Interindividual Variation in Gut Nitrergic Neuron Density Is Regulated By GDNF Levels and ETV1.

BACKGROUND & AIMS: The size and function of the enteric nervous system (ENS) can vary substantially between individuals. Because ENS function is involved in the etiology of a growing number of common human diseases, understanding mechanisms that regulate ENS variation is important. METHODS: We analyzed RNAseq data from 41 normal adult human colon biopsies and single-cell RNA-seq data from human and mouse developing gut. To establish cause-consequence relationship we used alleles in mice that allow levels change of the candidate effector molecule in the comparable range to human samples. We used siRNA and primary neuronal cultures to define downstream molecular events and characterized gut functional changes in mice where molecular phenotypes paralleled findings in humans. RESULTS: We found that glial cell line-derived neurotrophic factor (GDNF) levels in the human colon vary about 5-fold and correlate strongly with nitrergic marker expression. In mice, we defined that GDNF levels are regulated via its 3' untranslated region (3' UTR) in the gastrointestinal tract and observed similar correlation between GDNF levels and nitrergic lineage development. We identified miR-9 and miR-133 as evolutionarily conserved candidates for negative regulation of GDNF expression in the gastrointestinal tract. Functionally, an increase in inhibitory nitrergic innervation results in an increase in gastrointestinal tract transit time, stool size, and water content accompanied with modestly reduced epithelial barrier function. Mechanistically, we found that GDNF levels regulate nitrergic lineage development via induction of transcription factor ETV1, corroborated by single-cell gene expression data in human and mouse developing enteric neurons. CONCLUSIONS: Our results reveal how normal variation in GDNF levels influence ENS size, composition, and gut function, suggesting a mechanism for well-known interindividual variation among those parameters.

Calcium-mediated DAD in membrane potentials and triggered activity in atrial myocytes of ETV1f / fMyHCCre /+ mice.

The E-twenty-six variant 1 (ETV1)-dependent transcriptome plays an important role in atrial electrical and structural remodelling and the occurrence of atrial fibrillation (AF), but the underlying mechanism of ETV1 in AF is unclear. In this study, cardiomyocyte-specific ETV1 knockout (ETV1f/fMyHCCre/+, ETV1-CKO) mice were constructed to observe the susceptibility to AF and the underlying mechanism in AF associated with ETV1-CKO mice. AF susceptibility was examined by intraesophageal burst pacing, induction of AF was increased obviously in ETV1-CKO mice than WT mice. Electrophysiology experiments indicated shortened APD50 and APD90, increased incidence of DADs, decreased density of ICa,L in ETV1-CKO mice. There was no difference in VINACT,1/2 and VACT,1/2, but a significantly longer duration of the recovery time after inactivation in the ETV1-CKO mice. The recording of intracellular Ca2+ showed that there was significantly increased in the frequency of calcium spark, Ca2+ transient amplitude, and proportion of SCaEs in ETV1-CKO mice. Reduction of Cav1.2 rather than NCX1 and SERCA2a, increase RyR2, p-RyR2 and CaMKII was reflected in ETV1-CKO group. This study demonstrates that the increase in calcium spark and SCaEs corresponding to Ca2+ transient amplitude may trigger DAD in membrane potential in ETV1-CKO mice, thereby increasing the risk of AF.

CircPRELID2 functions as a promoter of renal cell carcinoma through the miR-22-3p/ETV1 cascade.

BACKGROUND: Emerging evidence has indicated that a number of circular RNAs (circRNAs) participate in renal cell carcinoma (RCC) carcinogenesis. Nevertheless, the activity and molecular process of circPRELID2 (hsa_circ_0006528) in RCC progression remain unknown. METHODS: CircPRELID2, miR-22-3p and ETS variant 1 (ETV1) levels were gauged by qRT-PCR. Effect of the circPRELID2/miR-22-3p/ETV1 axis was evaluated by detecting cell growth, motility, and invasion. Immunoblotting assessed related protein levels. The relationships of circPRELID2/miR-22-3p and miR-22-3p/ETV1 were confirmed by RNA immunoprecipitation (RIP), luciferase reporter or RNA pull-down assay. RESULTS: CircPRELID2 was up-regulated in RCC. CircPRELID2 silencing suppressed RCC cell growth, motility and invasion. Moreover, circPRELID2 silencing weakened M2-type macrophage polarization in THP1-induced macrophage cells. CircPRELID2 sequestered miR-22-3p, and circPRELID2 increased ETV1 expression through miR-22-3p. Moreover, the inhibitory impact of circPRELID2 silencing on RCC cell malignant behaviors was mediated by the miR-22-3p/ETV1 axis. Furthermore, circPRELID2 knockdown in vivo hampered growth of xenograft tumors. CONCLUSION: Our study demonstrates that circPRELID2 silencing can mitigate RCC malignant development through the circPRELID2/miR-22-3p/ETV1 axis, highlighting new therapeutic targets for RCC treatment.

