RASSF1A disrupts the NOTCH signaling axis via SNURF/RNF4‐mediated ubiquitination of HES1
2021; Springer Nature; Volume: 23; Issue: 2 Linguagem: Inglês
10.15252/embr.202051287
ISSN1469-3178
AutoresAngelos Papaspyropoulos, Andriani Angelopoulou, Ioanna Mourkioti, Aikaterini Polyzou, Daniela Paňková, Konstantinos Toskas, Simone Lanfredini, Αnastasia A. Pantazaki, Nefeli Lаgopati, Athanassios Kotsinas, Konstantinos Evangelou, Efstathios Chronopoulos, Eric O’Neill, Vassilis G. Gorgoulis,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoArticle13 December 2021free access Source DataTransparent process RASSF1A disrupts the NOTCH signaling axis via SNURF/RNF4-mediated ubiquitination of HES1 Angelos Papaspyropoulos Corresponding Author Angelos Papaspyropoulos [email protected] orcid.org/0000-0002-6178-9987 Department of Oncology, University of Oxford, Oxford, UK Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Biomedical Research Foundation, Academy of Athens, Athens, Greece Search for more papers by this author Andriani Angelopoulou Andriani Angelopoulou Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Biomedical Research Foundation, Academy of Athens, Athens, Greece Search for more papers by this author Ioanna Mourkioti Ioanna Mourkioti Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Search for more papers by this author Aikaterini Polyzou Aikaterini Polyzou Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Search for more papers by this author Daniela Pankova Daniela Pankova orcid.org/0000-0002-3478-8065 Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Konstantinos Toskas Konstantinos Toskas Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Simone Lanfredini Simone Lanfredini Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Anastasia A Pantazaki Anastasia A Pantazaki orcid.org/0000-0003-3791-6955 Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece Search for more papers by this author Nefeli Lagopati Nefeli Lagopati Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Biomedical Research Foundation, Academy of Athens, Athens, Greece Search for more papers by this author Athanassios Kotsinas Athanassios Kotsinas Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Search for more papers by this author Konstantinos Evangelou Konstantinos Evangelou Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Search for more papers by this author Efstathios Chronopoulos Efstathios Chronopoulos Laboratory for Research of the Musculoskeletal System, KAT General Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Eric O'Neill Corresponding Author Eric O'Neill [email protected] orcid.org/0000-0002-0060-6278 Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Vassilis Gorgoulis Corresponding Author Vassilis Gorgoulis [email protected] orcid.org/0000-0001-9001-4112 Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Biomedical Research Foundation, Academy of Athens, Athens, Greece Molecular and Clinical Cancer Sciences, Manchester Cancer Research Centre, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK Center for New Biotechnologies and Precision Medicine, Medical School, National and Kapodistrian University of Athens, Athens, Greece Faculty of Health and Medical Sciences, University of Surrey, Surrey, UK Search for more papers by this author Angelos Papaspyropoulos Corresponding Author Angelos Papaspyropoulos [email protected] orcid.org/0000-0002-6178-9987 Department of Oncology, University of Oxford, Oxford, UK Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Biomedical Research Foundation, Academy of Athens, Athens, Greece Search for more papers by this author Andriani Angelopoulou Andriani Angelopoulou Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Biomedical Research Foundation, Academy of Athens, Athens, Greece Search for more papers by this author Ioanna Mourkioti Ioanna Mourkioti Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Search for more papers by this author Aikaterini Polyzou Aikaterini Polyzou Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Search for more papers by this author Daniela Pankova Daniela Pankova orcid.org/0000-0002-3478-8065 Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Konstantinos Toskas Konstantinos Toskas Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Simone Lanfredini Simone Lanfredini Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Anastasia A Pantazaki Anastasia A Pantazaki orcid.org/0000-0003-3791-6955 Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece Search for more papers by this author Nefeli