ETV4 Expression Research Articles

Oncogenic ETS Factors in Prostate Cancer.

Prostate cancer is unique among carcinomas in that a fusion gene created by a chromosomal rearrangement is a common driver of the disease. The TMPRSS2/ERG rearrangement drives aberrant expression of the ETS family transcription factor ERG in 50% of prostate tumors. Similar rearrangements promote aberrant expression of the ETS family transcription factors ETV1 and ETV4 in another 10% of cases. Together, these three ETS factors are thought to promote tumorigenesis in the majority of prostate cancers. A goal of precision medicine is to be able to apply targeted therapeutics that are specific to disease subtypes. ETS gene rearrangement positive tumors represent the largest molecular subtype of prostate cancer, but to date there is no treatment specific to this marker. In this chapter we will review the latest findings regarding the molecular mechanisms of ETS factor function in the prostate. These molecular details may provide a path towards new therapeutic targets for this subtype of prostate cancer. Further, we will describe efforts to target the oncogenic functions of ETS family transcription factors directly as well as indirectly.

MicroRNA-322 Regulates Self-renewal of Mouse Spermatogonial Stem Cells through Rassf8.

Spermatogonial stem cells (SSCs) are essential for spermatogenesis and male fertility. MicroRNAs (miRs) are key regulators of gene expression involved in self-renewal, differentiation, and apoptosis. However, the function and mechanisms of individual miR in regulating self-renewal and differentiation of SSCs remain unclear. Here, we report for the first time that miR-322 regulates self-renewal of SSCs. Functional assays revealed that miR-322 was essential for SSC self-renewal. Mechanistically, miR-322 promoted SSC self-renewal by targeting RASSF8 (ras association domain family 8). Moreover, the WNT/β-catenin signaling pathway was involved in the miR-322-mediated regulation. Furthermore, miR-322 overexpression increased GFRα1, ETV5 and PLZF expression but decreased STRA8, C-KIT and BCL6 expression. Our study provides not only a novel insight into molecular mechanisms regulating SSC self-renewal but also a basis for the diagnosis, treatment, and prevention of male infertility.

Functional Screening for Pathways Regulating Chemoresistance and Sensitivity in Leukemia Stem Cells