Reduction of ETV1 is Identified as a Prominent Feature of Age-Related Cataract.

PURPOSE: To identify the inactive genes in cataract lenses and explore their function in lens epithelial cells (LECs). METHODS: Lens epithelium samples obtained from both age-related cataract (ARC) patients and normal donors were subjected to two forms of histone H3 immunoprecipitation: H3K9ac and H3K27me3 chromatin immunoprecipitation (ChIP), followed by ChIP-seq. The intersection set of "active genes in normal controls" and "repressed genes in cataract lenses" was identified. To validate the role of a specific gene, ETV1, within this set, quantitative polymerase chain reaction (qPCR), western blot, and immunofluorescence were performed using clinical lens epithelium samples. Small interference RNA (siRNA) was utilized to reduce the mRNA level of ETV1 in cultured LECs. Following this, transwell assay and western blot was performed to examine the migration ability of the cells. Furthermore, RNA-seq analysis was conducted on both cell samples with ETV1 knockdown and control cells. Additionally, the expression level of ETV1 in LECs was examined using qPCR under H2O2 treatment. RESULTS: Six genes were identified in the intersection set of "active genes in normal controls" and "repressed genes in ARC lenses". Among these genes, ETV1 showed the most significant fold-change decrease in the cataract samples compared to the control samples. After ETV1 knockdown by siRNA in cultured LECs, reduced cell migration was observed, along with a decrease in the expression of ß-Catenin and Vimentin, two specific genes associated with cell migration. In addition, under the oxidative stress induced by H2O2 treatment, the expression level of ETV1 in LECs significantly decreased. CONCLUSIONS: Based on the findings of this study, it can be concluded that ETV1 is significantly reduced in human ARC lenses. The repression of ETV1 in ARC lenses appears to contribute to the disrupted differentiation of lens epithelium, which is likely caused by the inhibition of both cell differentiation and migration processes.

The Value of Combined Detection of Serum PSA, MALAT1 and TMPRSS2-ETV1 in Evaluating the Progress and Prognosis of Prostate Cancer.

OBJECTIVE: To explore the prognostic value of combined detection of serum prostate specific antigen (PSA), lung cancer metastasis-associated transcript 1 (MALAT1), transmembrane serine protease 2 (TMPRSS2), and erythropoietin-specific transforming gene variant 1 (ETV1) in prostate cancer. METHODS: Ninety patients with prostate cancer who were treated in hospital were divided into two groups according to tumor node metastasis stage: Stage I-II group (n = 34) and stage III-IV group (n = 56). The serum levels of PSA, MALAT1, and TMPRSS2-ETV1 were detected in both groups and correlated with prostate cancer status to determine their value as indicators of disease progression and prognosis. RESULTS: Age, body mass index (BMI), and Gleason score differed significantly between the study group and the control group (p < 0.05). The expression levels of serum PSA and MALAT1 were higher in group III-IV than in group I-II, and the positive expression rate of TMPRSS2-ETV1 was significantly higher in group III-IV than in the control group (p < 0.05). Pearson's correlation analysis showed that serum PSA, MALAT1, and TMPRSS2-ETV1 were significantly correlated with prostate cancer (p < 0.05). Differences in PSA levels correlated with differences in age, BMI, type of pathology, and Gleason score, whereas differences in serum MALAT1 levels correlated with differences in age, BMI, and type of pathology. Gleason scores differed significantly between patients with positive and negative TMPRSS2-ETV1 indicators (p < 0.05). Multivariate logistic regression analysis showed that serum PSA, MALAT1, and TMPRSS2-ETV1 were independent risk factors affecting the prognosis of prostate cancer (p < 0.05). The areas under the curve (AUCs) of serum PSA, MALAT1, and TMPRSS2-ETV1 as prognostic predictors in prostate cancer were 0.692, 0.731, and 0.709, respectively, whereas the AUC of the combination was 0.819. Assessment of disease progression using the combination of indicators had a significantly higher prognostic value than single indicators (p < 0.05). CONCLUSIONS: Serum levels of PSA, MALAT1, and TMPRSS2-ETV1 were abnormal in patients with prostate cancer, and the combined detection of these factors provided a reference for assessing disease progression and predicting the prognosis of prostate cancer.

The Value of Combined Detection of Serum PSA, MALAT1 and TMPRSS2-ETV1 in Evaluating the Progress and Prognosis of Prostate Cancer

Objective: To explore the prognostic value of combined detection of serum prostate specific antigen (PSA), lung cancer metastasis-associated transcript 1 (MALAT1), transmembrane serine protease 2 (TMPRSS2), and erythropoietin-specific transforming gene variant 1 (ETV1) in prostate cancer. Methods: Ninety patients with prostate cancer who were treated in hospital were divided into two groups according to tumor node metastasis stage: Stage I−II group (n = 34) and stage III−IV group (n = 56). The serum levels of PSA, MALAT1, and TMPRSS2-ETV1 were detected in both groups and correlated with prostate cancer status to determine their value as indicators of disease progression and prognosis. Results: Age, body mass index (BMI), and Gleason score differed significantly between the study group and the control group (p < 0.05). The expression levels of serum PSA and MALAT1 were higher in group III–IV than in group I–II, and the positive expression rate of TMPRSS2-ETV1 was significantly higher in group III–IV than in the control group (p < 0.05). Pearson’s correlation analysis showed that serum PSA, MALAT1, and TMPRSS2-ETV1 were significantly correlated with prostate cancer (p < 0.05). Differences in PSA levels correlated with differences in age, BMI, type of pathology, and Gleason score, whereas differences in serum MALAT1 levels correlated with differences in age, BMI, and type of pathology. Gleason scores differed significantly between patients with positive and negative TMPRSS2-ETV1 indicators (p < 0.05). Multivariate logistic regression analysis showed that serum PSA, MALAT1, and TMPRSS2-ETV1 were independent risk factors affecting the prognosis of prostate cancer (p < 0.05). The areas under the curve (AUCs) of serum PSA, MALAT1, and TMPRSS2-ETV1 as prognostic predictors in prostate cancer were 0.692, 0.731, and 0.709, respectively, whereas the AUC of the combination was 0.819 (AU)

Development and characterization of an ETV1 rabbit monoclonal antibody for the immunohistochemical detection of ETV1 expression in cancer tissue specimens.

BACKGROUND: Aberrant ETV1 overexpression arising from gene rearrangements or mutations occur frequently in prostate cancer, round cell sarcomas, gastrointestinal stromal tumors, gliomas, and other malignancies. The absence of specific monoclonal antibodies (mAb) has limited its detection and our understanding of its oncogenic function. METHODS: An ETV1 specific rabbit mAb (29E4) was raised using an immunogenic peptide. Key residues essential for its binding were probed by ELISA and its binding kinetics were measured by surface plasmon resonance imaging (SPRi). Its selective binding to ETV1 was assessed by immunoblots and immunofluorescence assays (IFA), and by both single and double-immuno-histochemistry (IHC) assays on prostate cancer tissue specimens. RESULTS: Immunoblot results showed that the mAb is highly specific and lacked cross-reactivity with other ETS factors. A minimal epitope with two phenylalanine residues at its core was found to be required for effective mAb binding. SPRi measurements revealed an equilibrium dissociation constant in the picomolar range, confirming its high affinity. ETV1 (+) tumors were detected in prostate cancer tissue microarray cases evaluated. IHC staining of whole-mounted sections revealed glands with a mosaic staining pattern of cells that are partly ETV1 (+) and interspersed with ETV1 (-) cells. Duplex IHC, using ETV1 and ERG mAbs, detected collision tumors containing glands with distinct ETV1 (+) and ERG (+) cells. CONCLUSIONS: The selective detection of ETV1 by the 29E4 mAb in immunoblots, IFA, and IHC assays using human prostate tissue specimens reveals a potential utility for the diagnosis, the prognosis of prostate adenocarcinoma and other cancers, and the stratification of patients for treatment by ETV1 inhibitors.

A Transcription Factor Etv1/Er81 Is Involved in the Differentiation of Sweet, Umami, and Sodium Taste Cells.

Taste cells are maintained by continuous turnover throughout a lifetime, yet the mechanisms of taste cell differentiation, and how taste sensations remain constant despite this continuous turnover, remain poorly understood. Here, we report that a transcription factor Etv1 (also known as Er81) is involved in the differentiation of taste cells responsible for the preference for sweet, umami, and salty tastes. Molecular analyses revealed that Etv1 is expressed by a subset of taste cells that depend on Skn-1a (also known as Pou2f3) for their generation and express T1R genes (responsible for sweet and umami tastes) or Scnn1a (responsible for amiloride-sensitive salty taste). Etv1CreERT2/CreERT2 mice express Etv1 isoform(s) but not Etv1 in putative proprioceptive neurons as comparable to wild-type mice, yet lack expression of Etv1 or an isoform in taste cells. These Etv1CreERT2/CreERT2 mice have the same population of Skn-1a-dependent cells in taste buds as wild-type mice but have altered gene expression in taste cells, with regional differences. They have markedly decreased electrophysiological responses of chorda tympani nerves to sweet and umami tastes and to amiloride-sensitive salty taste evoked by sodium cation, but they have unchanged responses to bitter or sour tastes. Our data thus show that Etv1 is involved in the differentiation of the taste cells responsible for sweet, umami, and salty taste preferences.

ETV1 inhibition depressed M2 polarization of tumor-associated macrophage and cell process in gastrointestinal stromal tumor via down-regulating PDE3A.

M2-type polarization of tumor associated-macrophage (TAM) is involved in the malignancy of gastrointestinal stromal tumor (GIST) progression. ETS variant 1 (ETV1) has been previously validated to regulate GIST pathogenesis. Our study intended to explore the role and mechanism of ETV1 in mediating the M2-polarization of TAM in GIST progression. First, we analyzed the correlation between ETV1 expression and M2-polarization in GIST tissues. IL-4 was used to treat THP-1-derived TAM cells and IL-4-stimulated TAM were co-cultured with GIST-T1 cells to mimic the GIST microenvironment. A loss-of-function assay was performed to explore the role of ETV1. Results showed that ETV1 elevation was positively correlated with M2-polarization. IL-4-induced TAM promoted ETV1 expression, silencing ETV1 inhibited proliferation, invasion and KIT activation in IL-4-treated GIST cells, while cell apoptosis was enhanced. Besides, co-culture of ETV1-silenced GIST cells significantly depressed M2-polarization in TAM, presented as decreased levels of CD206, Agr-1 and cytokines, as well as the proportion of CD206-positive TAM. PDE3A was positively correlated with ETV1 and M2-polarization. Overexpressing PDE3A reversed the inhibitory effects of ETV1 silencing. Generally, ETV1 inhibition depressed M2-polarization of TAM in GIST and its promotion on pathological aggravation via down-regulating PDE3A. This evidence may provide a new target for GIST regulation.

From basic to clinical:Advances in research on ETV1 / 肿瘤

ETS variant gene 1(ETV1)is an oncogene that plays an important role in embryonic development and malignant progression of tumors.Chromosomal translocations,gene amplifications,and activation of the mitogen activated protein kinase(MAPK)signal pathways lead to the up-regulation of ETV1,and then ETV1,as a transcription factor,regulates the expression of downstream target genes by binding to their promoters or enhancers,thereby playing a pivotal role in the occurrence and development of prostate cancer,gastrointestinal stromal tumor(GIST)and breast cancer.ETV1 is also a potential tumor biomarker,which has clinical value in diagnosis and prognostic evaluation in various tumors,and strategies targeting ETV1 can provide new treatment options to tumor patients.This article discusses the mechanisms of ETV1 activation and pro-oncogenic mechanisms driven by ETV1,and summarizes and prospects the clinical application of ETV1 as a tumor biomarker and therapeutic target.

SH3BP2 Silencing Increases miRNAs Targeting ETV1 and Microphthalmia-Associated Transcription Factor, Decreasing the Proliferation of Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Gain of function in receptor tyrosine kinases type III, KIT, or PDGFRA drives the majority of GIST. Previously, our group reported that silencing of the adaptor molecule SH3 Binding Protein 2 (SH3BP2) downregulated KIT and PDGFRA and microphthalmia-associated transcription factor (MITF) levels and reduced tumor growth. This study shows that SH3BP2 silencing also decreases levels of ETV1, a required factor for GIST growth. To dissect the SH3BP2 pathway in GIST cells, we performed a miRNA array in SH3BP2-silenced GIST cell lines. Among the most up-regulated miRNAs, we found miR-1246 and miR-5100 to be predicted to target MITF and ETV1. Overexpression of these miRNAs led to a decrease in MITF and ETV1 levels. In this context, cell viability and cell cycle progression were affected, and a reduction in BCL2 and CDK2 was observed. Interestingly, overexpression of MITF enhanced cell proliferation and significantly rescued the viability of miRNA-transduced cells. Altogether, the KIT-SH3BP2-MITF/ETV1 pathway deserves to be considered in GIST cell survival and proliferation.

CIC-Rearranged Sarcomas: An Intriguing Entity That May Lead the Way to the Comprehension of More Common Cancers.

Capicua transcriptional repressor (CIC)-rearranged sarcoma, belonging to the undifferentiated round cells sarcoma family, is characterized by high metastatic rate and poor chemo response. CIC sarcoma represents a new entity harboring the recurrent chromosomal translocation between CIC and, in most of the cases, DUX4. CIC-DUX4 imposes a CIC-specific transcriptional signature, which drives cell transformation, proliferation, and migration. While the discovery of the fusion represented the first evidence of a role of CIC in cancer, a complete comprehension of CIC-rearranged activity is still required before providing new potential avenues for therapy. To date, a specific and effective treatment for CIC sarcoma has yet to be defined. In this review, we initially highlight the clinical features and pathogenesis of CIC-rearranged sarcomas along with current therapeutic approaches and then focus on the specific oncogenic mechanisms driven by the CIC-rearrangement. We discuss novel therapeutic options evoked by the aberrant relations of CIC-DUX4 with the IGF system, DUSP6, P300/CBP, and CCNE1. We also discuss how different mutations involving CIC might converge on a common upregulation of CIC-target genes across human cancers. A deeper understanding of the oncogenic mechanisms driven by the chimera CIC-DUX4 might provide novel therapeutic opportunities with a general impact in cancer.

The Etv1/Er81 transcription factor coordinates myelination-related genes to regulate Schwann cell differentiation and myelination.

Background: Axonal myelination is critical for the functioning of vertebrate nervous system. Myelin sheath malformation or degeneration can cause a variety of neurological diseases. Our previous study identified multiple potential myelination-related transcriptional factors (TFs), including expressed sequence tag (ETS) variant transcription factor 1 (Etv1)/Er81, via gene microarray analysis of Schwann cells (SCs) at various myelination stages. Etv1 is known to be involved in the regulation of neuronal specialization, muscle spindle differentiation, and sensorimotor connectivity. However, to our knowledge, to date, there are no relevant studies that Etv1 regulates SC myelination. Methods: To investigate the roles of Etv1 in SC re-myelination, an in vivo mouse myelination model was used, in which the sciatic nerve is crushed. Etv1 in nerves was knocked down via in situ injection of cholesterol-modified Etv1-small interfering (si)RNA. The expression of myelin-associated glycoprotein (MAG) was evaluated by Western blotting (WB) and immunohistochemistry (IHC). Myelination was assessed by transmission electron microscopy (TEM). The effects of Etv1 on SC proliferation, migration, and differentiation were assessed in vitro using the EdU cell proliferation kit, a culture-insert scratch assay, a SC aggregate sphere migration assay on the axons of dorsal root ganglions (DRGs), and a SC differentiation model. Chromatin immunoprecipitation (ChIP) united with quantitative real-time PCR (qPCR), known as ChIP-qPCR, and luciferase activity reporter assays were performed to explore the possible mechanisms by which Etv1 controls SC differentiation and myelination. Results: The results demonstrated that Etv1 promoted myelination by facilitating SC proliferation, migration, and differentiation. Etv1 expression in SCs was upregulated during re-myelination, and knocking down Etv1 expression dramatically abrogated SC re-myelination in the crushed sciatic nerves. Moreover, silencing of Etv1 by siRNA in SCs in vitro inhibited its migration, proliferation, and differentiation. The results of ChIP-qPCR and luciferase reporter assay showed that Etv1 may regulate SC differentiation and myelination by binding to the promoters of myelination-related genes, such as MAG and Runx2, to initiate their transcription. Conclusions: Taken together, these findings demonstrated a previously unknown role of Etv1 in SC differentiation and myelination, providing a candidate molecular target for clinical interventions in demyelinating diseases.

ETV1 Positively Correlated With Immune Infiltration and Poor Clinical Prognosis in Colorectal Cancer.

Objective: Numerous studies recently suggested that the immune microenvironment could influence the development of colorectal cancer (CRC). These findings implied that the infiltration of immune cells could be a promising prognostic biomarker for CRC. Methods: Furthermore, the Oncomine database and R2 platform analysis were applied in our research to validate CRC clinical prognosis via expression levels of polyoma enhancer activator 3 (PEA3) members. We explored the correlation of ETV1, ETV4, and ETV5 with tumor-infiltrating immune cells (TIICs) in CRC tumor microenvironments via the Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA). Immunohistochemistry (IHC) was used to validate our CRC clinical data. Results: Our findings indicated that the upregulation of PEA3 members including ETV1 and ETV5 was positively associated with poor prognosis in CRC patients. Meanwhile, ETV1 and ETV5 may play significant roles in the development progress of CRC. Furthermore, ETV1 tends to be associated with immune infiltration of CRC, especially with cancer-associated fibroblasts and M2 macrophages. Conclusion: These findings revealed that ETV1 and ETV5 played significant roles in the development of CRC. Moreover, ETV1 was significantly associated with the infiltration of cancer-associated fibroblasts and M2 macrophages in CRC. Targeting ETV1 can be a potential auspicious approach for CRC treatment.

Novel ETV1 mutation in small cell lung cancer transformation resistant to EGFR tyrosine kinase inhibitors.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients harboring mutations in the epidermal growth factor receptor (EGFR) gene respond dramatically to EGFR tyrosine kinase inhibitors (TKIs). However, these patients inevitably develop acquired resistance to EGFR-TKIs. Among them, small cell lung cancer (SCLC) transformation is a relatively rare mechanism. METHODS: We used a 639 cancer-relevant gene panel to detect genetic differences in tissues before and after EGFR-TKIs resistance caused by SCLC transformation. In vitro experiments were conducted to study the role of ETS variant transcription factor 1 (ETV1) on SCLC transformation and EGFR-TKIs resistance. RESULTS: We present two EGFR-mutant lung adenocarcinoma (LUAD) patients. One patient, with EGFR exon 19 deletion (Ex19del), accepted first-line gefitinib treatment and then received osimertinib treatment due to acquisition of an EGFR-T790M mutation. A novel ETV1 mutation (p.P159S) was detected in the SCLC tissue after osimertinib resistance when not coexisting with T790M. The other patient harbored an EGFR exon 21 mutation (p.L858R), and had a long-lasting response to first-line gefitinib, and then transformed to SCLC after TKI resistance. A previously unreported ETV1 mutation (p.E462Q) was detected in the SCLC tissue. In vitro, ETV1 p.E462Q and p.P159S mutations participated in neuroendocrine differentiation by inducing the expression of achaete-scute homolog 1 (ASCL1) and promoting the proliferation of H69 cells. ETV1 p.E462Q and p.P159S mutations were also resistant to gefitinib and osimertinib after introduction into H358 cells. CONCLUSIONS: Novel ETV1 p.E462Q and p.P159S mutations were found in the SCLC tissues of TKIs-resistant LUAD patients, providing a new understanding of ETV1 involvement in acquired resistance to EGFR-TKIs via SCLC transformation.

Sumoylation of transcription factor ETV1 modulates its oncogenic potential in prostate cancer.

The transcription factor ETS variant 1 (ETV1) is capable of promoting prostate tumorigenesis. We demonstrate that ETV1 can be posttranslationally modified by covalent attachment of small ubiquitin-like modifier 1 (SUMO1) onto four different lysine residues. In human embryonic kidney 293T cells, mutation of these sumoylation sites stimulated the transactivation potential of ETV1 at the matrix metalloproteinase 1 (MMP1), but not Yes-associated protein 1 gene promoter, while ETV1 protein stability and intracellular localization remained unchanged. In stark contrast, sumoylation-deficient ETV1 was repressed in its ability to stimulate the MMP1 promoter and to cooperate with a histone demethylase, JmjC domain-containing 2A (JMJD2A), in LNCaP prostate cancer cells. Mutation of sumoylation sites enhanced the ability of ETV1 to interact with the histone deacetylase (HDAC) 1, but had basically no impact on complex formation with HDAC3 or JMJD2A. Further, compared to non-sumoylated ETV1, its sumoylated forms were less able to bind to the transcription factor, SMAD family member 4. Lastly, in contrast to wild-type ETV1, sumoylation-deficient ETV1 repressed LNCaP cell growth. Altogether, these data suggest that sumoylation modulates ETV1 function in a cell type-specific manner, possibly by altering the spectrum of transcriptional cofactors being recruited. Notably, SUMO pathway components SUMO1, ubiquitin-like modifier activating enzyme 2 and ubiquitin conjugating enzyme 9 were upregulated in prostate tumors, implying that enhanced sumoylation indeed promotes ETV1's oncogenic activity during prostate cancer formation.

LncRNA PCGEM1 mediates oxaliplatin resistance in hepatocellular carcinoma via miR-129-5p/ETV1 axis in vitro.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most severe malignant cancers that leads to high death rate worldwide. Recent research revealed that long non-coding RNAs (lncRNAs) exert a critical role regarding chemoresistance in numerous cancers, including HCC. OBJECTIVES: Our research aimed to explore the function and molecular mechanism of lncRNA PCGEM1 on oxaliplatin resistance of HCC in vitro. MATERIAL AND METHODS: Expression of the lncRNA PCGEM1, together with miR-129-5p, and the mRNA level of ETV1 and drug resistance-related genes including LRPA, MDR1 and MDR3 were determined using quantitative real-time polymerase chain reaction (qRT-PCR) in an oxaliplatin-resistant HCC cell line (Hep3B/OXA). Cell Counting Kit-8 (CCK-8) was employed to assess the viability and cell survival rate, and transwell assays were performed to measure the number of migrated or invaded cells. In addition, the relation among lncRNA PCGEM1, miR-129-5p and ETV1 were determined using luciferase assay. RESULTS: Our data indicated that PCGEM1 and ETV1 expression were enhanced in Hep3B/OXA cells. Furthermore, knockdown of lncRNA PCGEM1 significantly decreased the migration, invasion and mRNA expressions of LRPA, MDR1 and MDR3, and the cell viability in Hep3B/OXA cells. The starBase online tool and luciferase assays verified that miR-129-5p targeted PCGEM1 and ETV1, signifying that PCGEM1 could enhance ETV1 expression via suppressing miR-129-5p. CONCLUSIONS: Our findings demonstrated that PCGEM1 modulated oxaliplatin resistance by targeting the miR-129-5p/ETV1 pathway in HCC in vitro, suggesting a potential strategy for the treatment of chemoresistant HCC.

Glucose-Regulated Protein 94 Mediates the Proliferation and Metastasis through the Regulation of ETV1 and MAPK Pathway in Colorectal Cancer.

