Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis.

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

Adenovirus small E1A directs activation of Alu transcription at YAP/TEAD- and AP-1-bound enhancers through interactions with the EP400 chromatin remodeler.

Alu retrotransposons, which form the largest family of mobile DNA elements in the human genome, have recently come to attention as a potential source of regulatory novelties, most notably by participating in enhancer function. Even though Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, their expression has long been known to increase in response to various types of stress, including viral infection. Here we show that, in primary human fibroblasts, adenovirus small e1a triggered derepression of hundreds of individual Alus by promoting TFIIIB recruitment by Alu-bound TFIIIC. Epigenome profiling revealed an e1a-induced decrease of H3K27 acetylation and increase of H3K4 monomethylation at derepressed Alus, making them resemble poised enhancers. The enhancer nature of e1a-targeted Alus was confirmed by the enrichment, in their upstream regions, of the EP300/CBP acetyltransferase, EP400 chromatin remodeler and YAP1 and FOS transcription factors. The physical interaction of e1a with EP400 was critical for Alu derepression, which was abrogated upon EP400 ablation. Our data suggest that e1a targets a subset of enhancer Alus whose transcriptional activation, which requires EP400 and is mediated by the e1a-EP400 interaction, may participate in the manipulation of enhancer activity by adenoviruses.

Inhibition of human adenovirus replication by TRIM35-mediated degradation of E1A.

Human adenovirus (HAdV) is ubiquitous in the human population, constituting a significant burden of global respiratory diseases. Children and individuals with low immunity are at risk of developing severe infections without approved antiviral treatment for HAdV. Our study demonstrated that TRIM35 inhibited HAdV-C5 early gene transcription, early protein expression, genome replication, and infectious virus progeny production. Furthermore, TRIM35 was found to inhibit HAdV replication by attenuating E1A expression. Mechanistically, TRIM35 interacts with and degrades E1A by promoting its K48-linked ubiquitination. Additionally, K253 and K285 are the key sites necessary for TRIM35 degradation. Moreover, an oncolytic adenovirus carrying shTRIM35 was constructed and observed to exhibit improved oncolysis in vivo, providing new ideas for clinical tumor treatment. Our results expand the broad antiviral activity of TRIM35 and mechanically support its application as a HAdV replication inhibitor. IMPORTANCE E1A is an essential human adenovirus (HAdV) protein responsible for the early replication of adenovirus while interacting with multiple host proteins. Understanding the interaction between HAdV E1A and TRIM35 helps identify effective antiviral therapeutic targets. The viral E1A protein is a crucial activator and regulator of viral transcription during the early infection stages. We first reported that TRIM35 interacts with E1A to resist adenovirus infection. Our study demonstrated that TRIM35 targets E1A to resist adenovirus, indicating the applicability of targeting virus-dependent host factors as a suitable antiviral strategy.

Targeting Cell Cycle Facilitates E1A-Independent Adenoviral Replication.

DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.

Transcription Factor ETV4 Activates AURKA to Promote PD-L1 Expression and Mediate Immune Escape in Lung Adenocarcinoma.

INTRODUCTION: The occurrence and progression of lung adenocarcinoma (LUAD) impair T-cell immune responses, causing immune escape and subsequently affecting the efficacy of immunotherapy in patients. Aurora kinase A (AURKA) is upregulated in varying cancers, but its role in LUAD immune escape is elusive. This work attempted to explore molecular mechanisms of AURKA regulation in LUAD immune escape. METHODS: Through bioinformatics analysis, AURKA level in LUAD was evaluated, and potential upstream transcription factors of AURKA were predicted using hTFtarget. ETS variant transcription factor 4 (ETV4) expression in LUAD was analyzed through The Cancer Genome Atlas. Pearson's correlation analysis was then utilized to test the correlation between AURKA and ETV4. Interaction and binding between AURKA and ETV4 were validated through dual-luciferase assay and chromatin immunoprecipitation. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) tested relative mRNA expression of AURKA and ETV4 in LUAD cells, cell counting kit-8 assayed cell viability, and Western blot analysis was conducted to determine the protein level of programmed death-ligand 1 (PD-L1). Coculture of LUAD cells with activated CD8+ T cells was carried out, and an LDH assay was used to assess the cytotoxicity of CD8+ T cells against LUAD cells. Interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) levels in the coculture system were assessed by enzyme-linked immunosorbent assay (ELISA). Western blot assessed protein levels of JAK2, p-JAK2, STAT3, and p-STAT3. RESULTS: Compared to normal tissues, AURKA and ETV4 were upregulated in tumor tissues, and AURKA presented a negative association with CD8+ T-cell immune infiltration but a positive association with PD-L1. qRT-PCR unveiled significantly upregulated mRNA of AURKA and ETV4 in LUAD cells compared to normal lung epithelial cells. Knockdown of AURKA significantly decreased cell viability and PD-L1 protein level in LUAD cells, enhanced cytotoxicity of CD8+ T cells against LUAD cells and IFN-γ, IL-2, and TNF-α expression, while overexpression of AURKA yielded opposite results. Furthermore, the knockdown of ETV4 could reverse the oncogenic characteristics of cells caused by AURKA overexpression. CONCLUSION: Our study illustrated that ETV4/AURKA axis promoted PD-L1 expression, suppressed CD8+ T-cell activity, and mediated immune escape in LUAD by regulating the JAK2/STAT3 signaling pathway.