INTRODUCTIONOn average 5 recurrent mutations are present in each patient with acute myeloid leukemia (AML). Many mutated genes are implicated as tumor suppressor genes, but their contribution to leukemia stem cell (LSC) survival and chemoresistance is often unknown. We hypothesized that ectopic expression of the wildtype sequence of these genes will restore the function of the tumor suppressor gene and will lead to reduced clonal expansion and increased chemosensitivity.AIMTo evaluate the contribution of recurrently mutated genes to leukemia stem cell survival and chemoresistance in human AML cells in vitro and in vivo.METHODSWe performed a loss-of-function screening in primary human AML cells and cell lines by lentiviral expression of a pool of 22 wildtype genes associated with AML pathogenesis, which can restore gene function of a repressed pathway or a dysfunctional tumor suppressor gene. The 22 full-length cDNAs were labelled with a genetic barcode, which can be amplified with a common primer for all 22 genes. The readout of the screening was reduced representation of the barcode DNA after in vitro culture or in vivo growth in patient-derived xenograft (PDX) models, which was amplified from DNA and quantified by next-generation sequencing (NGS).Five PDX models with favorable, normal or complex cytogenetics and 3-5 recurrent mutations per model were screened in order to identify cDNAs that could potentially limit the proliferative capacity of LSCs in vivo (5 mice per model). Nine to 16 weeks after transplantation, DNA from blood, bone marrow and spleen were analyzed by NGS. We also screened two CD34+-enriched cord blood samples in vitro. After transduction with the cDNA pool, cells were cultured for 11 days and the barcode representation was analyzed by NGS at days 2, 4, 7, 9 and 11.Finally, we screened the human leukemia cell lines U937and PB14, a newly established cell line from an AML patient with mutations in FLT3, NPM1, RAD21, GSE1, and ROBO2. Cells were treated with cytarabine, doxorubicin or venetoclax for a 3-day period, followed by a 4-day recovery period to allow outgrowth of resistant clones and accumulation of cDNAs that conferred drug resistance.RESULTSOur loss-of-function screening with overexpressed wildtype genes revealed that expression of ETV6 and PTPN11 depleted LSCs in 3 of 5 PDX models and that expression of CEBPA and KDM6A depleted the progeny of normal CD34+ cells in cord blood. ASXL1, EZH2, CUX1, SMC1A and SMC3 had a general negative effect on stem cell self-renewal in both leukemic and normal CD34+ cells.Relative frequencies of the leukemia-specific genes ETV6 and PTPN11 were reduced 3 to 16-fold and 2 to 3-fold, respectively. Both genes were not mutated in the 3 patients' diagnostic samples, but had reduced RNA expression by 10-70% compared to healthy control peripheral blood mononuclear cells. Relative frequencies of cord blood-specific CEBPA was reduced 4-fold and KDM6A 5-fold.We then evaluated whether activation of a repressed pathway can increase sensitivity to cytarabine, doxorubicin or venetoclax in U937 and PB14 cells after 1 or 2 weeks of treatment. All three drugs showed better cytotoxic effects upon p53 expression in both cell lines by a factor of 1.4 to 2.5 fold. Cytarabine and venetoclax improved elimination of leukemic cells that had been transduced with U2AF1 in PB14 cells, which are U2AF1 wildtype. Venetoclax improved elimination of U937 cells that had been transduced with ETV6 or KDM6A, which are wildtype for these genes, while RNA expression was reduced more than 50% in these cells compared to other leukemic cell lines (NB4 and MV4-11).CONCLUSIONFunctional cDNA screening in PDX models in vivo is feasible and can reveal selective vulnerabilities of leukemic compared to normal stem cells. Our approach was validated by the finding that p53 expression improved chemosensitivity for all drugs tested in two leukemia cell lines, which is expected from the known function of p53 as a critical tumor suppressor gene.Similarly, overexpression of the transcriptional corepressor ETV6 in leukemia cells with low ETV6 expression was found to inhibit leukemia stem cell proliferation in vivo and to sensitize U937 cells to venetoclax. Therefore, activation of ETV6 should be explored as a novel strategy to inhibit LSCs and improve treatment response. DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Janssen: Consultancy; StemLine Therapeutics: Consultancy; Bayer Pharma AG: Consultancy, Research Funding; Tetralogic: Research Funding; Sunesis: Research Funding; Daiichi Sankyo: Research Funding; Karyopharm: Research Funding; BergenBio: Research Funding; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding.

Establishment of a transgenic mouse to model ETV7 expressing human tumors

The ETS transcription factor ETV7 has been characterized as a hematopoietic oncoprotein, which requires cooperating mutations for its leukemogenic activity. Although the ETV7 gene is highly conserved among vertebrates, part of the rodents, including Mus musculus, deleted the Etv7 gene locus. Many human hematopoietic malignancies upregulate ETV7 expression but contrary to ETV7’s role in oncogenesis, its physiological role in normal tissues is unknown. To determine the physiological function of ETV7 in vivo and determine its role in tumorigenesis in a mouse model, we have generated an ETV7 transgenic mouse that carries a single copy of human BAC DNA containing the ETV7 gene locus and its regulatory sequences. ETV7 heterozygous (ETV7Tg+/WT) mice were fertile, normal in size and born at a normal Mendelian frequency. They had a normal blood count, did not display any gross physical or behavioral abnormalities, and were not tumor-prone. The ETV7 expression pattern in hematopoietic cells of ETV7Tg+/WT mice is very similar to that in human hematopoietic cells. To examine the oncogenic potential of ETV7 in vivo, we crossed ETV7Tg+/WT mice with tumor-prone mouse models. ETV7 greatly accelerated loss of Pten (phosphatase and tensin homolog)-evoked leukemogenesis in PtenΔ/ΔETV7Tg+/WT mice after deletion of the conditional Pten allele. Consistent with this observation, ETV7 expression enhanced the colony-forming and self-renewal activities of primary myeloid Pten−/− cells. In this study we established a transgenic mouse in which we can more accurately model ETV7-associated human tumorigenesis in vivo.

Maldevelopment of the submandibular gland in a mouse model of apert syndrome.

