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• W1973548313 abstract "Article16 June 2005free access Transforming activity of MECT1-MAML2 fusion oncoprotein is mediated by constitutive CREB activation Lizi Wu Corresponding Author Lizi Wu Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Jingxuan Liu Jingxuan Liu Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Ping Gao Ping Gao Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Makoto Nakamura Makoto Nakamura Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Yang Cao Yang Cao Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Huangxuan Shen Huangxuan Shen Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author James D Griffin James D Griffin Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Lizi Wu Corresponding Author Lizi Wu Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Jingxuan Liu Jingxuan Liu Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Ping Gao Ping Gao Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Makoto Nakamura Makoto Nakamura Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Yang Cao Yang Cao Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Huangxuan Shen Huangxuan Shen Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author James D Griffin James D Griffin Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Lizi Wu 1,2, Jingxuan Liu1,2, Ping Gao1,2, Makoto Nakamura1,2, Yang Cao1,2, Huangxuan Shen1,2 and James D Griffin1,2 1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA 2Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA *Corresponding author. Department of Medical Oncology, Dana-Farber Cancer Institute, Mayer 540, 44 Binney Street, Boston, MA 02115, USA. Tel.: +1 617 632 5451; Fax: +1 617 632 4388; E-mail: [email protected] The EMBO Journal (2005)24:2391-2402https://doi.org/10.1038/sj.emboj.7600719 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Salivary gland tumors, a group of histologically diverse benign and malignant neoplasms, represent a challenging problem for diagnosis and treatment. A specific recurring t(11;19)(q21;p13) translocation is associated with two types of salivary gland tumors, mucoepidermoid carcinomas and Warthin's tumors. This translocation generates a fusion protein comprised of the N-terminal CREB (cAMP response element-binding protein)-binding domain of the CREB regulator MECT1 (Mucoepidermoid carcinoma translocated-1) and the C-terminal transcriptional activation domain of the Notch coactivator Mastermind-like 2 (MAML2). Here, we demonstrate that the MECT1-MAML2 fusion protein induces expression of multiple genes known to be CREB transcriptional targets. MECT1-MAML2 was found to bind to CREB, recruit p300/CBP into the CREB complex through a binding domain on MAML2, and constitutively activate CREB-dependent transcription. The transforming activity of MECT1-MAML2 was markedly reduced by blocking CREB DNA binding. Thus, this fusion oncogene mimics constitutive activation of cAMP signaling, by activating CREB directly. This study has identified a novel, critical mechanism of transformation for an oncogene associated very specifically with salivary gland tumors, and identified potential targets for the development of novel therapies. Introduction Salivary gland tumors are a group of highly heterogeneous benign and malignant neoplasms arising from the major or minor salivary glands, and account for approximately 5% of all head-and-neck cancers. The clinical management of these tumors is complicated by their varied biological behaviors and relative infrequency (McGurk and Renehan, 2001). Understanding the molecular changes in different types of salivary gland tumors is important for both the treatment and diagnosis of these diseases. Mucoepidermoid carcinomas (MEC) are the most common human malignant salivary gland and the second most common bronchial gland tumors, while Warthin's tumors are the second most common benign parotid gland tumors. Although these two types of tumors are histologically distinct, both are associated with a specific recurring chromosomal t(11;19)(q14–21;p12–13) translocation, suggesting that the chromosomal rearrangement is directly involved in their pathogenesis (Martins et al, 1996, 1997, 2004). The genes involved in the t(11;19) translocation in these tumors have been identified, and consist of a novel gene at 19p13 termed Mucoepidermoid carcinoma translocated-1 (MECT1, also called TORC1) and a member of the Mastermind-like gene family (MAML2) at 11q21 (Conkright et al, 2003; Tonon et al, 2003; Enlund et al, 2004). Intriguingly, the