Sin3b interacts with Myc and decreases Myc levels.

Myc expression is deregulated in many human cancers. A yeast two-hybrid screen has revealed that the transcriptional repressor Sin3b interacts with Myc protein. Endogenous Myc and Sin3b co-localize and interact in the nuclei of human and rat cells, as assessed by co-immunoprecipitation, immunofluorescence, and proximity ligation assay. The interaction is Max-independent. A conserved Myc region (amino acids 186-203) is required for the interaction with Sin3 proteins. Histone deacetylase 1 is recruited to Myc-Sin3b complexes, and its deacetylase activity is required for the effects of Sin3b on Myc. Myc and Sin3a/b co-occupied many sites on the chromatin of human leukemia cells, although the presence of Sin3 was not associated with gene down-regulation. In leukemia cells and fibroblasts, Sin3b silencing led to Myc up-regulation, whereas Sin3b overexpression induced Myc deacetylation and degradation. An analysis of Sin3b expression in breast tumors revealed an association between low Sin3b expression and disease progression. The data suggest that Sin3b decreases Myc protein levels upon Myc deacetylation. As Sin3b is also required for transcriptional repression by Mxd-Max complexes, our results suggest that, at least in some cell types, Sin3b limits Myc activity through two complementary activities: Mxd-dependent gene repression and reduction of Myc levels.

Plasminogen activator inhibitor 1--insulin-like growth factor binding protein 3 cascade regulates stress-induced senescence.

Cellular senescence is widely believed to play a key role in tumor suppression, but the molecular pathways that regulate senescence are only incompletely understood. By using a secretome proteomics approach, we identified insulin-like growth factor binding protein 3 (IGFBP3) as a secreted mediator of breast cancer senescence upon chemotherapeutic drug treatment. The senescence-inducing activity of IGFBP3 is inhibited by tissue-type plasminogen activator-mediated proteolysis, which is counteracted by plasminogen activator inhibitor 1 (PAI-1), another secreted mediator of senescence. We demonstrate that IGFBP3 is a critical downstream target of PAI-1-induced senescence. These results suggest a role for an extracellular cascade of secreted proteins in the regulation of cellular senescence.

Proteomic Analysis of the EWS-Fli-1 Interactome Reveals the Role of the Lysosome in EWS-Fli-1 Turnover.

Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly Fli-1. EWS-Fli-1 fusion accounts for 85% of cases. The growth and survival of Ewing sarcoma cells are critically dependent on EWS-Fli-1. A large body of evidence has established that EWS-Fli-1 functions as a DNA-binding transcription factor that regulates the expression of a number of genes important for cell proliferation and transformation. However, little is known about the biochemical properties of the EWS-Fli-1 protein. We undertook a series of proteomic analyses to dissect the EWS-Fli-1 interactome. Employing a proximity-dependent biotinylation technique, BioID, we identified cation-independent mannose 6-phosphate receptor (CIMPR) as a protein located in the vicinity of EWS-Fli-1 within a cell. CIMPR is a cargo that mediates the delivery of lysosomal hydrolases from the trans-Golgi network to the endosome, which are subsequently transferred to the lysosomes. Further molecular cell biological analyses uncovered a role for lysosomes in the turnover of the EWS-Fli-1 protein. We demonstrate that an mTORC1 active-site inhibitor, torin 1, which stimulates the TFEB-lysosome pathway, can induce the degradation of EWS-Fli-1, suggesting a potential therapeutic approach to target EWS-Fli-1 for degradation.

Roles of USP1 in Ewing sarcoma.

Ewing sarcoma is a cancer of bone and soft tissue in children and young adults that is driven by the EWS-ETS fusion transcription factor, most commonly EWS-FLI1. We previously reported that Ewing sarcoma harbors two populations of cells, the CD133high population displaying higher growth rate and the CD133low population displaying chemotherapy resistance. We now find that the ubiquitin-specific protease 1 (USP1) is a transcriptional target of the EWS-FLI1 fusion oncoprotein, expressed at high and low levels in the CD133high and the CD133low populations, respectively, and determines chemo-sensitivity. We also find that USP1 inhibits cdc42, increases EWS-FLI1 transcriptional output, and simulates Ewing sarcoma growth. We show that chemo-sensitization by USP1 is independent of cdc42. A pharmacological inhibitor of USP1 was able to activate cdc42 and inhibit Ewing sarcoma growth. These results uncover critical roles for USP1 in Ewing sarcoma, which regulates growth and chemo-sensitivity via distinct mechanisms.

Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma.

Ewing sarcoma is a cancer of bone and soft tissue in children and young adults primarily driven by the EWS-FLI1 fusion oncoprotein, which has been undruggable. Here, we report that Ewing sarcoma depends on secreted sphingomyelin phosphodiesterase 1 (SMPD1), a ceramide-generating enzyme, and ceramide. We find that G-protein-coupled receptor 64 (GPR64)/adhesion G-protein-coupled receptor G2 (ADGRG2) responds to ceramide and mediates critical growth signaling in Ewing sarcoma. We show that ceramide induces the cleavage of the C-terminal intracellular domain of GPR64, which translocates to the nucleus and restrains the protein levels of RIF1 in a manner dependent on SPOP, a substrate adaptor of the Cullin3-RING E3 ubiquitin ligase. We demonstrate that both SMPD1 and GPR64 are transcriptional targets of EWS-FLI1, indicating that SMPD1 and GPR64 are EWS-FLI1-induced cytokine-receptor dependencies. These results reveal the SMPD1-ceramide-GPR64 pathway, which drives Ewing sarcoma growth and is amenable to therapeutic intervention.

Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma.

Retinoblastoma is a cancer of the infant retina primarily driven by loss of the Rb tumor suppressor gene, which is undruggable. Here, we report an autocrine signaling, mediated by secreted frizzled-related protein 2 (SFRP2), which suppresses nitric oxide and enables retinoblastoma growth. We show that coxsackievirus and adenovirus receptor (CXADR) is the cell-surface receptor for SFRP2 in retinoblastoma cells; that CXADR functions as a "dependence receptor," transmitting a growth-inhibitory signal in the absence of SFRP2; and that the balance between SFRP2 and CXADR determines nitric oxide production. Accordingly, high SFRP2 RNA expression correlates with high-risk histopathologic features in retinoblastoma. Targeting SFRP2 signaling by SFRP2-binding peptides or by a pharmacological inhibitor rapidly induces nitric oxide and profoundly inhibits retinoblastoma growth in orthotopic xenograft models. These results reveal a cytokine signaling pathway that regulates nitric oxide production and retinoblastoma cell proliferation and is amenable to therapeutic intervention.

Redundant Signaling as the Predominant Mechanism for Resistance to Antibodies Targeting the Type-I Insulin-Like Growth Factor Receptor in Cells Derived from Childhood Sarcoma.

Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R-targeted antibodies. Sensitivity to the IGF-1R-targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1-resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (∼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R-targeted antibodies tested against this model.

The interaction of the von Hippel-Lindau tumor suppressor and heterochromatin protein 1.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor is associated with renal carcinoma, hemangioblastoma and pheochromocytoma. The VHL protein is a component of a ubiquitin ligase complex that ubiquitinates and degrades hypoxia inducible factor-α (HIF-α). Degradation of HIF-α by VHL is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in tumor suppression and other biological processes. Using GST-VHL pull-down experiment and mass spectrometry, we detected an interaction between VHL and heterochromatin protein 1 (HP1). We identified a conserved HP1-binding motif (PXVXL) in the ß domain of VHL, which is disrupted in a renal carcinoma-associated P81S mutant. We show that the VHL P81S mutant displays reduced binding to HP1, yet retains the ability to interact with elongin B, elongin C, and cullin 2 and is fully capable of degrading HIF-α. We also demonstrate that HP1 increases the chromatin association of VHL. These results suggest a role for the VHL-HP1 interaction in VHL chromatin targeting.

Slit2 signaling stimulates Ewing sarcoma growth.