Apert syndrome is characterized by craniosynostosis and bony syndactyly of the hands and feet. The cause of Apert syndrome is a single nucleotide substitution mutation (S252W or P253R) in fibroblast growth factor receptor 2 (FGFR2). Clinical experience suggests increased production of saliva by Apert syndrome patients, but this has not been formally investigated. FGFR2 signaling is known to regulate branching morphogenesis of the submandibular glands (SMGs). With the Apert syndrome mouse model (Ap mouse), we investigated the role of FGFR2 in SMGs and analyzed the SMG pathology of Apert syndrome. Ap mice demonstrated significantly greater SMG and sublingual gland (SMG/SLG complex) mass/body weight and percentage of parenchyma per unit area of the SMG compared with control mice. Furthermore, gene expression of Fgf1, Fgf2, Fgf3, Pdgfra, Pdgfrb, Mmp2, Bmp4, Lama5, Etv5, and Dusp6 was significantly higher in the SMG/SLG complex of Ap mice. FGF3 and BMP4 exhibited altered detection patterns. The numbers of macrophages were significantly greater in SMGs of Ap mice than in controls. Regarding functional evaluations of the salivary glands, no significant differences were observed. These results suggest that the gain-of-function mutation in FGFR2 in the SMGs of Ap mice enhances branching morphogenesis. Developmental Dynamics 247:1175-1185, 2018. © 2018 Wiley Periodicals, Inc.

Genome wide mapping of ETV6 binding sites in pre-B leukemic cells

Genetic alterations in the transcriptional repressor ETV6 are associated with hematological malignancies. Notably, the t(12;21) translocation leading to an ETV6-AML1 fusion gene is the most common genetic alteration found in childhood acute lymphoblastic leukemia. Moreover, most of these patients also lack ETV6 expression, suggesting a tumor suppressor function. To gain insights on ETV6 DNA-binding specificity and genome wide transcriptional regulation capacities, we performed chromatin immunoprecipitation experiments coupled to deep sequencing in a t(12;21)-positive pre-B leukemic cell line. This strategy led to the identification of ETV6-bound regions that were further associated to gene expression. ETV6 binding is mostly cell type-specific as only few regions are shared with other blood cell subtypes. Peaks localization and motif enrichment analyses revealed that this unique binding profile could be associated with the ETV6-AML1 fusion protein specific to the t(12;21) background. This study underscores the complexity of ETV6 binding and uncovers ETV6 transcriptional network in pre-B leukemia cells bearing the recurrent t(12;21) translocation.

Ubiquitin ligase COP1 coordinates transcriptional programs that control cell type specification in the developing mouse brain

The E3 ubiquitin ligase CRL4COP1/DET1 is active in the absence of ERK signaling, modifying the transcription factors ETV1, ETV4, ETV5, and c-JUN with polyubiquitin that targets them for proteasomal degradation. Here we show that this posttranslational regulatory mechanism is active in neurons, with ETV5 and c-JUN accumulating within minutes of ERK activation. Mice with constitutive photomorphogenesis 1 (Cop1) deleted in neural stem cells showed abnormally elevated expression of ETV1, ETV4, ETV5, and c-JUN in the developing brain and spinal cord. Expression of c-JUN target genes Vimentin and Gfap was increased, whereas ETV5 and c-JUN both contributed to an expanded number of cells expressing genes associated with gliogenesis, including Olig1, Olig2, and Sox10. The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 contributed to the perinatal lethality, as several Cop1-deficient mice also lacking c-Jun and Etv5, or lacking Etv5 and heterozygous for Etv1, were viable.

The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells

The ETS family of transcription factors is involved in several normal remodeling events and pathological processes including tumor progression. ETS transcription factors are divided into subfamilies based on the sequence and location of the ETS domain. ETV5 (Ets variant gene 5; also known as ERM) is a member of the PEA3 subfamily. Our meta-analysis of normal, benign, and malignant thyroid samples demonstrated that ETV5 expression is upregulated in papillary thyroid cancer and was predominantly associated with BRAF V600E or RAS mutations. However, the precise role of ETV5 in these lesions is unknown. In this study, we used the KTC1 cell line as a model for human advanced papillary thyroid cancer (PTC) because the cells harbor the heterozygous BRAF (V600E) mutation together with the C250T TERT promoter mutation. The role of ETV5 in PTC proliferation was tested using RNAi followed by high-throughput screening. Signaling pathways driving ETV5 expression were identified using specific pharmacological inhibitors. To determine if ETV5 influences the expression of epithelial-to-mesenchymal (EMT) markers in these cells, an EMT PCR array was used, and data were confirmed by qPCR and ChIP-qPCR. We found that ETV5 is critical for PTC cell growth, is expressed downstream of the MAPK pathway, and directly upregulates the transcription factor TWIST1, a known marker of intravasation and metastasis. Increased ETV5 expression could therefore be considered as a marker for advanced PTCs and a possible future therapeutic target.

In This Issue

HomeCirculation ResearchVol. 123, No. 5In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published16 Aug 2018https://doi.org/10.1161/RES.0000000000000225Circulation Research. 2018;123:507is related toSafety and Efficacy of Intracoronary Infusion of Allogeneic Human Cardiac Stem Cells in Patients With ST-Segment Elevation Myocardial Infarction and Left Ventricular DysfunctionTransplanted Mesenchymal Stem Cells Reduce Autophagic Flux in Infarcted Hearts via the Exosomal Transfer of miR-125bThe Transcription Factor ETV1 Induces Atrial Remodeling and ArrhythmiaETV1 and Atrial Remodeling (p 550)Download figureDownload PowerPointIncreased ETVI levels drive arrhythmia in mice, report Rommel et al.Although advanced age, high blood pressure, and alcohol abuse have been recognized as important risk factors for developing atrial fibrillation (AF), the molecular pathology underlying the development of AF remains largely unknown. Rommel and colleagues have now discovered that the transcription factor ETV1 is upregulated in the atria of patients with permanent AF. To examine the potential role of this transcription factor in the development of AF, they created transgenic mice with cardiac myocyte-specific overexpression of ETV1. They found that at first the hearts of these animals appeared normal, but by 12 weeks of age, the mice had developed arrhythmias, and their atria were enlarged and dilated. Survival rates of these animals were significantly reduced. The team then created mice in which cardiac myocyte expression of ETV1 was ablated and found that these animals had survival rates comparable to wild-type controls and that they were protected against induced AF-like atrial remodeling. Transcriptome and chromatin analyses of the transgenic and control animals revealed 170 genes that were specifically targeted by ETV1. Further investigations of the ETV1 target genes could lead to novel insights and new treatments for AF.MSCs Protect MI Through Exosome-Mediated Autophagy (p 564)Download figureDownload PowerPointMesenchymal stem cell–derived exosomes protect infarcted hearts from excess autophagy, say Xiao et al.Autophagy—the intracellular process of degrading and recycling damaged organelles—is activated by mild ischemia and, under this condition, can preserve cell viability. During severe ischemia, however, autophagy can be activated chronically and lead to excessive cell death. It is thought that reduced cell death is one of the potential benefits of stem cell treatments for myocardial infarction. However, it is unclear whether stem cells treatments influence autophagy. Xiao and colleagues now report that treatment of mice with mesenchymal stem cells (MSCs), after myocardial infarction, leads to reduced cardiac myocyte autophagy compared with that seen in untreated animals. Furthermore, direct inhibition of autophagy prior to infarction resulted in mice with better cardiac performance and ventricular remodeling. In vitro, coculturing experiments confirmed the autophagy-suppressing effect of MSCs on cardiac myocytes. And the team went on to show that exosomes packed with the microRNA miR-125b-5p were the MSC-derived mediators of autophagy suppression. By identifying pathways and molecules that control autophagy, these results uncover new targets for postinfarction therapies that may bypass the need for cell therapy.Allogeneic Cardiac Cells for STEMI (p 579)Download figureDownload PowerPointFernandéz-Avilés et al find allogenic stem cells offer no clinical benefits to myocardial infarction patients.Stem cells of various types are under investigation as potential therapies for preventing heart failure following myocardial infarction. While the use of autologous stem cells avoids the possibility of immunorejection, these cells can be less effective if the patients are elderly or suffer from multiple comorbities. Allogenic cells from healthy donors avoid this problem, and can be readily produced in large quantities. To examine the safety and efficacy of allogenic human cardiac stem cells, Fernandéz-Avilés and colleagues conducted a phase I/II clinical trial (CAREMI). In this trial, donor-derived allogenic cardiac stem cells were administered via intracoronary infusions to 33 patients ≈1 week after myocardial infarction, while another 16 patients received a placebo. After a 12-month follow up, there were no deaths or major adverse cardiac events, indicating that the procedure was safe. However, there was no evidence that the stem cells reduced infarct size or improved left ventricle remodeling or the patients’ quality of life. These results could inform the design of future cell therapy trials. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesSafety and Efficacy of Intracoronary Infusion of Allogeneic Human Cardiac Stem Cells in Patients With ST-Segment Elevation Myocardial Infarction and Left Ventricular DysfunctionFrancisco Fernández-Avilés, et al. Circulation Research. 2018;123:579-589Transplanted Mesenchymal Stem Cells Reduce Autophagic Flux in Infarcted Hearts via the Exosomal Transfer of miR-125bChangchen Xiao, et al. Circulation Research. 2018;123:564-578The Transcription Factor ETV1 Induces Atrial Remodeling and ArrhythmiaCarolin Rommel, et al. Circulation Research. 2018;123:550-563 August 17, 2018Vol 123, Issue 5 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000225PMID: 30355147 Originally publishedAugust 16, 2018 PDF download Advertisement