MECT1 gene product was recently characterized as a binding protein for CREB (cAMP response element-binding protein) transcription factor (Conkright et al, 2003; Iourgenko et al, 2003). CREB belongs to the class of basic domain-leucine zipper transcription factors and binds as a dimer to cAMP-responsive elements (CRE), 8-bp palindromic sequences TGACGTCA (Johannessen et al, 2004). In response to cAMP signaling and activation of protein kinase A (PKA), CREB is phosphorylated at Ser133 resulting in the recruitment of the coactivators CREB-binding protein (CBP) and p300, thereby activating the transcription of cAMP-responsive genes. In normal cells, MECT1 acts as a CREB regulator, functioning by binding to CREB and enhancing transcription of a CRE-containing reporter independently of CREB phosphorylation (Conkright et al, 2003; Iourgenko et al, 2003). The MAML2 gene, on the other hand, encodes a transcriptional coactivator for Notch receptors (Wu et al, 2002). Notch signaling is an evolutionarily conserved mechanism in which cell–cell interactions may determine cell fates, and is important in the development of many tissues (Artavanis-Tsakonas et al, 1999). MAML2 is part of the Notch transcriptional complex, which involves the transcription factor CSL and the nuclear activated form of Notch receptor (ICN) after ligand stimulation, and is required for transcriptional activation of the Notch target genes (Wu et al, 2002). The consequence of this chromosome translocation is the creation of a novel fusion protein, MECT1-MAML2, which is composed of the 42 aa CREB-binding domain from MECT1 with the 981 aa transcriptional activation domain (TAD) from MAML2 (Tonon et al, 2003; Enlund et al, 2004). The MECT1-MAML2 fusion has transforming activity, as indicated by the finding that it independently induces colony formation in the E1A-immortalized rat kidney epithelial cell line, RK3E (Tonon et al, 2003). However, the mechanism of transformation is unknown. Interestingly, several known Notch target genes are induced independent of Notch ligand stimulation, including Hairy-Enhancer of Split-1 (HES-1) and HES-related protein (HERP). Since previous studies indicate that Notch signaling is important in salivary gland development in Drosophila (Haberman et al, 2003), it was postulated that altered Notch signaling might affect gland cell differentiation and therefore contribute to the transforming activity of MECT1-MAML2. However, the mechanism of activation of Notch target gene expression by MECT1-MAML2 is unknown, and we have previously shown that this activity does not require the normal Notch transcription factor (CSL)-binding sites in the Notch target HES-1 promoter (Tonon et al, 2003). While activation of Notch target genes is still an attractive mechanism responsible for its transforming activity, Conkright et al (2003) suggested that interference of the CREB pathway by the MECT1-MAML2 fusion may also be important. First, the fusion retains the CREB-binding domain from the MECT1 translocation partner and is able to bind CREB. Second, the fusion induces activation of a reporter containing the CRE site, and a dominant-negative CREB expression plasmid (A-CREB), known to interfere with the CREB DNA binding, blocked such activation. Since the CREB complex mediates basic cellular processes including cell growth and survival in response to a variety signals, and is also implicated in a number of cancers including endocrine tumors, melanoma, and leukemias (Yu and Melmed, 2001; Poser and Bosserhoff, 2004; Shankar and Sakamoto, 2004), targeting of the CREB pathway might also be critical in mediating MECT1-MAML2 fusion's transforming activity. In preliminary studies with expression arrays, we found that MECT1-MAML2 induced expression of multiple genes previously shown to be responsive to cAMP signaling in epithelial cells. In an effort to understand the role of the CREB pathway in transformation by the fusion protein, we have examined the role of the CREB pathway in cellular transformation and analyzed the mechanism of activation of this pathway by MECT1-MAML2. Results Transforming activity of the MECT1-MAML2 fusion protein requires both the CREB-binding domain from MECT1 and the TAD domain from MAML2 MECT1-MAML2 is a fusion of the N-terminal CREB-binding domain of MECT1 (42 aa) with the C-terminal TAD of the Notch coactivator MAML2 (981 aa). Previously, we showed that the MECT1-MAML2 fusion efficiently induces foci formation in E1A-immortalized RK3E cells, while the fusion partner MAML2 has no activity (Tonon et al, 