MYC Acts Via the PTEN Tumor Suppressor to Elicit Autoregulation and Genome-Wide Gene Repression by Activation of the Ezh2 Methyltransferase
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Published OnlineFirst November 7, 2012; DOI: 10.1158/0008-5472.CAN-12-2522 Cancer Molecular and Cellular Pathobiology Research MYC Acts via the PTEN Tumor Suppressor to Elicit Autoregulation and Genome-Wide Gene Repression by Activation of the Ezh2 Methyltransferase Mandeep Kaur1 and Michael D. Cole1,2 Abstract The control of normal cell growth is a balance between stimulatory and inhibitory signals. MYC is a pleiotropic transcription factor that both activates and represses a broad range of target genes and is indispensable for cell growth. Whereas much is known about gene activation by MYC, there is no established mechanism for the majority of MYC-repressed genes. We report that MYC transcriptionally activates the PTEN tumor suppressor in normal cells to inactivate the phosphoinositide 3-kinase (PI3K) pathway, thus suppressing AKT activation. Suppression of AKT enhances the activity of the EZH2 histone methyltransferase, a subunit of the epigenetic repressor Polycomb Repressive Complex 2 (PRC2), while simultaneously stabilizing the protein. MYC-mediated enhancement in EZH2 protein level and activity results in local and genome-wide elevation in the repressive H3K27me3 histone modification, leading to widespread gene repression including feedback autoregulation of the MYC gene itself. Depletion of either PTEN or EZH2 and inhibition of the PI3K/AKT pathway leads to gene derepression. Importantly, expression of a phospho-defective EZH2 mutant is sufficient to recapitulate nearly half of all MYC-mediated gene repression. We present a novel epigenetic model for MYC-mediated gene repression and propose that PTEN and MYC exist in homeostatic balance to control normal growth, which is disrupted in cancer cells. Cancer Res; 73(2); 1–11. Ó2012 AACR. Introduction been made in understanding the role of Myc as a transcrip- Cancer is driven by the activation of oncogenes and the loss tional activator (4). In addition to activating target genes, Myc of tumor suppressors. One tumor suppressor that is frequently also represses an almost equal number of genes (3), and this inactivated in diverse cancers is the phosphatase and tensin repression is important for Myc-mediated cell proliferation homolog gene (PTEN; reviewed in ref. 1). PTEN acts as a and transformation (5, 6). However, despite major advances in fi negative regulator of the phosphoinositide 3-kinase (PI3K) the Myc eld, there is no uniform mechanism of repression that pathway by converting PIP3 to PIP2, which in turn prevents satisfactorily accounts for the majority of Myc repressed genes. fi activation of the Akt pathway downstream of PI3K. The PI3K/ In fact, the rst Myc-regulated gene ever described was the MYC Akt pathway is an important component of cell signaling that gene itself, through what is generally considered an regulates a myriad of biologic processes including growth, autoregulatory feedback loop (7, 8). Autoregulation is postu- fi proliferation, and apoptosis (1). Cancer cells often exhibit lated to be important in ne-tuning the amount of Myc in a cell, fi mutations in the PI3K/Akt pathway, including loss of PTEN, as small changes in Myc expression are suf cient to shift the ultimately resulting in activation of the pathway and its balance from normal to aberrant growth. Notably, many cancer downstream effectors (2). cells have lost the ability to autoregulate (9, 10), and more Myc The MYC gene is the most frequently amplified gene in is advantageous for growth and proliferation of cancer. MYC human cancer, and deregulated expression of MYC is a hall- One pathway implicated in autoregulation of the Drosophila mark of 70% of all cancers (3). Myc is a well-established ortholog in (dmyc) is the Polycomb group of pleiotropic transcription factor, and significant progress has proteins (11). The Polycomb Repressive Complex 2 (PRC2) epigenetically silences genes by trimethylating lysine 27 on histone H3 (H3K27me3), a function carried out by the methyl- Authors' Affiliations: Departments of 1Pharmacology and 2Genetics, Dartmouth Medical School, Norris Cotton Cancer Center, Lebanon, New transferase Enhancer of Zeste 2 (Ezh2), which is an integral Hampshire component of the complex (12). The Ezh2 gene is frequently fi Note: Supplementary data for this article are available at Cancer Research ampli ed or overexpressed in many tumors and was described Online (http://cancerres.aacrjournals.org/). as an E2F-responsive oncogene (13). Corresponding Author: Michael D. Cole, Dartmouth Medical School, In this study, we show that Myc suppresses the PI3K/Akt Norris Cotton Cancer Center, Lebanon, NH 03756. Phone: 603-653- pathway via transcriptional upregulation of the PTEN tumor 9975; Fax: 603-653-9952; E-mail: [email protected] suppressor. Significantly, suppression of Akt results in Ezh2- doi: 10.1158/0008-5472.CAN-12-2522 mediated gene repression in 2 mammalian systems, including Ó2012 American Association for Cancer Research. autoregulation. Activation of Ezh2 is both necessary and www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst November 7, 2012; DOI: 10.1158/0008-5472.CAN-12-2522 Kaur and Cole sufficient to account for nearly half of all Myc-repressed genes. request. The data are presented as percentage binding com- We propose a general mechanism for Myc-mediated repres- pared with input for each sample. All experiments were carried sion and autoregulation linked to an important tumor sup- out 3 to 4 times and error bars represent SD. pressor pathway. Luciferase assay Materials and Methods Luciferase assays were conducted with the Dual luciferase Cell lines, drug treatments, and western blotting Reporter Kit (Promega). The pRL vector constitutively expres- À/À The c-myc (HO 15.19) and Phoenix cells were maintained sing Renilla luciferase was used to normalize for transfection in Dulbecco's Modified Eagle's Medum (DMEM) supplemented efficiency. A total of 2 Â 105 IMECs were plated in 12-well with 10% FBS. Immortalized mammary epithelial cells (IMEC; dishes 24 hours before transfections. On the day of transfec- ref. 14) were cultured in DMEM:F12 50:50 media supplemented tion, each well was transfected with pRL, pGL3 Basic (to assess with EGF, insulin, hydrocortisone, and 5% FBS. To generate basal reporter activity) or MYC promoter-GL3 and the indi- stable cell lines, retroviral vectors were used to create poly- cated plasmids. Twenty-four hours later, luciferase activity was clonal populations. For LY294002 (Cayman Chemical; #70920) measured using the Wallac 1450 MicroBeta TriLux system treatments, cells were plated subconfluently 12 to 16 hours (Perkin Elmer). Experiments were carried out 3 times in before treatment. Treatments lasted for 2 hours followed by triplicates, and error bars represent SD. radioimmunoprecipitation assay (RIPA) lysis for immunoblot- ting. Antibody information used for immunoblotting and Soft agar assay chromatin immunoprecipitation (ChIP) can be found in Sup- In 6-well plates, 10,000 cells (ÆsiRNA treatment for 48 hours) plementary Information. were plated in 0.3% agar, layered over a 0.6% agar base layer. Wells were re-fed with 200 mL complete media every other day. Plasmids and RNA interference Fourteen days after plating, colonies were counted and imaged. To generate the myc promoter luciferase reporter, a 2.5-kb fragment containing the human MYC promoters (P1 and P2) Results and upstream regulatory elements was cloned into the pGL3 Myc suppresses AKT through activation of PTEN Basic vector. Ezh2/S21A and Ezh2/S21E mutants were gener- expression ated using the QuikChange II Site Directed Mutagenesis Kit The PTEN tumor suppressor was previously shown to be a (Stratagene) as per manufacturer's protocol. pUSE MYR-AKT direct Myc target gene with an E-box that is occupied by Myc in was a gift from Dr. G. Lienhard (Dartmouth, Hanover, NH). vivo (16, 17). We analyzed PTEN expression and found that it is Silencer Select Pre-designed siRNAs were obtained from Myc-activated at both the protein and mRNA levels in human À/À Ambion (Applied Biosystems) and transfected using Lipofec- mammary epithelial cells (IMECs) and myc rat fibroblast tamine RNAi Max (Invitrogen; 10 nmol/L siRNA). At the cells (Fig. 1A and B). Therefore, we decided to explore the indicated time point, cells were harvested for protein or RNA. functional consequences of PTEN activation by Myc. siRNA sequences are listed in Supplementary Information PTEN is a dual lipid and protein phosphatase that acts as a (Supplementary Table S2). Silencer Select negative control negative regulator of the PI3K/Akt pathway (1). As PTEN siRNA was used as a transfection control and to account for dephosphorylates PIP3 to PIP2, thus attenuating Akt activa- nonspecific effects. tion, we tested whether PTEN activation had an effect on Akt signaling. Activation of Akt involves phosphorylation of Serine Real-time reverse transcription PCR 473 (pS473), so we assessed the levels of pS473 in response to Total RNA was harvested from log phase cultures with exogenous Myc expression. Notably, we found that Myc inhibits À/À TRIzol (Invitrogen), and cDNA was synthesized using the Super Akt S473 phosphorylation in both IMECs and myc rat Script kit from Invitrogen. Two-step real-time PCR was carried fibroblasts (Fig. 1C). In IMECs, there was a basal level of pS473 out using the SYBR Green Mix (Bio-Rad) on a Bio-Rad C1000 in the parental/vector cells that becomes undetectable with Thermal cycler. The expression of glyceraldehyde-3-phosphate Myc overexpression. In rat fibroblasts, there is no detectable þ þ dehydrogenase (GAPDH) or actin was used for normalization. pS473 in myc / cells, which express native levels of Myc. In À/À Primer sequences are available upon request. contrast, myc cells derived by genetic knockout have highly elevated pS473, which is subsequently suppressed to undetect- Chromatin immunoprecipitation able levels by reconstitution of Myc. Repression of Akt pS473 is Log phase cultures were fixed with 1% formaldehyde for 10 not evident with a Myc mutant lacking the conserved Myc Box 2 minutes and subjected to ChIP assays with minor modifica- transactivation domain (DMB2; Fig.