ETV7-Mediated DNAJC15 Repression Leads to Doxorubicin Resistance in Breast Cancer Cells

Breast cancer treatment often includes Doxorubicin as adjuvant as well as neoadjuvant chemotherapy. Despite its cytotoxicity, cells can develop drug resistance to Doxorubicin. Uncovering pathways and mechanisms involved in drug resistance is an urgent and critical aim for breast cancer research oriented to improve treatment efficacy. Here we show that Doxorubicin and other chemotherapeutic drugs induce the expression of ETV7, a transcriptional repressor member of ETS family of transcription factors. The ETV7 expression led to DNAJC15 down-regulation, a co-chaperone protein whose low expression was previously associated with drug resistance in breast and ovarian cancer. There was a corresponding reduction in Doxorubicin sensitivity of MCF7 and MDA-MB-231 breast cancer cells. We identified the binding site for ETV7 within DNAJC15 promoter and we also found that DNA methylation may be a factor in ETV7-mediated DNAJC15 transcriptional repression. These findings of an inverse correlation between ETV7 and DNAJC15 expression in MCF7 cells in terms of Doxorubicin resistance, correlated well with treatment responses of breast cancer patients with recurrent disease, based on our analyses of reported genome-wide expression arrays. Moreover, we demonstrated that ETV7-mediated Doxorubicin-resistance involves increased Doxorubicin efflux via nuclear pumps, which could be rescued in part by DNAJC15 up-regulation. With this study, we propose a novel role for ETV7 in breast cancer, and we identify DNAJC15 as a new target gene responsible for ETV7-mediated Doxorubicin-resistance. A better understanding of the opposing impacts of Doxorubicin could improve the design of combinatorial adjuvant regimens with the aim of avoiding resistance and relapse.

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

BackgroundThe 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.MethodsDifferent 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.ResultsOur 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.ConclusionMMP13 potentiates the effects of the ETV4 oncogene during breast cancer genesis and progression.

ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes

Rapid impulse propagation is a defining attribute of the pectinated atrial myocardium and His-Purkinje system (HPS) that safeguards against atrial and ventricular arrhythmias, conduction block, and myocardial dyssynchrony. The complex transcriptional circuitry that dictates rapid conduction remains incompletely understood. Here, we demonstrate that ETV1 (ER81)-dependent gene networks dictate the unique electrophysiological characteristics of atrial and His-Purkinje myocytes. Cardiomyocyte-specific deletion of ETV1 results in cardiac conduction abnormalities, decreased expression of rapid conduction genes (Nkx2–5, Gja5, and Scn5a), HPS hypoplasia, and ventricularization of the unique sodium channel properties that define Purkinje and atrial myocytes in the adult heart. Forced expression of ETV1 in postnatal ventricular myocytes (VMs) reveals that ETV1 promotes a HPS gene signature while diminishing ventricular and nodal gene networks. Remarkably, ETV1 induction in human induced pluripotent stem cell-derived cardiomyocytes increases rapid conduction gene expression and inward sodium currents, converting them towards a HPS phenotype. Our data identify a cardiomyocyte-autonomous, ETV1-dependent pathway that is responsible for specification of rapid conduction zones in the heart and demonstrate that ETV1 is sufficient to promote a HPS transcriptional and functional program upon VMs.