Ewing sarcoma is a cancer of bone and soft tissue in children driven by EWS::ETS fusion, most commonly EWS::FLI1. Because current cytotoxic chemotherapies are not improving the survival of those with metastatic or recurrent Ewing sarcoma cases, there is a need for novel and more effective targeted therapies. While EWS::FLI1 is the major driver of Ewing sarcoma, EWS::FLI1 has been difficult to target. A promising alternative approach is to identify and target the molecular vulnerabilities created by EWS::FLI1. Here we report that EWS::FLI1 induces the expression of Slit2, the ligand of Roundabout (Robo) receptors implicated in axon guidance and multiple other developmental processes. EWS::FLI1 binds to the Slit2 gene promoter and stimulates the expression of Slit2. Slit2 inactivates cdc42 and stabilizes the BAF chromatin remodeling complexes, enhancing EWS::FLI1 transcriptional output. Silencing of Slit2 strongly inhibited anchorage-dependent and anchorage-independent growth of Ewing sarcoma cells. Silencing of Slit2 receptors, Robo1 and Robo2, inhibited Ewing sarcoma growth as well. These results uncover a new role for Slit2 signaling in stimulating Ewing sarcoma growth and suggest that this pathway can be targeted therapeutically.

Proteomic dissection of the von Hippel-Lindau (VHL) interactome.

The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex containing elongin B, elongin C, cullin 2, and Rbx1, which acts as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the alpha subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in the maintenance of primary cilium, extracellular matrix formation, and tumor suppression. We undertook a series of proteomic analyses to gain a comprehensive picture of the VHL-interacting proteins. We found that the ARF tumor suppressor interacts with VHL30, a longer VHL isoform, but not with VHL19, a shorter VHL isoform. ARF was found to release VHL30 from the E3 ligase complex, promoting the binding of VHL30 to a protein arginine methyltransferase, PRMT3. Our analysis of the VHL19 interactome also uncovered that VHL19 displays an affinity to collagens and their biosynthesis enzymes.

Quantitative proteomic identification of the BRCA1 ubiquitination substrates.

Mutation of the BRCA1 tumor suppressor gene predisposes women to hereditary breast and ovarian cancers. BRCA1 forms a heterodimer with BARD1. The BRCA1/BARD1 heterodimer has ubiquitin ligase activity, considered to play crucial roles in tumor suppression and DNA damage response. Nevertheless, relevant BRCA1 substrates are poorly defined. We have developed a new approach to systematically identify the substrates of ubiquitin ligases by identifying proteins that display an enhanced incorporation of His-tagged ubiquitin upon ligase coexpression; using this method, we identified several candidate substrates for BRCA1. These include scaffold attachment factor B2 (SAFB2) and Tel2 as well as BARD1. BRCA1 was found to enhance SAFB protein expression and induce Tel2 nuclear translocation. Identification of the ubiquitination substrates has been a major obstacle to understanding the functions of ubiquitin ligases. The quantitative proteomics approach we devised for the identification of BRCA1 substrates will facilitate the identification of ubiquitin ligase-substrate pairs.

EZH2 suppresses endogenous retroviruses and an interferon response in cancers.

Ewing sarcoma is an aggressive cancer of bone and soft tissue in children. It is characterized by the chromosomal translocation between EWS and an Ets family transcription factor, most commonly FLI1. We recently reported that Ewing sarcoma depends on the autocrine signaling mediated by a cytokine, NELL2. NELL2 signaling stimulates the transcriptional output of EWS-FLI1 through the BAF chromatin remodeling complexes. While studying the impact of NELL2 silencing on Ewing sarcoma, we found that suppression of NELL2 signaling induces the expression of endogenous retroviruses (ERVs) and LINE-1 retrotransposons, an interferon response, and growth arrest. We determined that a histone methyltransferase, EZH2, is the critical downstream target of NELL2 signaling in suppressing ERVs, LINE-1, an interferon response, and growth arrest. We show that EZH2 inhibitors induce ERVs, LINE-1, and an interferon response in a variety of cancer types. These results uncover the role for NELL2-EZH2 signaling in suppressing endogenous virus-like agents and an antiviral response, and suggest the potential utility of EZH2 inhibitors in enhancing anti-tumor immunity.

NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growth.

BAF chromatin remodeling complexes play important roles in chromatin regulation and cancer. Here, we report that Ewing sarcoma cells are dependent on the autocrine signaling mediated by NELL2, a secreted glycoprotein that has been characterized as an axon guidance molecule. NELL2 uses Robo3 as the receptor to transmit critical growth signaling. NELL2 signaling inhibits cdc42 and upregulates BAF complexes and EWS-FLI1 transcriptional output. We demonstrate that cdc42 is a negative regulator of BAF complexes, inducing actin polymerization and complex disassembly. Furthermore, we identify NELL2highCD133highEWS-FLI1high and NELL2lowCD133lowEWS-FLI1low populations in Ewing sarcoma, which display phenotypes consistent with high and low NELL2 signaling, respectively. We show that NELL2, CD133, and EWS-FLI1 positively regulate each other and upregulate BAF complexes and cell proliferation in Ewing sarcoma. These results reveal a signaling pathway regulating critical chromatin remodeling complexes and cancer cell proliferation.

Loss of MGA repression mediated by an atypical polycomb complex promotes tumor progression and invasiveness.

MGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated Mga in two mouse models of non-small cell lung cancer using a CRISPR-based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of non-canonical Polycomb ncPRC1.6 targets, including genes involved in metastasis and meiosis. Moreover, MGA deletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA-MAX, E2F6, and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes these ncPRC1.6 subunits. Lastly, we report that MGA loss also induces a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F target genes mediated by MGA-MAX associated with a non-canonical Polycomb complex.

GDF6-CD99 Signaling Regulates Src and Ewing Sarcoma Growth.

We report here that the autocrine signaling mediated by growth and differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family of cytokines, maintains Ewing sarcoma growth by preventing Src hyperactivation. Surprisingly, Ewing sarcoma depends on the prodomain, not the BMP domain, of GDF6. We demonstrate that the GDF6 prodomain is a ligand for CD99, a transmembrane protein that has been widely used as a marker of Ewing sarcoma. The binding of the GDF6 prodomain to the CD99 extracellular domain results in recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity. GDF6 silencing causes hyperactivation of Src and p21-dependent growth arrest. We demonstrate that two GDF6 prodomain mutants linked to Klippel-Feil syndrome are hyperactive in CD99-Src signaling. These results reveal a cytokine signaling pathway that regulates the CSK-Src axis and cancer cell proliferation and suggest the gain-of-function activity for disease-causing GDF6 mutants.

MYC Regulation of D2HGDH and L2HGDH Influences the Epigenome and Epitranscriptome.

Mitochondrial D2HGDH and L2HGDH catalyze the oxidation of D-2-HG and L-2-HG, respectively, into αKG. This contributes to cellular homeostasis in part by modulating the activity of αKG-dependent dioxygenases. Signals that control the expression/activity of D2HGDH/L2HGDH are presumed to broadly influence physiology and pathology. Using cell and mouse models, we discovered that MYC directly induces D2HGDH and L2HGDH transcription. Furthermore, in a manner suggestive of D2HGDH, L2HGDH, and αKG dependency, MYC activates TET enzymes and RNA demethylases, and promotes their nuclear localization. Consistent with these observations, in primary B cell lymphomas MYC expression positively correlated with enhancer hypomethylation and overexpression of lymphomagenic genes. Together, these data provide additional evidence for the role of mitochondria metabolism in influencing the epigenome and epitranscriptome, and imply that in specific contexts wild-type TET enzymes could demethylate and activate oncogenic enhancers.

Identification and characterization of SAP25, a novel component of the mSin3 corepressor complex.

The transcriptional corepressor mSin3 is associated with histone deacetylases (HDACs) and is utilized by many DNA-binding transcriptional repressors. We have cloned and characterized a novel mSin3A-binding protein, SAP25. SAP25 binds to the PAH1 domain of mSin3A, associates with the mSin3A-HDAC complex in vivo, and represses transcription when tethered to DNA. SAP25 is required for mSin3A-mediated, but not N-CoR-mediated, repression. SAP25 is a nucleocytoplasmic shuttling protein, actively exported from the nucleus by a CRM1-dependent mechanism. A fraction of SAP25 is located in promyelocytic leukemia protein (PML) nuclear bodies, and PML induces a striking nuclear accumulation of SAP25. An isotope-coded affinity tag quantitative proteomic analysis of the SAP25 complex revealed that SAP25 is associated with several components of the mSin3 complex, nuclear export machinery, and regulators of transcription and cell cycle. These results suggest that SAP25 is a novel core component of the mSin3 corepressor complex whose subcellular location is regulated by PML.

EWS-FLI-1 creates a cell surface microenvironment conducive to IGF signaling by inducing pappalysin-1.

Ewing sarcoma is an aggressive cancer of bone and soft tissue in children with poor prognosis. It is characterized by the chromosomal translocation between EWS and an Ets family transcription factor, most commonly FLI-1. EWS-FLI-1 fusion accounts for 85% of Ewing sarcoma cases. EWS-FLI-1 regulates the expression of a number of genes important for sarcomagenesis, can transform NIH3T3 and C3H10T1/2 cells, and is necessary for proliferation and tumorigenicity of Ewing sarcoma cells, suggesting that EWS-FLI-1 is the causative oncoprotein. Here we report that EWS-FLI-1 induces the expression of pappalysin-1 (PAPPA), a cell surface protease that degrades IGF binding proteins (IGFBPs) and increases the bioavailability of IGF. EWS-FLI-1 binds to the pappalysin-1 gene promoter and stimulates the expression of pappalysin-1, leading to degradation of IGFBPs and enhanced IGF signaling. Silencing of pappalysin-1 strongly inhibited anchorage-dependent and anchorage-independent growth as well as xenograft tumorigenicity of Ewing sarcoma cells. These results suggest that EWS-FLI-1 creates a cell surface microenvironment conducive to IGF signaling by inducing pappalysin-1, which emerged as a novel target to inhibit IGF signaling in Ewing sarcoma.

Role of galactose in cellular senescence.

Cellular senescence has been proposed to play critical roles in tumor suppression and organismal aging, but the molecular mechanism of senescence remains incompletely understood. Here we report that a putative lysosomal carbohydrate efflux transporter, Spinster, induces cellular senescence in human primary fibroblasts. Administration of d-galactose synergistically enhanced Spinster-induced senescence and this synergism required the transporter activity of Spinster. Intracellular d-galactose is metabolized to galactose-1-phosphate by galactokinase. Galactokinase-deficient fibroblasts, which accumulate intracellular d-galactose, displayed increased baseline senescence. Senescence of galactokinase-deficient fibroblasts was further enhanced by d-galactose administration and was diminished by restoration of wild-type galactokinase expression. Silencing galactokinase in normal fibroblasts also induced senescence. These results suggest a role for intracellular galactose in the induction of cellular senescence.

FGF19 functions as autocrine growth factor for hepatoblastoma.

Hepatoblastoma is the most common liver cancer in children, accounting for over 65% of all childhood liver malignancies. Hepatoblastoma is distinct from adult liver cancer in that it is not associated with hepatitis virus infection, cirrhosis, or other underlying liver pathology. The paucity of appropriate cell and animal models has been hampering the mechanistic understanding of hepatoblastoma pathogenesis. Consequently, there is no molecularly targeted therapy for hepatoblastoma. To gain insight into cytokine signaling in hepatoblastoma, we employed mass spectrometry to analyze the proteins secreted from Hep293TT hepatoblastoma cell line we established and identified the specific secretion of fibroblast growth factor 19 (FGF19), a growth factor for liver cells. We determined that silencing FGF19 by shRNAs or neutralizing secreted FGF19 by anti-FGF19 antibody inhibits the proliferation of hepatoblastoma cells. Furthermore, blocking FGF19 signaling by an FGF receptor kinase inhibitor suppressed hepatoblastoma growth. RNA expression analysis in hepatoblastoma tumors revealed that the high expression of FGF19 signaling pathway components as well as the low expression of FGF19 signaling repression targets correlates with the aggressiveness of the tumors. These results suggest the role of FGF19 as autocrine growth factor for hepatoblastoma.