Lagopati Nefeli Lagopati Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Biomedical Research Foundation, Academy of Athens, Athens, Greece Search for more papers by this author Athanassios Kotsinas Athanassios Kotsinas Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Search for more papers by this author Konstantinos Evangelou Konstantinos Evangelou Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Search for more papers by this author Efstathios Chronopoulos Efstathios Chronopoulos Laboratory for Research of the Musculoskeletal System, KAT General Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Eric O'Neill Corresponding Author Eric O'Neill [email protected] orcid.org/0000-0002-0060-6278 Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Vassilis Gorgoulis Corresponding Author Vassilis Gorgoulis [email protected] orcid.org/0000-0001-9001-4112 Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece Biomedical Research Foundation, Academy of Athens, Athens, Greece Molecular and Clinical Cancer Sciences, Manchester Cancer Research Centre, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK Center for New Biotechnologies and Precision Medicine, Medical School, National and Kapodistrian University of Athens, Athens, Greece Faculty of Health and Medical Sciences, University of Surrey, Surrey, UK Search for more papers by this author Author Information Angelos Papaspyropoulos *,1,2,3, Andriani Angelopoulou2,3,†, Ioanna Mourkioti2,†, Aikaterini Polyzou2,†, Daniela Pankova1, Konstantinos Toskas1, Simone Lanfredini1, Anastasia A Pantazaki4, Nefeli Lagopati2,3, Athanassios Kotsinas2, Konstantinos Evangelou2, Efstathios Chronopoulos5, Eric O'Neill *,1 and Vassilis Gorgoulis *,2,3,6,7,8 1Department of Oncology, University of Oxford, Oxford, UK 2Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens (NKUA), Athens, Greece 3Biomedical Research Foundation, Academy of Athens, Athens, Greece 4Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece 5Laboratory for Research of the Musculoskeletal System, KAT General Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece 6Molecular and Clinical Cancer Sciences, Manchester Cancer Research Centre, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK 7Center for New Biotechnologies and Precision Medicine, Medical School, National and Kapodistrian University of Athens, Athens, Greece 8Faculty of Health and Medical Sciences, University of Surrey, Surrey, UK † These authors contributed equally to this work *Corresponding author. Tel: +30 210 746 2174; E-mail: [email protected] *Corresponding author. Tel: +44 01865 617321; E-mail: [email protected] *Corresponding author. Tel: +30 210 746 2352; E-mail: [email protected] EMBO Reports (2022)23:e51287https://doi.org/10.15252/embr.202051287 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract RASSF1A promoter methylation has been correlated with tumor dedifferentiation and aggressive oncogenic behavior. Nevertheless, the underlying mechanism of RASSF1A-dependent tumor dedifferentiation remains elusive. Here, we show that RASSF1A directly uncouples the NOTCH-HES1 axis, a key suppressor of differentiation. Interestingly, the crosstalk of RASSF1A with HES1 occurs independently from the signaling route connecting RASSF1A with the Hippo pathway. At the molecular level, we demonstrate that RASSF1A acts as a scaffold essential for the SUMO-targeted E3 ligase SNURF/RNF4 to target HES1 for degradation. The reciprocal relationship between RASSF1A and HES1 is evident across a wide range of human tumors, highlighting the clinical significance of the identified pathway. We show that HES1 upregulation in a RASSF1A-depleted environment renders cells non-responsive to the downstream effects of γ-secretase inhibitors (GSIs) which restrict signaling at the level of the NOTCH receptor. Taken together, we report a mechanism through which RASSF1A exerts autonomous regulation of the critical Notch effector HES1, thus classifying RASSF1A expression as an integral determinant of the clinical effectiveness of Notch inhibitors. Synopsis The RASSF1A tumor suppressor uncouples the NOTCH-HES1 axis by triggering SNURF/RNF4-mediated HES1 ubiquitination. Loss of RASSF1A promotes cancer stemness via NOTCH-independent HES1 stabilization and confers resistance to γ-secretase inhibitors. A RASSF1A/RNF4/HES1 protein complex is responsible for RNF4-mediated HES1 degradation in cancer cells. RASSF1A promotes cancer stemness through HES1-dependent activation of the core pluripotency network. RASSF1A expression inversely correlates with HES1 levels across the vast majority of human tumour types. HES1 stabilization in a RASSF1A-depleted environment renders cancer cells non-responsive to γ-secretase inhibitors. Introduction RASSF1A epigenetic inactivation correlates with poor clinicopathological characteristics in the vast majority of human solid malignancies (Grawenda & O'Neill, 2015). RASSF1A is a key mediator of Hippo pathway activity and facilitates transcription factor selection for the Hippo effector YAP (Hamilton et al, 2009; van der Weyden et al, 2012; Pefani et al, 2016). In addition to its tumor-suppressive role, RASSF1A undergoes stringent epigenetic regulation throughout early embryonic development to allow smooth transition from pluripotency to differentiation (Papaspyropoulos et al, 2018). Tumorigenesis and somatic cell reprogramming are governed by similar mechanisms, as tumor progression is the outcome of dedifferentiation processes (Friedmann-Morvinski & Verma, 2014). Tumor dedifferentiation has been linked with upregulation of stem cell (SC) markers, such as OCT4, NANOG, and SOX2 and their target genes, which are normally enriched in embryonic stem cells (ESCs) (Friedmann-Morvinski & Verma, 2014; Hepburn et al, 2019). Current findings support that Notch signaling holds a crucial role in inducing tumor dedifferentiation. Activation of the NOTCH1-HES1 axis in solid tumors results in increased epithelial-to-mesenchymal transition (EMT) accompanied by upregulation of stemness genes (Reedijk et al, 2008; Fender et al, 2015; Jin et al, 2017; Fendler et al, 2020). The NOTCH-HES1 axis can be effectively disrupted by γ-secretase inhibitors (GSIs) preventing the proteolytic cleavage of the NOTCH receptor and subsequent translocation of its intracellular domain (NCID) to the nucleus, thereby abrogating both Notch signaling and SC marker expression (Chu et al, 2013). In this study, we present a novel mechanism through which RASSF1A prevents tumor dedifferentiation by triggering HES1 degradation in a Hippo-independent manner. The significance of the identified pathway is depicted in a wide spectrum of human malignancies, whereas employing NOTCH inhibitors defines the precise clinical setting for potential therapeutic intervention. Results and Discussion RASSF1A levels inversely correlate with HES1 expression across human tumor types We previously showed that NOTCH signaling negatively correlates with RASSF1A in human tumors such as breast and lung (Pefani et al, 2016). The Notch target HES1 is a basic helix-loop-helix (bHLH) transcriptional regulator (Artavanis-Tsakonas et al, 1999) which promotes CSC self-renewal (Gao et al, 2014). To investigate a potential RASSF1A-HES1 regulation, we analyzed RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) portal deriving from 32 different human tumor types arising in different organs (n = 9,527 samples). Interestingly, we found that RASSF1A and HES1 are reciprocally expressed across whole patient cohorts for tumors with both high (Fig 1A and Dataset EV1) and low (Fig 1B and Dataset EV1) levels of RASSF1A. Along the same lines, a correlative analysis implementing again RNA-seq data from the TCGA and GTEx cancer databases demonstrated a statistically significant (P < 0.01) negative correlation of RASSF1A with HES1 levels across all tumor types (Fig 1C and Dataset EV1). In line with our clinical data (Fig 1D), upon silencing of RASSF1A, HES1 levels were significantly increased in both human osteosarcoma (U2OS) and cervical cancer (HeLa) cells (Fig 1E and F), implying that RASSF1A may functionally regulate HES1. RASSF1A and HES1 Transcripts Per Million (TPMs) in normal tissues (Fig 1D) were only included to provide a rough estimation of how deregulated the expression of each marker may be in cancer compared to normal tissue. A limitation to this estimation may derive from the small sample size of two normal tissue types (the respective of cervical cancer-CESC and sarcoma-SARC) which relates to their poor representation by the TCGA database. Figure 1. The RASSF1A expression pattern is reciprocal to HES1 across human tumor types Correlation heatmaps depicting an opposite RASSF1A-HES1 expression pattern across the tumors with highest expression of RASSF1A. RNA seq data were retrieved from the TCGA database. Of the 32 tumor types of the TCGA database included in the analysis, tumor types with an average of > 4.2 (median) RASSF1A Transcripts per million (TPM) were classified as RASSF1Ahigh, whereas tumor types with an average of < 4.2 RASSF1A TPM were classified as RASSF1Alow. See also Dataset EV1. Same as A, for the TCGA tumors with the lowest expression of RASSF1A. See also Dataset EV1. Correlation analysis between RASSF1A and HES1 levels across 32 human tumor types (n indicates total number of tumor samples). RNA seq data were retrieved from the TCGA and GTEx databases and analyzed using the GEPIA/GEPIA2 online tools (Tang et al, 2017, 2019). See also Dataset EV1. RASSF1A (bottom) and HES1 (top) transcripts per million (TPM) in the indicated types of human tumors and respective normal tissue. Data were extracted from the TCGA and GTEx databases using the GEPIA/GEPIA2 online tool for normal and cancer gene expression profiling and interactive analyses. The number of patients is indicated in each tumor type. See also Dataset EV1. qPCR for HES1 mRNA levels in siCTRL (non-targeting) and siRASSF1A-transfected U2OS and HeLa cells. U2OS cells were transfected with either siCTRL or siRASSF1A and lysates were immunoblotted for HES1 levels. U2OS cells Tet-On inducibly expressing FLAG-RASSF1A were treated with doxycycline at a concentration of 0.5 μg/ml for 24 h. The cells were fractionated in order to acquire the nuclear and cytoplasmic extracts, which were subsequently Western blotted and probed with the indicated antibodies. Data information: Tumor type abbreviations: ACC: Adrenocortical carcinoma; BLCA: Breast invasive carcinoma; BRCA: Breast invasive carcinoma; CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: Cholangiocarcinoma; COAD: Colon adenocarcinoma; DLBC: Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; ESCA: Esophageal carcinoma; KICH: Kidney Chromophobe; KIRC: Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; KIPAN: Pan-kidney cohort (KIRP + KIRC + KICH); LAML: Acute Myeloid Leukemia; LIHC: Liver hepatocellular carcinoma; LUSC: Lung squamous cell carcinoma; OV: Ovarian serous cystadenocarcinoma; PRAD: Prostate adenocarcinoma; SARC: Sarcoma; THCA: Thyroid carcinoma; UVM: Uveal melanoma. ***P < 0.001 of Student's t-test. Error bars indicate s.e.m. Data shown are representative of three biological replicates (n = 3). Source data are available online for this figure. Source Data for Figure 1 [embr202051287-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint To identify potential RASSF1A-dependent regulators of HES1, we analyzed TCGA data from clinical samples expressing high and low RASSF1A levels (Fig EV1A and Dataset EV1). One of the top hits significantly correlating with RASSF1A levels was the transcription factor GATA1 (P < 0.00001; Fig EV1A), a HES1 transcriptional repressor (Ross et al, 2012). Indeed, GATA1 depletion led to HES1 upregulation in our system (Fig EV1B). Interestingly, GATA1 levels were found decreased upon silencing of the Hippo pathway component Mst1/2 in Xenopus embryos (Nejigane et al, 2013), likely indicating dependency on RASSF1A which functions as an MST2 activator (Matallanas et al, 2007). In support of this, RASSF1A depletion and overexpression in U2OS cells resulted in GATA1 reduction and increase, respectively (Fig EV1C and D), indicating that GATA1 may be responsible for HES1 regulation by RASSF1A at the transcriptional level. Click here to expand this figure. Figure EV1. Transcriptional regulation of HES1 by RASSF1A Analysis of TCGA RNA-seq data from clinical samples expressing highest (LAML) and lowest (ESCA) RASSF1A levels to identify transcription factors following RASSF1A expression. One of the most prominent hits was the HES1 repressor GATA1 (P < 0.00001). Top: qPCR for HES1 levels in U2OS cells transfected with non-targeting (siCTRL) or GATA1-targeting (siGATA1) siRNA. Bottom: qPCR for GATA1 levels verifying successful KD. qPCR for GATA1 levels in U2OS cells transfected with either siCTRL or RASSF1A-targeting (siRASSF1A) siRNA. Western blotting for RASSF1A levels demonstrates knockdown efficiency. qPCR for GATA1 levels in U2OS cells transfected with pCDNA3 or FLAG-RASSF1A. Western blotting demonstrates FLAG-RASSF1A induction. Data information: Tumor type abbreviations: LAML: Acute Myeloid Leukemia; ESCA: Esophageal carcinoma. **P < 0.01, of Student's t-test. Error bars indicate s.e.m. Data shown are representative of three biological replicates (n = 3). Source data are available online for this figure. Download figure Download PowerPoint The observation that HES1 mRNA levels follow the HES1 protein stabilization pattern after RASSF1A modulation (Fig 1E and F) is in keeping with the presence of deeply characterized feedback loops in the case of HES1, where the amount of HES1 protein or its