Differential Splicing of Human Adenovirus 5 E1A RNA Expressed in cis versus in trans.

Human adenovirus (HAdV) is used extensively as a vector for gene delivery for a variety of purposes, including gene therapy and vaccine development. Most adenoviral vectors used for these approaches have a deletion of early region 1 (E1), which is complemented by the cell line. Most commonly, these are 293 cells for HAdV serotype 2 or 5. The 293 cells have the left end of HAdV5 integrated into chromosome 19 and express the E1 genes and protein IX. We observed that viruses with the E1 region deleted often grow less well on 293 cells than E1 wild-type viruses. Therefore, we investigated whether this poor growth is caused by splicing differences between the E1A RNA provided by the cell line (in trans) and the E1A RNA provided by the infecting viral genome (in cis). We observed that E1A RNA that was expressed from the genomes of 293 cells was spliced differently during infection with an E1A-deleted dl312 virus than E1A RNA from the same cells infected with dl309 or wt300. Importantly, 293 cells were not able to fully complement the late E1A transcripts, specifically 11S, 10S, and 9S RNA, which express the E1A217R, E1A171R, and E1A55R isoforms, respectively. We observed that these splicing differences likely arise due to different subnuclear localizations of E1A RNA. E1A RNA expressed from the viral genome was localized to viral replication centers, while E1A RNA expressed from the cell's genome was not. This loss of the late E1A mRNAs and their associated proteins impacts viral growth, gene expression, and protein levels. Complementation of the late E1A mRNAs in 293 cells restored some of the growth defect observed with dl312 and resulted in higher virus growth.IMPORTANCE Human adenovirus has become an important tool for medicine and research, and 293 cells and various similar cell lines are used extensively for virus production in situations where high viral yields are important. Such complementing cell lines are used for the production of viral vectors and vaccines, which often have deletions and replacements in various viral genes. Deletions in essential genes, such as E1, are often complemented by the cell line that is used for virus propagation in trans Here, we show that even complete genetic complementation of a viral gene does not result in full protein complementation, a defect that compromises virus growth. This is particularly important when high viral yields are crucial, as in virus production for vaccine development or gene therapy.

Absent in melanoma 2 enhances anti-tumour effects of CAIX promotor controlled conditionally replicative adenovirus in renal cancer.

Conditionally replicative adenoviruses (CRAds) were promising approach for solid tumour treatment, but its oncolytic efficiency and toxicity are still not satisfactory for further clinical application. Here, we developed the CAIX promotor (CAIXpromotor )-controlled CRAd armed with a tumour suppressor absent in melanoma 2 (AIM2) to enhance its oncolytic potency. The CAIXpromotor -AIM2 adenoviruses (Ad-CAIXpromotor -AIM2) could efficiently express E1A and AIM2 in renal cancer cells. Compared with Ad-CAIXpromotor , Ad-CAIXpromotor -AIM2 significantly inhibited cell proliferation and enhanced cell apoptosis and cell killing, thus resulting in the oncolytic efficiency in 786-O cells or OSRC-2 cells. To explore the therapeutic effect, various Ads were intratumourally injected into OSRC-2-xenograft mice. The tumour growth was remarkably inhibited in Ad-CAIXpromotor -AIM2-treated group as demonstrated by reduced tumour volume and weight with a low toxicity. The inflammasome inhibitor YVAD-CMK resulted in the reduction of anti-tumour activity by Ad-CAIXpromotor -AIM2 in vitro or in vivo, suggesting that inflammasome activation response was required for the enhanced therapeutic efficiency. Furthermore, lung metastasis of renal cancer mice was also suppressed by Ad-CAIXpromotor -AIM2 treatment accompanied by the decreased tumour fossil in lung tissues. These results indicated that the tumour-specific Ad-CAIXpromotor -AIM2 could be applied for human renal cancer therapy. The therapeutic strategy of AIM2-based CRAds could be a potential and promising approach for the therapy of primary solid or metastasis tumours.