Colorectal cancer (CRC) is a worldwide health problem. Glucose-regulated protein 94 (GRP94) is known as an important endoplasmic reticulum-stress response protein that shows correlation with aggressive cancer behavior. However, the role of GRP94 in CRC is still unclear. Our results showed that silencing GRP94 (GRP94-KD) reduced cell proliferation, invasion and migration of CRC cells and suppressed tumorigenesis in the xenograft mouse model. Rescue assay showed that ETV1 overexpression reversed the effect of GRP94 on cell proliferation and migration. In the molecular mechanism, we found that knockdown of GRP94 inhibited the level of MAPK pathway, including ERK/p-ERK, JNK/p-JNK, and p38/p-p38 signals. Cyclooxygenase-2 and epithelial-mesenchymal transformation biomarkers, such as N-cadherin, vimentin, and ß-catenin were suppressed in GRP94 knockdown cells. Treatment of specific inhibitors of MAPK pathway showed that ERK/p-ERK, and p38/p-p38 inhibitors significantly influenced ETV1 expression as compared to JNK/p-JNK inhibitor. Our results indicated that silencing GRP94 repressed the ability of EMT process, cancer cell proliferation, metastasis, and CRC tumorigenesis. Therefore, GRP94 may play an important role in CRC by regulating ETV1 and MAPK pathway.

Frequent copy number gains of SLC2A3 and ETV1 in testicular embryonal carcinomas.

Testicular germ cell tumours (TGCTs) appear as different histological subtypes or mixtures of these. They show similar, multiple DNA copy number changes, where gain of 12p is pathognomonic. However, few high-resolution analyses have been performed and focal DNA copy number changes with corresponding candidate target genes remain poorly described for individual subtypes. We present the first high-resolution DNA copy number aberration (CNA) analysis on the subtype embryonal carcinomas (ECs), including 13 primary ECs and 5 EC cell lines. We identified recurrent gains and losses and allele-specific CNAs. Within these regions, we nominate 30 genes that may be of interest to the EC subtype. By in silico analysis of data from 150 TGCTs from The Cancer Genome Atlas (TCGA), we further investigated CNAs, RNA expression, somatic mutations and fusion transcripts of these genes. Among primary ECs, ploidy ranged between 2.3 and 5.0, and the most common aberrations were DNA copy number gains at chromosome (arm) 7, 8, 12p, and 17, losses at 4, 10, 11, and 18, replicating known TGCT genome characteristics. Gain of whole or parts of 12p was found in all samples, including a highly amplified 100 kbp segment at 12p13.31, containing SLC2A3. Gain at 7p21, encompassing ETV1, was the second most frequent aberration. In conclusion, we present novel CNAs and the genes located within these regions, where the copy number gain of SLC2A3 and ETV1 are of interest, and which copy number levels also correlate with expression in TGCTs.

Androgen up-regulation of Twist1 gene expression is mediated by ETV1.

Twist1, a basic helix-loop-helix transcription factor that regulates a number of genes involved in epithelial-to-mesenchymal transition (EMT), is upregulated in prostate cancer. Androgen regulation of Twist1 has been reported in a previous study. However, the mechanism of androgen regulation of the Twist1 gene is not understood because the Twist1 promoter lacks androgen receptor (AR)-responsive elements. Previous studies have shown that the Twist1 promoter has putative binding sites for PEA3 subfamily of ETS transcription factors. Our lab has previously identified Ets Variant 1 (ETV1), a member of the PEA3 subfamily, as a novel androgen-regulated gene that is involved in prostate cancer cell invasion through unknown mechanism. In view of these data, we hypothesized that androgen-activated AR upregulates Twist1 gene expression via ETV1. Our data confirmed the published work that androgen positively regulates Twist1 gene expression and further showed that this positive effect was directed at the Twist1 promoter. The positive effect of androgen on Twist1 gene expression was abrogated upon disruption of AR expression by siRNA or of AR activity by Casodex. More importantly, our data show that disruption of ETV1 leads to significant decrease in both androgen-mediated upregulation as well as basal level of Twist1, which we are able to rescue upon re-expression of ETV1. Indeed, we are able to show that ETV1 mediates the androgen upregulation of Twist1 by acting on the proximal region of Twist1 promoter. Additionally, our data show that Twist1 regulates prostate cancer cell invasion and EMT, providing a possible mechanism by which ETV1 mediates prostate cancer cell invasion. In conclusion, in this study we report Twist1 as an indirect target of AR and androgen regulation through ETV1.