Regulation of NOTCH Signaling by Reciprocal Inhibition of HES1 and Deltex 1 and Its Role in Osteosarcoma Invasiveness
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Oncogene (2010) 29, 2916–2926 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness P Zhang, Y Yang, R Nolo, PA Zweidler-McKay and DPM Hughes Department of Pediatrics Research, Children’s Cancer Hospital, University of Texas MD Anderson Cancer Center, Houston, TX, USA The highly conserved NOTCH signaling pathway has function as an oncogene, for example, in T-cell acute many essential functions in the development of diverse lymphoblastic leukemia or mammary adenocarcinoma, cells, tissues and organs from Drosophila to humans, and or as a tumor suppressor, for example, in neuroendo- dysregulated NOTCH signaling contributes to several crine and B-cell malignancies (Allenspach et al., 2002; disorders, including vascular and bone defects, as well as Weng et al., 2004; Zweidler-McKay and Pear, 2004; several cancers. Here we describe a novel mechanism of Zweidler-McKay et al., 2005). NOTCH regulation by reciprocal inhibition of two Ligand (Jagged1/2, Dll1/3/4) binding to membrane- NOTCH downstream effectors: Deltex1 and HES1. This localized NOTCH1–4 triggers a conformational change mechanism appears to regulate invasion of osteosarcoma of NOTCH, exposing cleavage sites for ADAM10 and cells, as Deltex1 blocks osteosarcoma invasiveness by g-secretase (Fortini, 2002). After cleavage, the intracel- downregulating NOTCH/HES1 signaling. The inhibitory lular domain of NOTCH (ICN) is released into the effect of endogenous Deltex1 on NOTCH signaling is cytoplasm and translocates into the nucleus, binding to mediated through binding with the intracellular domain of transcription factors CSL (CBF1, Su(H) and LAG-1), NOTCH and ubiquitination and degradation of NOTCH also known as RBPjk (Struhl and Adachi, 1998), receptors. Conversely, we show that the NOTCH target initiating transcription. The primary targets of NOTCH gene HES1 causes transcriptional inhibition of Deltex1 by are two basic helix-loop-helix transcriptional repressor directly binding to the promoter of Deltex1. An HES1 families: the Hairy/enhancer-of-split (HES) and the binding site is identified 400 bp upstream of the transcrip- Hairy/enhancer of split with YRWP motif (HERP) tion start site of Deltex1. HES1-mediated repression of families (Iso et al., 2003). HES and HERP proteins Deltex1 requires the C-terminal H3/H4 and WRPW normally form homo- or heterodimers, inhibiting domains of HES1, which associate with the TLE/Groucho expression of specific downstream target genes in a corepressors. Taken together, we define a molecular tissue-specific manner. ICN also directly activates other mechanism regulating NOTCH signaling by reciprocal target genes in a CSL-independent manner, including inhibition of the NOTCH target genes HES1 and Deltex1 MAPK phosphatase LIP1, pre-Ta and cyclin D1 in mammalian cells. This mechanism may have important (Allenspach et al., 2002). clinical implications for targeting NOTCH signaling in NOTCH induces expression of Deltex1 (DTX1), a osteosarcoma and other cancers. human ortholog of Drosophila Deltex (Matsuno et al., Oncogene (2010) 29, 2916–2926; doi:10.1038/onc.2010.62; 2002), but DTX1 expression is not strictly NOTCH published online 8 March 2010 dependent (Matsuno et al., 1998). DTX1 contains a RING finger and two WWE domains, indicating that Keywords: NOTCH; HES1; Deltex1(DTX1); osteo- DTX1 may be an E3 ubiquitin ligase (Jackson et al., sarcoma; invasion 2000). DTX1 can regulate T-cell activation through ubiquitination and degradation of MEKK1 in T cells (Liu and Lai, 2005). Depending on the cellular context, Deltex can either promote or inhibit NOTCH signaling. Introduction In Drosophila, Deltex promotes NOTCH-dependent transcriptional activity, and human DTX1 can have a NOTCH signaling affects fundamental processes in cell- similar effect (Matsuno et al., 1998). However, Deltex also fate determination, including stem cell maintenance, forms trimeric NOTCH–Deltex–Kurz (b-arrestin) protein differentiation, proliferation and apoptosis (Matsuno complexes, mediating degradation of NOTCH receptors et al., 2002). The functions of NOTCH signaling in through a ubiquitination-dependent pathway (Mukherjee cancer vary depending on cellular context. NOTCH may et al., 2005). DTX1 also can block NOTCH-dependent transcription in mammalian lymphoid cells and neuron Correspondence: Dr DPM Hughes, Department of Pediatrics cells (Sestan et al., 1999; Itoh et al., 2003). Izon et al. Research, Children’s Cancer Hospital, University of Texas MD Anderson (2002) suggested that DTX1 suppresses NOTCH1-in- Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. duced gene expression in lymphoid progenitor cells by E-mail: [email protected] Received 15 August 2009; revised 27 January 2010; accepted 1 February blocking coactivator recruitment. No clear function has 2010; published online 8 March 2010 been established for DTX1 in malignancy. NOTCH regulation by HES1 and Deltex1 P Zhang et al 2917 We previously reported that NOTCH/HES1 signaling