2003). To determine if the other fusion partner, MECT1, might have transforming activity, MECT1 was introduced into RK3E cells and focus formation was quantified. In contrast to MECT1-MAML2, the MECT1 gene product itself was unable to induce focus formation in RK3E cells (Figure 1A). Therefore, only the MECT1-MAML2 fusion, but not the two genes involved in the translocation, is able to transform. Figure 1.Both the CREB-binding domain of MECT1 and the TAD domain of MAML2 are required for the MECT1-MAML2 fusion to induce foci formation in RK3E cells. (A) RK3E cells were transfected with the indicated expression plasmids, and stained with crystal violet for foci formation at 3 weeks post-transfection. (B) Diagram of the constructs used in focus assays, and the number of foci generated from the transfection of each of these constructs. The number of foci shown was obtained from the 10 cm plates based on the triplicate experiments. M-M2, M2, M, M1, M-M1, and M-VP stand for MECT1-MAML2 fusion, MAML2, MECT1, MAML1, MECT1-MAML1, and MECT1-VP16, respectively. All these genes were cloned into the pFLAG-CMV2 expression vector, and expressed as FLAG-tagged proteins. Download figure Download PowerPoint The motifs within MECT1-MAML2 fusion required for transformation were evaluated by using chimeric proteins including MECT1-MAML1 and MECT1-VP16, generated by replacing the TAD domain of MAML2 in MECT1-MAML2 fusion with the corresponding TAD domain of MAML1, another member of the MAML family (Wu et al, 2002), and the unrelated activation domain of VP16, respectively (Tonon et al, 2003). Colony formation was observed for MECT1-MAML1, but not for MECT1-VP16 nor the full-length MAML1 (Figure 1B). These results indicate that the MAML1 TAD domain can functionally replace MAML2 TAD, suggesting that this TAD domain of the MAML family has unique properties contributing to MECT1-MAML2-mediated transformation. Moreover, the TAD domain of wild-type MAML2 did not produce colonies, indicating that the domain is not sufficient for transformation; therefore, the N-terminal 42 residues of the MECT1 gene are also required for the transforming activity of the fusion protein. In conclusion, the transforming activity of MECT1-MAML2 fusion results from the unique activity generated by both the MECT1 CREB-binding domain and the MAML2 TAD, which are brought together by the t(11;19) translocation. Microarray analysis identified downstream targets differentially regulated by the MECT1-MAML2 fusion oncoprotein including those normally regulated by cAMP signaling For a systematic analysis of potential MECT1-MAML2 target genes contributing to transformation, we used Affymetrix human U133A array to compare the transcription profiles of HeLa cells that were transiently transfected with the MECT1-MAML2 fusion, MAML2, MECT1, or vector. Expression of MECT1-MAML2 fusion and its fusion partners was confirmed (Supplementary Figure S1). With at least two-fold changes at the transcript levels considered as significant, we found that there is a unique set of genes differentially regulated by the MECT1-MAML2 fusion compared to expression of either fusion partner alone (see Table I). These genes are clearly involved in many critical cellular processes including cell growth, differentiation, and apoptosis, suggesting that the fusion protein may interfere with multiple cellular processes that lead to cell transformation. The identified set of target genes (Table I) included seven genes previously reported to be regulated by cAMP, including the transcription factors, ATF3 (Hai and Hartman, 2001), CREM (Daniel et al, 2000), FOS (Ahn et al, 1998), and two members of the nuclear receptor subfamily 4, NR4A1 (Nur77) and NR4A2 (Nurr1) (Kovalovsky et al, 2002); inhibitor of DNA-binding protein, Id2 (Scobey et al, 2004); and stanniocalcin 1 (Wong et al, 2002). Table 1. Target genes regulated by the MECT1-MAML2 fusion oncoprotein in HeLa cells Gene symbol Description M-M2 fold change MAML2 fold change MECT1 fold change Family ADAMTS5 A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2) 2.5 −0.5 1.6 Protease ATF3 Activating transcription factor 3 6.6 1.5 1.3 Transcription regulator BNIP3L BCL2/adenovirus E1B 19 kDa interacting protein 3-like 3.7 0.2 −0.5 — CARD14 Caspase recruitment domain family, member 14 2.8 0.1 1.2 — CLK1 CDC-like kinase 1 3.0 0.4 1.7 Kinase CREM cAMP responsive element modulator 2.0 −0.8 1.8 Transcription regulator CYP26B1 Cytochrome P450, family 26, subfamily B, polypeptide 1 2.5 0.0 1.9 Cytochrome P450 CYR61 Cysteine-rich, angiogenic inducer, 61 