targets in the cell determine HES1 gene transcription rate (Kobayashi et al, 2009; Roese-Koerner et al, 2017; Duan et al, 2019). Similarly to HES1, which is expressed in cells and tissues in an oscillatory fashion (Kobayashi et al, 2009; Roese-Koerner et al, 2017), RASSF1A levels have also been described to oscillate through the cell cycle via degradation by SCF E3 ligase (Song et al, 2008) suggesting that these regulatory processes may be linked. Given that HES1 can be both nuclear and cytoplasmic (Sturrock et al, 2014), we next asked whether RASSF1A-HES1 regulation may differentially affect HES1 protein levels in the nuclear and cytoplasmic fraction. We employed a U2OS Tet-On cell line inducibly expressing FLAG-RASSF1A (U2OSTet ON – FLAG-RASSF1A) upon doxycycline treatment and assessed HES1 protein levels following subcellular fractionation. Interestingly, consistent with a recently described RASSF1A function in nucleocytoplasmic protein transfer and nuclear RASSF1A localization (Chatzifrangkeskou et al, 2019), RASSF1A expression reduced only the nuclear HES1 pool while cytoplasmic HES1 levels remained unaffected (Figs 1G and EV2A). Click here to expand this figure. Figure EV2. RASSF1A-mediated ubiquitination of HES1 Densitometry on Fig 1G showing that only nuclear HES1 levels are significantly reduced upon RASSF1A induction, based on the nuclear HES1/LAMIN B1 ratio. Cytoplasmic HES1 levels remain unaffected based on the cytoplasmic HES1/GAPDH ratio. In vivo ubiquitination assay in HES1 immunoprecipitates from nuclear and cytoplasmic fractions of U2OS cells Tet-On inducibly expressing FLAG-RASSF1A versus Control. Doxycycline (DOX) was used at a concentration of 0.5 μg/ml for 24 h. Immunoprecipitates and Input lysates are probed with displayed antibodies. In vivo ubiquitination assay in HES1 immunoprecipitates from nuclear and cytoplasmic fractions of U2OS cells transfected with either siCTRL or siRASSF1A. Immunoprecipitates and Input lysates are probed with displayed antibodies. Data information: **P < 0.01, of Student's t-test. Error bars indicate s.e.m. Data shown are representative of three biological replicates (n = 3). Source data are available online for this figure. Download figure Download PowerPoint RASSF1A stabilizes the E3 ligase RNF4 to inactivate HES1 through formation of a RASSF1A-RNF4-HES1 complex Apart from RASSF1A regulating HES1 expression, a proteomic screen for RASSF1A binding partners surprisingly identified HES1 itself as a potential interactor (Dataset EV2), suggesting that RASSF1A-HES1 regulation may expand to the post-translational level thereby affecting HES1 stability. In our proteomic screen (Dataset EV2), we also identified the E3 ubiquitin ligase SNURF/RNF4, whose Drosophila ortholog Degringolade (Dgrn) was shown to target HES proteins for SUMO-independent ubiquitination in vitro and in vivo (Abed et al, 2011; Barry et al, 2011). Additionally, a Sleeping Beauty genetic screen carried out in Rassf1a−/− mice identified RNF4 among the potentially most frequently inactivated genes synergizing with RASSF1A loss in tumorigenesis (van der Weyden et al, 2012). HES1 has been found to be degraded by additional E3 ligases in different contexts. For example, the E3 ubiquitin ligase SCFFBXL14 complex targets HES1 for proteolysis, thereby enabling neuronal differentiation (Chen et al, 2017). As previously published reports (Abed et al, 2011; Barry et al, 2011; van der Weyden et al, 2012) and our proteomic data strongly pointed to RNF4 as a potential player affecting RASSF1A-HES1 regulation in cancer cells, we were urged to explore a possible connection. Interestingly, RASSF1A expression in U2OS cells led to significant stabilization of RNF4 protein levels (Fig 2A). HES1 and RNF4 were both identified as co-immunoprecipitating in U2OS cells supporting the regulatory connection, while the HES1/RNF4 ratio was decreased in the presence of FLAG-RASSF1A, suggesting the interaction is supported by RASSF1A (Fig 2B). Moreover, HES1 was found associated with both RNF4 and RASSF1A, confirming the presence of a RASSF1A-RNF4-HES1 complex, concomitant with a reduction of HES1 protein levels (Fig 2B). To confirm that RNF4 was directly responsible for the decrease of HES1 upon RASSF1A activation, we overexpressed RASSF1A in the presence or absence of an RNF4-targeting siRNA and assessed HES1 ubiquitination levels in HES1 immunoprecipitates (Fig 2C). In accordance with data above, RASSF1A overexpression stabilized RNF4 with a concomitant reduction in HES1 levels due to increased HES1 ubiquitination (Fig 2C). However, depletion of RNF4 rescued HES1 despite RASSF1A overexpression (Fig 2C), confirming that RNF4 is required for RASSF1A-mediated HES1 regulation. In keeping with previous results suggesting that RASSF1A-HES1 regulation is nuclear, we found that HES1 was ubiquitinated predominantly in the nuclear fraction of U2OS cells, upon RASSF1A induction (Fig EV2B and C). Our observations in cells were further validated in an in vitro ubiquitination assay showing increased RNF4-mediated HES1 ubiquitination in the presence of RASSF1A (Fig 2D). Taken together, our data uncover a novel mechanism through which RASSF1A regulates the NOTCH effector HES1, via the E3 ligase RNF4 (Fig 2E). Figure 2. RASSF1A-dependent upregulation of E3 ligase SNURF/RNF4 destabilizes HES1 through the formation of a RASSF1A-RNF4-HES1 complex U2OS cells were transfected with either pCDNA3 or FLAG-RASSF1A. Cell extracts were collected and Western blotted with the indicated antibodies. qPCR from the same extracts demonstrated no significant differences in RNF4 mRNA levels. HES1 immunoprecipitation in U2OS cells treated with either pCDNA3 or FLAG-RASSF1A and immunoblotting with the indicated antibodies. Densitometry shows the HES1/RNF4 ratio is inversed upon RASSF1A induction. A RASSF1A-RNF4 complex destabilizes HES1 through direct binding. In vivo ubiquitination assay in HES1 immunoprecipitates from U2OS cells transfected with either pCDNA3/siCTRL, FLAG-RASSF1A/siCTRL, or FLAG-RASSF1A/siRNF4 to assess Ub chain incorporation. Immunoprecipitates and Input lysates are probed with displayed antibodies. In the absence of RNF4, HES1 levels remain stable despite RASSF1A expression. See also Fig EV2B and C. In vitro ubiquitination assay using purified proteins for HES1, RASSF1A, and RNF4. RNF4 served as the E3 ligase of the reaction. Schematic illustrating the RASSF1A-mediated regulation of HES1 stability via the E3 ligase RNF4. Data information: ***P < 0.001 of Student's t-test; n.s., non-significant. Error bars indicate s.e.m. Data shown are representative of three biological replicates (n = 3). Source data are available online for this figure. Source Data for Figure 2 [embr202051287-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint RASSF1A regulates HES1 activity in a Hippo pathway-independent manner RASSF1A epigenetic inactivation has been found to directly induce the expression of the core pluripotency gene Pou5f1/Oct4 in mouse ESCs and pre-implantation embryogenesis through the Hippo pathway effector YAP (Papaspyropoulos et al, 2018). Although mouse models have proven vital tools in recapitulating human disorders, substantial differences occur between the two species, rendering confirmation in human settings essential (Perlman, 2016). Therefore, we sought to investigate whether RASSF1A may exert a similar regulation of pluripotency genes in human cancer cells. To this end, we silenced RASSF1A in HeLa and U2OS cells and found that the core pluripotency markers were upregulated at the protein (Fig EV3A, B and D) and mRNA (Fig EV3C) level. To verify that those effects were RASSF1A-mediated, we induced RASSF1A expression in U2OSTet ON – FLAG-RASSF1A cells and confirmed that RASSF1A induction was indeed sufficient to restrict endogenous expression of all core pluripotency markers (Fig EV3E). Click here to expand this figure. Figure EV3. RASSF1A regulates the core pluripotency markers in cancer Western blotting and densitometry for core pluripotency marker expression in HeLa cells transfected with either non-targeting siRNA (siCTRL) or siRNA against RASSF1A (siRASSF1A). Immunofluorescence for core pluripotency marker expression in HeLa cells transfected with either siCTRL or siRASSF1A. qPCR for core stem cell marker levels in U2OS and HeLa cells in response to siRASSF1A versus siCTRL. Immunoblotting and densitometry for core pluripotency marker expression in U2OS cells transfected with either siCTRL or siRASSF1A. U2OS cells Tet-On inducibly expressing FLAG-RASSF1A were treated with 0.5 μg/ml of doxycycline for 24 h. Cells were subsequently lysed and lysates were immunoblotted with the indicated antibodies. Densitometry is provided for displayed Western blot images. Data information: Scale bars: 20 μm. *P < 0.05, **P < 0.01, and ***P < 0.001, of Student's t-test. Error bars indicate s.e.m. Data shown are representative of three biological replicates (n = 3). Source data are available online for this figure. Download figure Download PowerPoint In ac
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