PO-290 ETV7 regulates breast cancer stem cells content and chemoresistance

IntroductionCancer stem cells (CSCs) are considered the population of cells within the tumour able to drive tumorigenesis and known to be highly resistant to conventional chemotherapy. ETV7 is a poorly studied transcription factor member of ETS large family, known to be an interferon-stimulated gene. It has been recently found over-expressed in breast cancer (BC), with higher expression levels in the more aggressive BC subtypes. In this work, we investigated the effects of ETV7 increased expression on breast CSCs population and resistance to chemotherapy in BC cells.Material and methodsWe generated MCF7 and T47D BC-derived cells stably over-expressing ETV7 and obtained ETV7 KO in MDA-MB-231 BC cells using CRISPR/Cas9 technology. We analysed breast CSCs content via CD44/CD24 staining and FACS analysis, as well as mammospheres formation assay. We measured expression of ABC transporters and anti-apoptotic proteins via RT-qPCR and western blot. We finally assessed sensitivity to Doxorubicin and 5-Fluorouracil (5-FU) via MTT assay and AnnexinV/PI staining at FACS.Results and discussionsWe observed that the expression of ETV7 could be induced by various stimuli, particularly by chemotherapeutic drugs able to induce DNA damage. We then analysed the impact of ETV7 expression on the sensitivity to Doxorubicin and 5-FU and we could observe a significantly decreased sensitivity to these drugs upon ETV7 over-expression. We could also appreciate an increase in ABC transporters and BCL2 anti-apoptotic protein expression following ETV7 over-expression. We further observed that alteration of ETV7 expression could significantly affect the population of breast cancer stem cells (CD44+/CD24low cells) in different BC cell lines.ConclusionWe propose a novel role for ETV7 in breast cancer stem cells plasticity and associated resistance to conventional chemotherapy. We finally suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs targeted therapies and to the improvement of combinatorial regimens with the aim of avoiding resistance and relapse in breast cancer.

Graph complexity analysis identifies an ETV5 tumor-specific network in human and murine low-grade glioma.

Conventional differential expression analyses have been successfully employed to identify genes whose levels change across experimental conditions. One limitation of this approach is the inability to discover central regulators that control gene expression networks. In addition, while methods for identifying central nodes in a network are widely implemented, the bioinformatics validation process and the theoretical error estimates that reflect the uncertainty in each step of the analysis are rarely considered. Using the betweenness centrality measure, we identified Etv5 as a potential tissue-level regulator in murine neurofibromatosis type 1 (Nf1) low-grade brain tumors (optic gliomas). As such, the expression of Etv5 and Etv5 target genes were increased in multiple independently-generated mouse optic glioma models relative to non-neoplastic (normal healthy) optic nerves, as well as in the cognate human tumors (pilocytic astrocytoma) relative to normal human brain. Importantly, differential Etv5 and Etv5 network expression was not directly the result of Nf1 gene dysfunction in specific cell types, but rather reflects a property of the tumor as an aggregate tissue. Moreover, this differential Etv5 expression was independently validated at the RNA and protein levels. Taken together, the combined use of network analysis, differential RNA expression findings, and experimental validation highlights the potential of the computational network approach to provide new insights into tumor biology.

FGF2 and EGF induce epithelial-mesenchymal transition in malignant pleural mesothelioma cells via a MAPKinase/MMP1 signal.

Malignant pleural mesothelioma (MPM), an aggressive malignancy affecting pleural surfaces, occurs in three main histological subtypes. The epithelioid and sarcomatoid subtypes are characterized by cuboid and fibroblastoid cells, respectively. The biphasic subtype contains a mixture of both. The sarcomatoid subtype expresses markers of epithelial-mesenchymal transition (EMT) and confers the worst prognosis, but the signals and pathways controlling EMT in MPM are not well understood. We demonstrate that treatment with FGF2 or EGF induced a fibroblastoid morphology in several cell lines from biphasic MPM, accompanied by scattering, decreased cell adhesion and increased invasiveness. This depended on the MAP-kinase pathway but was independent of TGFβ or PI3-kinase signaling. In addition to changes in known EMT markers, microarray analysis demonstrated differential expression of MMP1, ESM1, ETV4, PDL1 and BDKR2B in response to both growth factors and in epithelioid versus sarcomatoid MPM. Inhibition of MMP1 prevented FGF2-induced scattering and invasiveness. Moreover, in MPM cells with sarcomatoid morphology, inhibition of FGF/MAP-kinase signaling induced a more epithelioid morphology and gene expression pattern. Our findings suggest a critical role of the MAP-kinase axis in the morphological and behavioral plasticity of mesothelioma.