OS187 OS187 promotes osteosarcoma invasion and metastasis (Zhang V DTX1 V DTX1 et al., 2008). In this report, we present a novel regulatory mechanism of NOTCH signaling in human cells. First, DTX1 DTX1 we show that endogenous DTX1 negatively regulates ICN1 NOTCH1 NOTCH-dependent transcriptional activity in osteosar- coma cells and that DTX1 physically binds to ICN1, HES1 HES1 inducing ubiquitination and degradation of ICN1. Next we show that the CSL-dependent NOTCH target gene actin actin HES1 transcriptionally inhibits DTX1 through direct binding to the promoter of DTX1. We identify an HES1 DTX1 binding site (N-box) in the promoter region of DTX1 Notch1 HES1 1.2 and show physical interaction of HES1 with this N-box. 1.2 9 * Finally, we show that this reciprocal inhibition of HES1 and DTX1 within the NOTCH pathway controls osteosarcoma invasiveness. Taken together, our results 0.8 0.8 * 6 provide a novel mechanism of NOTCH signaling regulation by its downstream target genes HES1 and DTX1. This mechanism may have important clinical 0.4 0.4 3 implications to determine whether modulation of NOTCH signaling might benefit patients with osteosar- Relative expression coma and other types of cancer. 0 0 0 V DTX1 V DTX1 V DTX1 OS187 OS187 Results 140 * DTX1 negatively regulates NOTCH1/HES1 signaling 300 and osteosarcoma invasiveness To investigate whether DTX1 antagonizes NOTCH 100 signaling in osteosarcoma, we overexpressed DTX1 in * 200 OS187 cells. DTX1 overexpression reduced ICN1 and HES1 protein (Figure 1a). DTX1 can be a transcription regulator or a ubiquitin ligase (Wu et al., 2001; Matsuno 60 et al., 2002), so we wished to know which mechanism was downregulating NOTCH protein levels. Reverse Invasive cell number 100 transcriptase (RT)–PCR and Q-PCR measured expres- Relative luciferase activity 20 * sion NOTCH1, HES1, DTX1 and actin mRNA in OS187 cells expressing DTX1 or empty vector (Figures 0 1b and c). NOTCH1 mRNA level was not significantly V DTX1 0 V DTX1 DTX1 decreased by DTX1 overexpression, whereas HES1 CSL reporter HES1 mRNA level was downregulated (Po0.05), suggesting that DTX1 downregulated NOTCH1 by affecting Figure 1 Deltex1 (DTX1) negatively regulates NOTCH1/HES1 signaling and suppresses invasion of osteosarcoma cells. Immuno- protein stability rather than gene transcription. Q-PCR blot (a) and PCR (b) analysis of DTX1, NOTCH1/ICN1, HES1 showed that HES1 mRNA level was significantly and actin in OS187 cells stably expressing vector control or DTX1 decreased (30%, Po0.05) in cells overexpressing gene. To confirm that the band identified by the western blot DTX1, presumably resulting from decreased ICN1 for ICN1 in fact represents the cleaved intracellular fragment protein. Because HES1 expression is affected by several of NOTCH1, we cultured OS 187 cells in 10 or 50 mM compound E, a g-secretase inhibitor (GSI) and showed that the cleaved protein is promoters and not just by ICN1, the reduction in reduced with GSI treatment (Supplemental Figure 1). (c) Histo- expression by only 30% was not surprising. To measure grams represent quantitative PCR analysis of NOTCH1, HES1 and directly the impact of DTX1 on ICN1-mediated gene DTX1 from the same cells. Ct values were normalized to actin; activation, we co-transfected the CSL-responsive pro- vector control is defined as a relative expression of 1 (*Po0.05). (d) Histograms represent luciferase emission from a CSL reporter moter (HES-AB) with Renilla luciferase control vector (that is, NOTCH responsive) in OS187 cells transduced with DTX1 into OS187 cells stably overexpressing human DTX1 or empty vector (*Po0.05). (e) Histograms represent Matrigel gene or empty vector. The HES-AB promoter is the invasion of OS187 cells stably expressing empty vector, DTX1 or 500 bp promoter region upstream of the HES1 gene DTX1 and HES1. Stably transduced OS187 cells (1 Â 104) were transcription start site, containing two ICN1-responsive seeded in each transwell and cell invasiveness was quantified by the invasive cell number after 48 h incubation. Average of three wells, CSL binding sites (Jarriault et al., 1995). Overexpression with error bars showing standard deviation, is shown (*Po0.05). of DTX1 decreased the activity of the CSL-responsive promoter by 30% in OS187 cells (Po0.05), indicating that DTX1 inhibits NOTCH/CSL signaling (Figure 1d). Oncogene NOTCH regulation by HES1 and Deltex1 P Zhang et al 2918 Likewise, Matrigel invasion was reduced by overexpres- regulatory function, we measured DTX1 interaction sion of DTX1 in OS187 cells, in parallel with down- with ICN1 and induction of proteasome-dependent regulation of NOTCH signaling and HES1 (Figure 1e). ICN1 downregulation. We treated COL cells, which However, constitutive HES1 expression in OS187 cells have high endogenous DTX1, with 5 mM EDTA for overexpressing DTX1 reversed the DTX1 inhibition on 15 min to induce NOTCH1 cleavage and ICN inter- cell invasion (Figure 1e). This result shows that DTX1- nalization, then immunoprecipated DTX1 from the mediated inhibition of the NOTCH pathway operates treated cells (Figure 2a). Immunoprecipitation of upstream of HES1.