2.5 2.1 0.0 — DKFZp451J1719 Hypothetical DKFZp451J1719 5.6 0.6 −0.1 — DNAJB9 DnaJ (Hsp40) homolog, subfamily B, member 9 7.6 −1.2 −1.6 Transcription regulator DUSP1 Dual specificity phosphatase 1 2.2 1.0 −1.0 Phosphatase FKBP8 FK506 binding protein 8, 38 kDa 2.7 1.4 1.6 — FOS v-fos FBJ murine osteosarcoma viral oncogene homolog 3.6 −2.3 1.3 Transcription regulator FOSB FBJ murine osteosarcoma viral oncogene homolog B 6.2 −0.7 0.1 Transcription regulator FOSL2 FOS-like antigen 2 2.4 −0.7 1.1 Transcription regulator GABARAPL1 GABA(A) receptor-associated protein like 1 3.0 0.8 −1.1 — GEM GTP binding protein overexpressed in skeletal muscle 3.9 0.5 1.6 Enzyme HOXA5 Homeo box A5 6.4 −0.5 1.2 Transcription regulator ID2 Inhibitor of DNA binding 2, dominant negative helix–loop–helix protein 4.6 −0.3 1.4 — INSL4 Insulin-like 4 (placenta) 2.4 0.6 1.5 — KLF4 Kruppel-like factor 4 (gut) 2.8 0.7 1.3 Transcription regulator KRT17 Keratin 17 2.5 −0.4 1.4 — MT1X Metallothionein 1X 2.3 1.3 1.3 — NDRG1 N-myc downstream regulated gene 1 2.8 −0.1 0.1 — NR4A1 Nuclear receptor subfamily 4, group A, member 1 (Nur77) 3.9 0.8 −1.6 Ligand-dependent nuclear receptor NR4A2 Nuclear receptor subfamily 4, group A, member 2 (Nurr1) 6.7 −0.1 1.7 Ligand-dependent nuclear receptor PIGA Phosphatidylinositol glycan, class A (paroxysmal nocturnal hemoglobinuria) 4.1 0.7 0.2 Enzyme PMP22 Peripheral myelin protein 22 2.1 −0.7 1.3 — SAP18 sin3-associated polypeptide, 18 kDa 2.6 0.6 0.1 Transcription regulator SLC16A6 Solute carrier family 16 (monocarboxylic acid transporters), member 6 2.4 1.1 1.4 — SLC20A1 Solute carrier family 20 (phosphate transporter), member 1 2.0 1.1 0.2 Transporter SLC2A3 Solute carrier family 2 (facilitated glucose transporter), member 3 2.4 1.3 1.8 Transporter SNRK SNF-1 related kinase 2.6 1.4 1.1 Kinase STC1 Stanniocalcin 1 5.2 1.3 1.9 — TFF1 Trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed) 10.1 1.6 0.2 Growth factor TM4SF1 Transmembrane 4 superfamily member 1 2.1 0.6 1.2 — YPEL5 Yippee-like 5 (Drosophila) 3.0 −0.3 1.3 — ZBED1 Zinc finger, BED domain containing 1 2.3 −0.7 1.5 — The bold lines indicate genes previously reported to be regulated by camp. To validate these expression array data, we performed real-time PCR assays for five target genes, including two known cAMP-responsive genes, Nurr1 and ATF3, and three genes previously unknown to be regulated by cAMP, DUSP1, TFF1, and NDRG1, and showed that all these genes are indeed upregulated when the fusion is expressed (Figure 2A). Figure 2.MECT1-MAML2 fusion induces expression of CREB transcriptional target genes. (A) Expression levels of five target genes (both known and previously unknown regulated by cAMP signaling) in MECT1-MAML2-transfected cells, relative to the empty vector-transfected cells, were determined by SYBR green real-time PCR. (B) Induction of two known CREB targets, ATF3 and Nurr1 genes, wasanalyzed by Western blot analyses in human immortalized parotid HSY cells transfected with the indicated expression constructs. Transfected FLAG-tagged proteins except MECT1-VP16 (its band overlaps with IgG light chain, and the expression is shown on the whole-cell lysate blot) were detected after immunoprecipitation (IP) with anti-FLAG antibodies. M2, M, M-M2, M-M1, and M-VP stand for MAML2, MECT1, MECT1-MAML2, MECT1-MAML1, and MECT1-VP16, respectively. Download figure Download PowerPoint MECT1-MAML2 induces expression of multiple targets of the cAMP signaling pathway; thus, we investigated mechanisms of activation of the cAMP signaling pathway via the fusion oncogene. Consistent with the microarray and real-time PCR data, Western blot analysis revealed that the MECT1-MAML2 fusion induced the expression of these two known target genes in the CREB pathway, Nurr1 and ATF3, at much higher levels than did either MAML2 or MECT1 alone in the human immortalized parotid cell line HSY (Figure 2B). Overexpression of the MECT1 gene caused only a modest induction of Nurr1 and ATF3 genes, despite the fact that its expression level was much higher than that of MECT1-MAML2. This might be explained in part by the difference in their subcellullar distribution: MECT1 is primarily localized in the cytoplasm (Supplementary Figure S2), while MECT1-MAML2 is localized in the nucleus when expressed as GFP-tagged proteins (Tonon et al, 2003). It was recently shown that increasing intracellular cAMP or calcium levels induces nuclear transport of MECT1 (TORC1), which is subsequently sufficient to activate CRE-dependent transcription (Bittinger et al, 2004). Thus, these data