Increased ETV4 expression correlates with estrogen-enhanced proliferation and invasiveness of cholangiocarcinoma cells

BackgroundCholangiocarcinoma (CCA) is one of the worst prognosis cancer. The survival time of CCA patients is related to serum estrogen levels and estrogen has been found to enhance the proliferation and invasiveness of CCA cells in vitro. This has led to the suggestion that estrogen may play an important role in the progression of CCA. This study tests the relevance of the previous in vitro findings in vivo using a mouse xenograft model of CCA, and investigates possible signaling mechanisms involved.MethodsKKU-213 and KKU-139 CCA cell lines were used in the experiments, xenografted to nude mice and treated with a potent estrogenic agent, 17β-estradiol (E2), and/or with tamoxifen (TAM), an estrogen antagonist.ResultsThe results demonstrated that E2 could accelerate growth of the xenograft-tumor and the effect was inhibited by TAM. PCR array screening of E2 responsive genes suggested ETV4 as a promising candidate intracellular mediator. ETV4-knockdown CCA cells were generated and these showed a diminished responsiveness to E2 in both cell and spheroid proliferation assays, and in invasion tests. These results point to ETV4 as a possible mediator of E2-activated CCA progression and as a potential target of TAM-mediated inhibition.ConclusionsFinally, TAM may be suggested as an adjunctive treatment of CCA to improve the conventional cytotoxic method with more patient toleration.

ETV4 Facilitates Cell-Cycle Progression in Pancreatic Cells through Transcriptional Regulation of Cyclin D1.

The ETS family transcription factor ETV4 is aberrantly expressed in a variety of human tumors and plays an important role in carcinogenesis through upregulation of relevant target gene expression. Here, it is demonstrated that ETV4 is overexpressed in pancreatic cancer tissues as compared with the normal pancreas, and is associated with enhanced growth and rapid cell-cycle progression of pancreatic cancer cells. ETV4 expression was silenced through stable expression of a specific short hairpin RNA (shRNA) in two pancreatic cancer cell lines (ASPC1 and Colo357), while it was ectopically expressed in BXPC3 cells. Silencing of ETV4 in ASPC1 and Colo357 cells reduced the growth by 55.3% and 38.9%, respectively, while forced expression of ETV4 in BXPC3 cells increased the growth by 46.8% in comparison with respective control cells. Furthermore, ETV4-induced cell growth was facilitated by rapid transition of cells from G1- to S-phase of the cell cycle. Mechanistic studies revealed that ETV4 directly regulates the expression of Cyclin D1 CCND1, a protein crucial for cell-cycle progression from G1- to S-phase. These effects on the growth and cell cycle were reversed by the forced expression of Cyclin D1 in ETV4-silenced pancreatic cancer cells. Altogether, these data provide the first experimental evidence for a functional role of ETV4 in pancreatic cancer growth and cell-cycle progression.Implications: The functional and mechanistic data presented here regarding ETV4 in pancreatic cancer growth and cell-cycle progression suggest that ETV4 could serve as a potential biomarker and novel target for pancreatic cancer therapy. Mol Cancer Res; 16(2); 187-96. ©2017 AACR.

Abstract B24: Molecular and functional dissection of the CIC-DUX4 fusion in undifferentiated round cell sarcoma

Abstract CIC-DUX4 soft-tissue tumors are an aggressive subset of undifferentiated round cell sarcomas that arise in children and young adults. The cytogenetic hallmark, (t(4;19) or t(10;19)), of this tumor is a chromosomal translocation that fuses the transcription factor, Capicua (CIC), with the double homeobox 4 protein, DUX4. Despite morphologic similarities to Ewing sarcoma, CIC-DUX4 sarcomas are clinically distinct with rapid development of lethal metastatic disease and chemoinsensitivity. The unique cytogenetic and clinical features that distinguish CIC-DUX4 sarcomas from other small round cell tumors provide a unique opportunity to identify and rationally target this population to improve clinical outcomes. To meet this need, we are taking a systematic functional approach to identify critical CIC-DUX4 targets that promote tumor progression and metastasis. Leveraging a comparative transcriptional analysis between cytogenetically distinct small round cell sarcomas, we reveal that the CIC-DUX4 fusion activates highly conserved molecular networks to control tumor growth and metastatic capacity. Using an orthotopic soft-tissue sarcoma model, we show that CIC-DUX4 induces ETV4 expression to drive invasion and metastasis. Genetic silencing of ETV4 in CIC-DUX4 expressing tumors limits tumor dissemination without a profound impact on tumor cell growth. These findings suggest that CIC-DUX4 enhances metastasis and tumor growth through distinct transcriptional repertoires. Consistent with this, we identify multiple components of the cell cycle machinery that are transcriptionally regulated by CIC-DUX4 expression. Specifically, the CIC-DUX4 fusion co-opts native CIC-binding specificity to transcriptionally activate these cell cycle target genes. Altogether, our molecular and functional dissection of the CIC-DUX4 fusion reveals highly conserved CIC-regulated transcriptional targets that promote tumor growth and metastasis. We aim to pharmacologically exploit these key CIC-DUX4-dependent transcriptional nodes to improve outcomes for this population with few therapeutic options. Citation Format: Ross A. Okimoto, Wei Wu, Trever G. Bivona. Molecular and functional dissection of the CIC-DUX4 fusion in undifferentiated round cell sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B24.