Open chromatin profiling identifies AP1 as a transcriptional regulator in oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is one of the ten most prevalent forms of cancer and is showing a rapid increase in incidence and yet exhibits poor survival rates. Compared to many other common cancers, the molecular changes that occur in this disease are relatively poorly understood. However, genes encoding chromatin remodeling enzymes are frequently mutated in OAC. This is consistent with the emerging concept that cancer cells exhibit reprogramming of their chromatin environment which leads to subsequent changes in their transcriptional profile. Here, we have used ATAC-seq to interrogate the chromatin changes that occur in OAC using both cell lines and patient-derived material. We demonstrate that there are substantial changes in the regulatory chromatin environment in the cancer cells and using this data we have uncovered an important role for ETS and AP1 transcription factors in driving the changes in gene expression found in OAC cells.

Soluble syntaxin 3 functions as a transcriptional regulator.

Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that Stx3 (syntaxin 3) unexpectedly also functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform that we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5 (RAN-binding protein 5), targets to the nucleus, and interacts physically and functionally with several transcription factors, including ETV4 (ETS variant 4) and ATF2 (activating transcription factor 2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified, suggesting that nuclear signaling is a conserved, novel function common among these membrane-trafficking proteins.

Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters.

How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.

Mimicry of Cellular A Kinase-Anchoring Proteins Is a Conserved and Critical Function of E1A across Various Human Adenovirus Species.

The E1A proteins of the various human adenovirus (HAdV) species perform the critical task of converting an infected cell into a setting primed for virus replication. While E1A proteins differ in both sequence and mechanism, the evolutionary pressure on viruses with limited coding capacity ensures that these proteins often have significant overlap in critical functions. HAdV-5 E1A is known to use mimicry to rewire cyclic AMP (cAMP) signaling by decoupling protein kinase A (PKA) from cellular A kinase-anchoring proteins (AKAPs) and utilizing PKA to its own advantage. We show here that E1As from other species of HAdV also possess this viral AKAP (vAKAP) function and examine how they manipulate PKA. E1A from most species of HAdV examined contain a small AKAP-like motif in their N terminus which targets the docking-dimerization domain of PKA as the binding interface for a conserved protein-protein interaction. This motif is also responsible for an E1A-mediated relocalization of PKA regulatory subunits from the cytoplasm into the nucleus, with species-specific E1A proteins having preference for one particular isoform of PKA subunit over another. Importantly, we showed that these newly characterized vAKAPs can integrate into cAMP-responsive transcription as well as contribute to viral genome replication and infectious progeny production for several distinct HAdV species.IMPORTANCE These data enhance the mechanistic knowledge on how HAdV E1A manipulates cellular PKA to benefit infection. The work establishes that mimicry of AKAPs and subversion of PKA-mediated cAMP signaling are conserved features for numerous human adenoviruses. This study also highlights the molecular determinants conferring selective protein-protein interactions between distinct PKA regulatory subunits and the different E1A proteins of these viruses. Additionally, it further emphasizes the utility of using viral proteins like E1A as tools for studying the molecular biology of cellular regulatory pathways.

Adenovirus 5 E1A-Mediated Suppression of p53 via FUBP1.