suggest that MECT1-MAML2 is a constitutive activator for CREB-mediated transcription, while MECT1 requires additional signaling for such transcriptional activation. Interestingly, while the chimeric protein MECT1-MAML1 activates expression of Nurr1 and ATF3 at high levels, the MECT1-VP16 chimeric protein does not (Figure 2B). These data were consistent with the differential activities of these proteins to activate the CRE promoter reporter (Supplementary Figure S3). Combined with our results from the colony formation studies (Figure 1), the ability of the MECT1-MAML2 fusion to activate the CREB pathway appears to correlate with its ability to induce colonies in RK3E cells. Disruption of CREB activity reduces the transforming activity of MECT1-MAML2 The importance of activation of the CREB pathway in mediating MECT1-MAML2 transforming activity was then evaluated using a well-characterized dominant-negative mutant, A-CREB, which specially blocks CREB binding to DNA by heterodimerizing with CREB (Ahn et al, 1998). RK3E cells transduced with A-CREB viruses (expressing A-CREB plus GFP), or control viruses (expressing GFP only) were transfected with MECT1-MAML2 to examine the expression of CREB target genes at 48 h post-transfection and to score for colonies after 3 weeks. We first confirmed by Western blot analysis that A-CREB is expressed in RK3E cells infected with A-CREB viruses (Figure 3A). The induction of Nurr1 and ATF3 proteins by MECT1-MAML2 was significantly reduced in RK3E cells expressing A-CREB, as compared to the control cells (Figure 3B). Importantly, expression of A-CREB also led to a significantly decreased number of colonies (Figure 3C). However, we found that A-CREB expression did not affect the number of colonies induced by an activating mutation of B-RAF proto-oncogene (V599E). Thus, these data indicate that interference of CREB activity reduced the activation of the CREB pathway, and also decreased the transforming activity of the fusion protein. Moreover, by knocking down the endogenous CREB level with RNAi in 293T cells, we found by Western blot analysis and/or real-time PCR that there are reduced expression levels of known CREB target genes, Nurr1 and ATF3, as well as three other target genes, DUSP1, TFF1, and NDRG1, previously unknown to be regulated by CREB, suggesting that induction of these genes might be dependent on CREB (Figures 3D and E). Taken together, CREB activity is important for activation of the CREB pathway and cellular transformation via the MECT1-MAML2 fusion. Figure 3.Disruption of CREB activity reduced the abilities of the MECT1-MAML2 fusion to activate the CREB pathway and to induce colony formation in RK3E. (A) Expression of A-CREB in RK3E cells transduced with A-CREB viruses was determined by Western blot analysis. (B) RK3E cells expressing A-CREB and GFP, and the control cells expressing GFP alone, were transfected with either vector or the MECT1-MAML2 expression construct. The expression levels of two CREB target genes, ATF3 and Nurr1, were analyzed by Western blot analysis. (C) A-CREB-expressing RK3E cells and the control cells were transfected with either vector only or the expression construct encoding MECT1-MAML2 fusion, and the colonies were scored 3 weeks after transfection. (D) 293T cells were cotransfected with the MECT1-MAML2 expression vector and CREB RNAi (or control RNAi), and the levels of ATF3, Nurr1, CREB, and transfected MECT1-MAML2 fusion were analyzed 48 h after transfection by Western blotting. (E) Expression levels of CREB, and five fusion target genes in 293T cells cotransfectd with MECT1-MAML2 and CREB RNAi, relative to cells cotransfected with MECT1-MAML2 and control RNAi were determined by SYBR green real-time PCR. Download figure Download PowerPoint Next, we asked if CREB activity is essential for the growth of MECT1-MAML2 fusion-expressing MEC cell lines. Two MECT1-MAML2-expressing MEC cell lines, H292 and H3118, along with the immortalized MECT1-MAML2-negative cell line HSY were transduced either with A-CREB or control GFP viruses. The multiplicity of infection (MOI) was optimized to obtain an infection rate of 30–50%, and then the percentage of GFP-positive populations was determined at 3 or 4 days intervals, over a total time period of 15 days. As shown in Figure 4A, the percentage of MECT1-MAML2-positive H3118 and H292 cells that expressed A-CREB was progressively reduced, while their control, GFP-only-expressing cells did not have a growth disadvantage. In contrast, the percentage of HSY cells (lacking expression of MECT1-MAML2) that