Berberine reinforces Sertoli cells niche and accelerates spermatogonial stem cells renewal in experimentally-induced varicocele condition in rats

BackgroundVaricocele is present in 10–20% of the male infertile population. Purpose: Present study was done to demonstrate the reinforcing effect of berberine (BBR), as an antioxidant and anti-inflammatory agent, on Sertoli cells-related niche and spermatogonial stem cells (SSCs) self-renewal in experimentally-induced VCL condition. Study design50 mature male Wistar rats were divided into control, control-sham, non-treated VCL-induced, 50 mg kg−1 and 100 mg kg−1 BBR-treated VCL-induced groups. MethodsThe Leydig and Sertoli cells distribution and Leydig cells steroidogenic activity, expression of glial cell line-derived neurotrophic factor (GDNF), proto-oncogene Rearranged during Transfection (c-RET) receptor, Ets variant gene 5 (Etv5) and B-cell chronic lymphocytic leukemia (CLL)/lymphoma 6, member B (Bcl-6b) at mRNA and protein levels were analyzed. The mRNA integrity and DNA fragmentation were assessed. Finally, the serum levels of testosterone, inhibin B and testicular total antioxidant capacity, total thiol molecules, catalase, and malondialdehyde were evaluated. ResultsObservations revealed that, the BBR significantly enhanced VCL-reduced Leydig and Sertoli cells population, maintained Leydig–Sertoli cells network, enhanced GDNF, c-RET Etv5 and Bcl6b expression, up-regulated testicular antioxidant and endocrine status. ConclusionThe BBR by boosting Leydig–Sertoli cells network up-regulates the GDNF, Etv5 and Bcl-6b expression/synthesis in SSCs, which in turn improves SSCs self-renewal activities. Thus, the BBR could be considered as an appropriate agent for antioxidant therapy of VCLs. However, more studies with bigger sample number and focus on BBR-induced effects on other genes involving in the self-renewal process are needed to have more deterministic results.

ETV4 collaborates with Wnt/β-catenin signaling to alter cell cycle activity and promote tumor aggressiveness in gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is the most common sarcoma, often resulting from a KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutation. The lineage transcription factor ETV1 is expressed similarly in GISTs regardless of malignant potential. Although the related transcription factor ETV4 has been associated with metastasis and tumor progression in other cancers, its role in GIST is unknown. In this study, we found that ETV4 levels were high in a subset of human GISTs and correlated with high mitotic rate. Through Gene Set Enrichment Analysis in selected human GISTs, we identified a relationship between ETV4 levels and β-catenin signaling, especially in advanced GISTs. GIST specimens with high ETV4 levels overexpressed cell cycle regulating genes and had aberrant activation of the canonical Wnt pathway. In human GIST cell lines, ETV4 RNA interference suppressed cell cycle genes and Wnt/β-catenin signaling. ETV4 knockdown also reduced tumor cell proliferation, invasion, and tumor growth in vivo. Conversely, ETV4 overexpression increased cyclin D1 expression and Wnt/β-catenin signaling. Moreover, we determined that ETV4 knockdown destabilized nuclear β-catenin and increased its degradation via COP1, an E3 ligase involved in both ETV4 and β-catenin turnover. Aberrant accumulation of ETV4 and nuclear β-catenin was found in patient derived xenografts created from metastatic GISTs that became resistant to tyrosine kinase inhibitors. Collectively, our findings highlight the significance of ETV4 expression in GIST and identify ETV4 as a biomarker in human GISTs.