Far-upstream element (FUSE) binding protein 1 (FUBP1) was originally identified as a regulator of the oncogene c-Myc via binding to the FUSE within the c-Myc promoter and activating the expression of the gene. Recent studies have identified FUBP1 as a regulator of transcription, translation, and splicing via its DNA and RNA binding activities. Here we report the identification of FUBP1 as a novel binding partner of E1A. FUBP1 binds directly to E1A via the N terminus (residues 1 to 82) and conserved region 3 (residues 139 to 204) of adenovirus 5 E1A. The depletion of FUBP1 via short interfering RNAs (siRNA) reduces virus growth and drives the upregulation of the cellular stress response by activating the expression of p53-regulated genes. During infection, FUBP1 is relocalized within the nucleus, and it is recruited to viral promoters together with E1A while at the same time being lost from the FUSE upstream of the c-Myc promoter. The depletion of FUBP1 affects viral and cellular gene expression. Importantly, in FUBP1-depleted cells, p53-responsive genes are upregulated, p53 occupancy on target promoters is enhanced, and histone H3 lysine 9 is hyperacetylated. This is likely due to the loss of the FUBP1-mediated suppression of p53 DNA binding. We also observed that E1A stabilizes the FUBP1-p53 complex, preventing p53 promoter binding. Together, our results identify, for the first time, FUBP1 as a novel E1A binding protein that participates in aspects of viral replication and is involved in the E1A-mediated suppression of p53 function.IMPORTANCE Viral infection triggers innate cellular defense mechanisms that have evolved to block virus replication. To overcome this, viruses have counterevolved mechanisms that ensure that cellular defenses are either disarmed or not activated to guarantee successful replication. One of the key regulators of cellular stress is the tumor suppressor p53 that responds to a variety of cellular stress stimuli and safeguards the integrity of the genome. During infection, many viruses target the p53 pathway in order to deactivate it. Here we report that human adenovirus 5 coopts the cellular protein FUBP1 to prevent the activation of the p53 stress response pathway that would block viral replication. This finding adds to our understanding of p53 deactivation by adenovirus and highlights its importance in infection and innate immunity.

A survivin-responsive, conditionally replicating adenovirus induces potent cytocidal effects in adult T-cell leukemia/lymphoma.

BACKGROUND: Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy caused by long-term human T-cell leukemia virus type I (HTLV-1) infection. Survivin-responsive, conditionally replicating adenoviruses regulated by multiple tumor-specific factors (Surv.m-CRAs), in which the expression of the adenoviral early region 1A gene is regulated by the survivin (BIRC5) promoter, can be used to treat several cancers. As survivin is overexpressed in ATL, we examined the effects of Surv.m-CRAs on ATL-selective replication and survival. METHODS: We tested two ATL cell lines and four HTLV-1-infected T-cell lines. The cells were subjected to infection with either E1-deleted, replication-defective adenoviruses or Surv.m-CRAs at various multiplicities of infection. RESULTS: Strong activation of survivin promoter was observed in all six cell lines. Moreover, the expression of the coxsackie and adenovirus receptor (CAR), which is important for adenoviral infection, was high in the cell lines. In contrast, we observed the absence of survivin promoter activity and a low expression of CAR in activated peripheral blood lymphocytes (PBLs) from healthy subjects. Surv.m-CRAs actively replicated and induced cytocidal effects in five out of six cell lines; conversely, we observed minimal viral replication and no marked cytotoxicity in normal activated PBLs. CONCLUSIONS: This is the first report demonstrating that Surv.m-CRAs constitute attractive potential anti-ATL agents.

Identification of TBX2 and TBX3 variants in patients with conotruncal heart defects by target sequencing.

BACKGROUND: Conotruncal heart defects (CTDs) are heterogeneous congenital heart malformations that result from outflow tract dysplasia; however, the genetic determinants underlying CTDs remain unclear. Increasing evidence demonstrates that dysfunctional TBX2 and TBX3 result in outflow tract malformations, implying that both of them are involved in CTD pathogenesis. We screened for TBX2 and TBX3 variants in a large cohort of CTD patients (n = 588) and population-matched healthy controls (n = 300) by target sequencing and genetically analyzed the expression and function of these variants. RESULTS: The probably damaging variants p.R608W, p.T249I, and p.R616Q of TBX2 and p.A192T, p.M65L, and p.A562V of TBX3 were identified in CTD patients, but none in controls. All altered amino acids were highly conserved evolutionarily. Moreover, our data suggested that mRNA and protein expressions of TBX2 and TBX3 variants were altered compared with those of the wild-type. We screened PEA3 and MEF2C as novel downstream genes of TBX2 and TBX3, respectively. Functional analysis revealed that TBX2R608W and TBX2R616Q variant proteins further activated HAS2 promoter but failed to activate PEA3 promoter and that TBX3A192T and TBX3A562V variant proteins showed a reduced transcriptional activity over MEF2C promoter. CONCLUSIONS: Our results indicate that the R608W and R616Q variants of TBX2 as well as the A192T and A562V variants of TBX3 contribute to CTD etiology; this was the first association of variants of TBX2 and TBX3 to CTDs based on a large population.