expressed A-CREB reduced only modestly. These results suggest that expression of A-CREB in MECT1-MAML2-positive MEC cells, but not in MECT1-MAML2-negative cells, resulted in a negative effect on cellular growth. In order to directly compare growth rates, GFP-positive cells were purified by fluorescence activated cell sorting. We found that A-CREB expression inhibited the growth of the MECT1-MAML2-positive MEC cells, but not the control HSY cells (Figure 4B). Finally, we investigated the effect of A-CREB expression on the cell cycle and apoptosis. We found that A-CREB expression caused an increased percentage of cells in the G1 phase in both H3118 and H292, as compared with their GFP control cells, where no significant difference was seen with HSY cells (Figure 4C). In addition, A-CREB expression did not lead to apoptosis in both M-M2-negative and -positive cells (not shown). Therefore, the inhibition of CREB activity via the dominant-negative A-CREB resulted in a significant inhibition of growth in the MECT1-MAML2-positive cells, but only marginally affected MECT1-MAML2-negative cells. These results demonstrate that CREB activity is critical for the growth of MECT1-MAML2-positive MEC cells. Figure 4.Disruption of CREB activity significantly suppressed the growth of two human MECT1-MAML2-positive MEC cell lines. (A) A representative diagram showing the changes in the percentage of GFP-positive cells between cell populations expressing A-CREB and GFP and their control counterparts expressing GFP only. Two MECT1-MAML2-expressing MEC cell lines, H292 and H3118, along with the immortalized normal parotid cell line HSY (MECT1-MAML2 negative), were transduced with A-CREB or control GFP viruses. The percentages of GFP-positive cells were determined by FACS analysis at 3–4 days intervals, for a total of 15 days. The percentage of GFP-positive cells at day 2 postinfection was considered as 100%, and the remaining data were normalized. (B) A representative growth curve of cells expressing A-CREB plus GFP versus controls expressing GFP only. The cell number was determined using crystal blue staining daily for 6 days. (C) Cell cycle distribution of HSY, H3118, and H292 cells transduced with A-CREB viruses or vector control viruses as determined by propidium iodide straining followed by FACS analysis. Download figure Download PowerPoint The ability of the MECT1-MAML2 fusion to recruit p300/CBP to the CRE transcriptional complex is partially responsible for activation of the CREB pathway and its transforming activity Signal-mediated activation of the cAMP/PKA pathway leads to phosphorylation of CREB at Ser133, which subsequently recruits p300/CBP coactivators (Johannessen et al, 2004). The recruitment of p300/CBP is a critical event for the CREB complex to activate transcription. We therefore considered the possibility that either MECT1-MAML2 leads to the phosphorylation of CREB, allowing the recruitment of p300/CBP, or that MECT1-MAML2 itself recruits transcriptional coactivators such as p300/CBP to CREB. Either of these mechanisms would allow the fusion to bypass cAMP signaling-dependent phosphorylation events. We found that the overexpression of the fusion in 293T cells did not induce phosphorylation of CREB at Ser133 (Supplementary Figure S4). Therefore, phosphorylation of CREB is unlikely to be the mechanism by which MECT1-MAML2 activates the CREB pathway. To test the possibility that MECT1-MAML2 recruits p300/CBP, we first determined whether p300/CBP colocalizes with the MECT1-MAML2 fusion. We found that p300 is localized in the nucleus with a diffuse staining pattern when expressed alone in U20S cells. However, p300 showed a nuclear dot pattern, and colocalized with the fusion protein when coexpressed with MECT1-MAML2 (Figure 5A). Figure 5.MECT1-MAML2 interacts with the CREB pathway regulatory molecule p300. (A) MECT1-MAML2 and p300 colocalize in the nuclei. U20S cells were transfected with the GFP-tagged MECT1-MAML2 and HA-tagged p300, and stained with an anti-HA antibody for p300 expression. The DAPI staining labels the nuclei of the same cells. (B) MECT1-MAML2 post-translationally modifies p300. 293T cells were transfected with HA-tagged p300 and either empty vector, FLAG-tagged MECT1-MAML2, MECT1, or MAML2 TA" @default.
• W1973548313 created "2016-06-24" @default.
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• W1973548313 title "Transforming activity of MECT1-MAML2 fusion oncoprotein is mediated by constitutive CREB activation" @default.
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