuan Z, Liang Y, et al. A microRNA component of the p53 tumour suppressor network. Nature. 2007a;447:1130–1134. doi: 10.1038/nature05939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. He L, He X, Lowe SW, Hannon GJ. microRNAs join the p53 network–another piece in the tumour-suppression puzzle. Nat Rev Cancer. 2007b;7:819–822. doi: 10.1038/nrc2232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S, et al. A microRNA polycistron as a potential human oncogene. Nature. 2005;435:828–833. doi: 10.1038/nature03552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Junttila MR, Evan GI. p53–a Jack of all trades but master of none. Nat Rev Cancer. 2009;9:821–829. doi: 10.1038/nrc2728. [DOI] [PubMed] [Google Scholar]
  27. Klein U, Lia M, Crespo M, Siegel R, Shen Q, Mo T, et al. The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell. 2010;17:28–40. doi: 10.1016/j.ccr.2009.11.019. [DOI] [PubMed] [Google Scholar]
  28. Kong YW, Cannell IG, de Moor CH, Hill K, Garside PG, Hamilton TL, et al. The mechanism of micro-RNA-mediated translation repression is determined by the promoter of the target gene. Proc Natl Acad Sci USA. 2008;105:8866–8871. doi: 10.1073/pnas.0800650105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lal A, Navarro F, Maher CA, Maliszewski LE, Yan N, O’Day E, et al. miR-24 Inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to “seedless” 3′UTR microRNA recognition elements. Mol Cell. 2009;35:610–625. doi: 10.1016/j.molcel.2009.08.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lin CH, Jackson AL, Guo J, Linsley PS, Eisenman RN. Myc-regulated microRNAs attenuate embryonic stem cell differentiation. EMBO J. 2009;28:3157–3170. doi: 10.1038/emboj.2009.254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lindstrom MS, Klangby U, Wiman KG. p14ARF homozygous deletion or MDM2 overexpression in Burkitt lymphoma lines carrying wild type p53. Oncogene. 2001;20:2171–2177. doi: 10.1038/sj.onc.1204303. [DOI] [PubMed] [Google Scholar]
  32. Lindstrom MS, Wiman KG. Role of genetic and epigenetic changes in Burkitt lymphoma. Semin Cancer Biol. 2002;12:381–387. doi: 10.1016/s1044-579x(02)00058-5. [DOI] [PubMed] [Google Scholar]
  33. Linsley PS, Schelter J, Burchard J, Kibukawa M, Martin MM, Bartz SR, et al. Transcripts targeted by the microRNA-16 family cooperatively regulate cell cycle progression. Mol Cell Biol. 2007;27:2240–2252. doi: 10.1128/MCB.02005-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lodygin D, Tarasov V, Epanchintsev A, Berking C, Knyazeva T, Korner H, et al. Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle. 2008;7:2591–2600. doi: 10.4161/cc.7.16.6533. [DOI] [PubMed] [Google Scholar]
  35. Luan S, Sun L, Huang F. MicroRNA-34a: a novel tumor suppressor in p53-mutant glioma cell line U251. Arch Med Res. 2010;41:67–74. doi: 10.1016/j.arcmed.2010.02.007. [DOI] [PubMed] [Google Scholar]
  36. Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell. 2006;126:1203–1217. doi: 10.1016/j.cell.2006.07.031. [DOI] [PubMed] [Google Scholar]
  37. Mu P, Han YC, Betel D, Yao E, Squatrito M, Ogrodowski P, et al. Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas. Genes Dev. 2009;23:2806–2811. doi: 10.1101/gad.1872909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Navarro F, Gutman D, Meire E, Caceres M, Rigoutsos I, Bentwich Z, et al. miR-34a contributes to megakaryocytic differentiation of K562 cells independently of p53. Blood. 2009;114:2181–2192. doi: 10.1182/blood-2009-02-205062. [DOI] [PubMed] [Google Scholar]
  39. O’Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature. 2005;435:839–843. doi: 10.1038/nature03677. [DOI] [PubMed] [Google Scholar]
  40. Olive V, Bennett MJ, Walker JC, Ma C, Jiang I, Cordon-Cardo C, et al. miR-19 is a key oncogenic component of mir-17-92. Genes Dev. 2009;23:2839–2849. doi: 10.1101/gad.1861409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ota A, Tagawa H, Karnan S, Tsuzuki S, Karpas A, Kira S, et al. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res. 2004;64:3087–3095. doi: 10.1158/0008-5472.can-03-3773. [DOI] [PubMed] [Google Scholar]
  42. Pajic A, Spitkovsky D, Christoph B, Kempkes B, Schuhmacher M, Staege MS, et al. Cell cycle activation by c-myc in a burkitt lymphoma model cell line. Int J Cancer. 2000;87:787–793. doi: 10.1002/1097-0215(20000915)87:6<787::aid-ijc4>3.0.co;2-6. [DOI] [PubMed] [Google Scholar]
  43. Raver-Shapira N, Marciano E, Meiri E, Spector Y, Rosenfeld N, Moskovits N, et al. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell. 2007;26:731–743. doi: 10.1016/j.molcel.2007.05.017. [DOI] [PubMed] [Google Scholar]
  44. Sala A, Bettuzzi S, Pucci S, Chayka O, Dews M, Thomas-Tikhonenko A. Advances in Cancer Research. In: Bettuzzi S, Pucci S, editors. Clusterin, part B. Vol. 105. Elsevier; USA: 2009. pp. 116–127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schuhmacher M, Kohlhuber F, Holzel M, Kaiser C, Burtscher H, Jarsch M, et al. The transcriptional program of a human B cell line in response to Myc. Nucl Acids Res. 2001;29:397–406. doi: 10.1093/nar/29.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sykes SM, Mellert HS, Holbert MA, Li K, Marmorstein R, Lane WS, et al. Acetylation of the p53 DNA-binding domain regulates apoptosis induction. Mol Cell. 2006;24:841–851. doi: 10.1016/j.molcel.2006.11.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tarasov V, Jung P, Verdoodt B, Lodygin D, Epanchintsev A, Menssen A, et al. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle. 2007;6:1586–1593. doi: 10.4161/cc.6.13.4436. [DOI] [PubMed] [Google Scholar]
  48. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. in vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science. 2004;303:844–848. doi: 10.1126/science.1092472. [DOI] [PubMed] [Google Scholar]
  49. Vaziri H, Dessain SK, Ng EE, Imai SI, Frye RA, Pandita TK, et al. hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell. 2001;107:149–159. doi: 10.1016/s0092-8674(01)00527-x. [DOI] [PubMed] [Google Scholar]
  50. Ventura A, Young AG, Winslow MM, Lintault L, Meissner A, Erkeland SJ, et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell. 2008;132:875–886. doi: 10.1016/j.cell.2008.02.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Welch C, Chen Y, Stallings RL. MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells. Oncogene. 2007;26:5017–5022. doi: 10.1038/sj.onc.1210293. [DOI] [PubMed] [Google Scholar]
  52. Wu X, Bayle JH, Olson D, Levine AJ. The p53-mdm-2 autoregulatory feedback loop. Genes Dev. 1993;7:1126–1132. doi: 10.1101/gad.7.7a.1126. [DOI] [PubMed] [Google Scholar]
  53. Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang B, Wang J, et al. Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat Immunol. 2008;9:405–414. doi: 10.1038/ni1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yamakuchi M, Ferlito M, Lowenstein CJ. miR-34a repression of SIRT1 regulates apoptosis. Proc Natl Acad Sci USA. 2008;105:13421–13426. doi: 10.1073/pnas.0801613105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yamakuchi M, Lowenstein CJ. MiR-34, SIRT1 and p53: the feedback loop. Cell Cycle. 2009;8:712–715. doi: 10.4161/cc.8.5.7753. [DOI] [PubMed] [Google Scholar]
  56. Yu D, Carroll M, Thomas-Tikhonenko A. p53 status dictates responses of B lymphomas to monotherapy with proteasome inhibitors. Blood. 2007;109:4936–4943. doi: 10.1182/blood-2006-10-050294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yu D, Dews M, Park A, Tobias JW, Thomas-Tikhonenko A. Inactivation of Myc in murine two-hit B lymphomas causes dormancy with elevated levels of interleukin 10 receptor and CD20: implications for adjuvant therapies. Cancer Res. 2005;65:5454–5461. doi: 10.1158/0008-5472.CAN-04-4197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yu D, Thomas-Tikhonenko A. A non-transgenic mouse model for B-cell lymphoma: in vivo infection of p53-null bone marrow progenitors by a Myc retrovirus is sufficient for tumorigenesis. Oncogene. 2002;21:1922–1927. doi: 10.1038/sj.onc.1205244. [DOI] [PubMed] [Google Scholar]

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