Structured and disordered regions cooperatively mediate DNA-binding autoinhibition of ETS factors ETV1, ETV4 and ETV5.

Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory domain that packs against the ETS domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein-protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.

ETS (E26 transformation-specific) up-regulation of the transcriptional co-activator TAZ promotes cell migration and metastasis in prostate cancer.

Prostate cancer is a very common malignant disease and a leading cause of death for men in the Western world. Tumorigenesis and progression of prostate cancer involves multiple signaling pathways, including the Hippo pathway. Yes-associated protein (YAP) is the downstream transcriptional co-activator of the Hippo pathway, is overexpressed in prostate cancer, and plays a vital role in the tumorigenesis and progression of prostate cancer. However, the role of the YAP paralog and another downstream effector of the Hippo pathway, transcriptional co-activator with PDZ-binding motif (TAZ), in prostate cancer has not been fully elucidated. Here, we show that TAZ is a basal cell marker for the prostate epithelium. We found that overexpression of TAZ promotes the epithelial-mesenchymal transition (EMT), cell migration, and anchorage-independent growth in the RWPE1 prostate epithelial cells. Of note, knock down of TAZ in the DU145 prostate cancer cells inhibited cell migration and metastasis. We also found that SH3 domain binding protein 1 (SH3BP1), a RhoGAP protein that drives cell motility, is a direct target gene of TAZ in the prostate cancer cells, mediating TAZ function in enhancing cell migration. Moreover, the prostate cancer-related oncogenic E26 transformation-specific (ETS) transcription factors, ETV1, ETV4, and ETV5, were required for TAZ gene transcription in PC3 prostate cancer cells. MAPK inhibitor U0126 treatment decreased TAZ expression in RWPE1 cells, and ETV4 overexpression rescued TAZ expression in RWPE1 cells with U0126 treatment. Our results show a regulatory mechanism of TAZ transcription and suggest a significant role for TAZ in the progression of prostate cancer.

ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis.

BACKGROUND: The ETS transcription factor ETV4 is involved in the main steps of organogenesis and is also a significant mediator of tumorigenesis and metastasis, such as in breast cancer. Indeed, ETV4 is overexpressed in breast tumors and is associated with distant metastasis and poor prognosis. However, the cellular and molecular events regulated by this factor are still misunderstood. In mammary epithelial cells, ETV4 controls the expression of many genes, MMP13 among them. The aim of this study was to understand the function of MMP13 during ETV4-driven tumorigenesis. METHODS: Different constructs of the MMP13 gene promoter were used to study the direct regulation of MMP13 by ETV4. Moreover, cell proliferation, migration, invasion, anchorage-independent growth, and in vivo tumorigenicity were assayed using models of mammary epithelial and cancer cells in which the expression of MMP13 and/or ETV4 is modulated. Importantly, the expression of MMP13 and ETV4 messenger RNA was characterized in 456 breast cancer samples. RESULTS: Our results revealed that ETV4 promotes proliferation, migration, invasion, and anchorage-independent growth of the MMT mouse mammary tumorigenic cell line. By investigating molecular events downstream of ETV4, we found that MMP13, an extracellular metalloprotease, was an ETV4 target gene. By overexpressing or repressing MMP13, we showed that this metalloprotease contributes to proliferation, migration, and anchorage-independent clonogenicity. Furthermore, we demonstrated that MMP13 inhibition disturbs proliferation, migration, and invasion induced by ETV4 and participates to ETV4-induced tumor formation in immunodeficient mice. Finally, ETV4 and MMP13 co-overexpression is associated with poor prognosis in breast cancer. CONCLUSION: MMP13 potentiates the effects of the ETV4 oncogene during breast cancer genesis and progression.

Inhibition of androgen receptor transactivation function by adenovirus type 12 E1A undermines prostate cancer cell survival.

BACKGROUND: Mutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance. METHODS: Luciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays. RESULTS: Adenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12. CONCLUSION: Ad12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.

Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes.

Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. IMPORTANCE: It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.

SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation.

Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. Here we show that SIRT7 is an NAD(+)-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo.