ELL-associated Factor Research Articles

EAF2 Downregulation Recruits Tumor-associated Macrophages in Prostate Cancer through Upregulation of MIF

BackgroundThe role of tumor inflammatory microenvironment in the advancement of cancer, particularly prostate cancer, is widely acknowledged. ELL-associated factor 2 (EAF2), a tumor suppressor that has been identified in the prostate, is often downregulated in prostate cancer. Earlier investigations have shown that mice with EAF2 gene knockout exhibited a substantial infiltration of inflammatory cells into the prostatic stroma.MethodsA cohort comprising 38 patients who had been diagnosed with prostate cancer and subsequently undergone radical prostatectomy (RP) was selected. These patients were pathologically graded according to the Gleason scoring system and divided into two groups. The purpose of this selection was to investigate the potential correlation between EAF2 and CD163 using immunohistochemistry (IHC) staining. Additionally, in vitro experimentation was conducted to verify the relationship between EAF2 expression, macrophage migration and polarization.ResultsOur study demonstrated that in specimens of human prostate cancer, the expression of EAF2 was notably downregulated, and this decrease was inversely associated with the number of CD163-positive macrophages that infiltrated the cancerous tissue. Cell co-culture experiments revealed that the chemotactic effect of tumor cells towards macrophages was intensified and that macrophages differentiated into tumor-associated macrophages (TAMs) when EAF2 was knocked out. Additionally, the application of cytokine protein microarray showed that the expression of chemokine macrophage migration inhibitory factor (MIF) increased after EAF2 knockout.ConclusionsOur findings suggested that EAF2 was involved in the infiltration of CD163-positive macrophages in prostate cancer via MIF.Graphical abstract

Mechanism of ELL-associated factor 2 and vasohibin 1 regulating invasion, migration, and angiogenesis in colorectal cancer.

As a novel endogenous anti-angiogenic molecule, vasohibin 1 (VASH1) is not only expressed in tumor stroma, but also in tumor tissue. Moreover, studies have shown that VASH1 may be a prognostic marker in colorectal cancer (CRC). Knockdown of VASH1 enhanced transforming growth factor-β1 (TGF-β1)/Smad3 pathway activity and type I/III collagen production. Our previous findings suggest that ELL-associated factor 2 (EAF2) may play a tumor suppressor and protective role in the development and progression of CRC by regulating signal transducer and activator of transcription 3 (STAT3)/TGF-β1 signaling pathway. However, the functional role and mechanism of VASH1-mediated TGF-β1 related pathway in CRC has not been elucidated. To investigate the expression of VASH1 in CRC and its correlation with the expression of EAF2. Furthermore, we studied the functional role and mechanism of VASH1 involved in the regulation and protection of EAF2 in CRC cells in vitro. We collected colorectal adenocarcinoma and corresponding adjacent tissues to investigate the clinical expression of EAF2 protein and VASH1 protein in patients with advanced CRC. Following, we investigated the effect and mechanism of EAF2 and VASH1 on the invasion, migration and angiogenesis of CRC cells in vitro using plasmid transfection. Our findings indicated that EAF2 was down-regulated and VASH1 was up-regulated in advanced CRC tissue compared to normal colorectal tissue. Kaplan-Meier survival analysis showed that the higher EAF2 Level group and the lower VASH1 Level group had a higher survival rate. Overexpression of EAF2 might inhibit the activity of STAT3/TGF-β1 pathway by up-regulating the expression of VASH1, and then weaken the invasion, migration and angiogenesis of CRC cells. This study suggests that EAF2 and VASH1 may serve as new diagnostic and prognostic markers for CRC, and provide a clinical basis for exploring new biomarkers for CRC. This study complements the mechanism of EAF2 in CRC cells, enriches the role and mechanism of CRC cell-derived VASH1, and provides a new possible subtype of CRC as a therapeutic target of STAT3/TGF-β1 pathway.

Overexpression of ELL-associated factor 2 suppresses invasion, migration, and angiogenesis in colorectal cancer.

BACKGROUNDThe androgen responsive gene, ELL-associated factor 2 (EAF2), expressed in benign prostate tissues, has been shown to play an important role in tumor suppression in a variety of malignant tumors. In addition, some scholars found that EAF2 frameshift mutations are associated with intratumor heterogeneity in colorectal cancer (CRC) and inactivation of EAF2 in microsatellite instability-high CRC. However, the molecular mechanism by which EAF2 is involved in CRC invasion and metastasis remains unclear.AIMTo determine the clinical value of expression of EAF2 protein in CRC, and to study the effects of EAF2 on the invasion, migration, and angiogenesis of CRC cells in vitro.METHODSIn this study, we collected colorectal adenocarcinoma and corresponding adjacent tissues to investigate the clinical expression of EAF2 protein in patients with advanced CRC. Subsequently, we investigated the effect of EAF2 on the invasion, migration, and angiogenesis of CRC cells in vitro using plasmid transfection.RESULTSEAF2 protein was lowly expressed in cancer tissues of patients with advanced CRC. Kaplan-Meier survival analysis showed that the survival rate of the high EAF2 level group was higher than that of the low EAF2 level group.CONCLUSIONOur results demonstrated that EAF2, as a tumor suppressor, may inhibit the invasion, metastasis, and angiogenesis of CRC cells by regulating the signal transducer and activator of transcription 3/transforming growth factor-β1 crosstalk pathway, and play a cancer suppressive and protective role in the occurrence and development of CRC. Our findings are of great significance to provide a new idea and theoretical basis for the targeted diagnosis and treatment of CRC.

Faster skin wound healing predicts survival after myocardial infarction

Skin wound healing and the cardiac healing response to myocardial infarction (MI) both progress through similar pathways involving inflammation, resolution, tissue repair, and scar formation. Due to the similarities, we hypothesized that the healing response to skin wounding would predict future response to MI. Mice were given a 3 mm skin wound using a disposable biopsy punch, and the skin wound was imaged daily until closure. Wound closure was quantified using Image J software. The same set of mice was given MI by permanent coronary artery ligation 28 days later and followed for 7 days. Cardiac physiology (volume, dimension, ejection fraction, fractional shortening, wall thickness) was measured by echocardiography at baseline, and 3 and 7 days post MI. Mice that survived until MI day 7 were grouped as survivors (30 mice, 68%), and animals that died from MI were grouped as non‐survivors (14 mice, 32%). Survivors had faster skin wound healing compared to non‐survivors. Faster skin wound healing predicted MI survival better than commonly used cardiac functional variables (e.g., infarct size, fractional shortening, and end diastolic dimension). We mapped the plasma N‐glycoproteome 3 days after skin wounding and 3 days after MI using mass spectrometry. The glycoproteome profile of MI day 3 plasma revealed alpha‐2‐macroglobulin and ELL‐associated factor 1 as strong predictors of future MI death and progression to heart failure. A second cohort of MI mice validated these findings. In patients, alpha‐2‐macroglobulin was 1.7±0.3‐fold elevated 48h after presentation of MI (p=0.009, 18 controls, 41 MI patients). The glycoproteome profile of plasma collected 3 days post skin wounding identified apolipoprotein D and vitamin D binding protein to mirror skin wound healing. Apolipoprotein D detrimentally regulated both skin and cardiac wound healing in part by promoting inflammation. Plasma levels of vitamin D binding protein and galectin 3 binding after skin wounding also predicted future MI dilation and progression to heart failure. Our results reveal that the skin is a mirror to the heart and common pathways link wound healing across organs.

Abstract 5864: Genetic screens reveal dependencies and chemotherapy modulators in NSD2-driven hematologic malignancies

Abstract The histone methyltransferase NSD2 contributes to the pathogenesis and chemoresistance of various hematologic cancers. In multiple myeloma (MM), NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance. Further, a gain-of-function mutation of NSD2 promotes oncogenic reprogramming and chemoresistance in acute lymphocytic leukemia (ALL). So far, attempts to develop specific and potent NSD2 inhibitors have not been successful. Here, we utilized genome-wide CRISPR-based loss-of-function genetic screens to explore molecular dependencies and modulators of chemotherapy response in hematologic malignancies with aberrant NSD2 activity. We assessed gene essentiality in KMS11-derived NSD2-high and NSD2-low isogenic MM cells. Differential essentiality analysis revealed multiple genes whose disruption is selectively detrimental in cells with NSD2 overexpression. These include the adenine nucleotide regulator Adenylate Kinase 2 (AK2) and the super elongation complex (SEC) component ELL-Associated Factor 1 (EAF1), which were validated by in vitro competition assays. We further found that NSD2-high cells exhibit higher dependency on the druggable anti-apoptotic factor BCL2 and demonstrated a stronger synergy between BCL2 inhibitors and the glucocorticoid dexamethasone in these cells compared to NSD2-low cells. This finding suggests that BCL2 inhibitors can be used to potentiate dexamethasone response in MM patients with t(4;14) chromosomal translocation, where NSD2 is overexpressed. We also utilized the CRISPR-based genetic approach to investigate cellular processes influencing sensitivity of NSD2-driven MM cells to dexamethasone. Our study identified multiple cellular pathways as modulators of dexamethasone response in MM. For example, we showed that the IL10-JAK-STAT pathway mediates dexamethasone resistance whereas certain components of mRNA degradation function as dexamethasone sensitizers. How these pathways and other uncovered key players alter MM cell response to dexamethasone is still under investigation. We previously demonstrated that an activating NSD2 mutation leads to dexamethasone resistance in ALL. We sought to explore the mechanisms by which mutant NSD2 drives dexamethasone resistance and the cellular processes that can be targeted to restore dexamethasone sensitivity in ALL cells. Genome-wide CRISPR screens in RCH-ACV, a dexamethasone-resistant B-ALL cell line harboring the NSD2 mutation, uncovered multiple modulators of dexamethasone response. Intriguingly, we found that disruption of any component of a transcriptional complex involving LMO2, LDB1 and LYL1 sensitizes RCH-ACV cells to dexamethasone. Targeting this complex could be relevant to overcoming dexamethasone resistance in relapsed pediatric ALL. Citation Format: Amin Sobh, Charlotte Kaestner, Alberto Riva, Jianping Li, Richard Bennett, Jonathan Licht. Genetic screens reveal dependencies and chemotherapy modulators in NSD2-driven hematologic malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5864.

Conditional Deletion of Eaf1 Induces Murine Prostatic Intraepithelial Neoplasia in Mice

ELL-associated factor 1 is a transcription elongation factor that shares significant homology and functional similarity to the androgen-responsive prostate tumor suppressor ELL-associated factor 2. EAF2 is frequently down-regulated in advanced prostate cancer and Eaf2 deletion in the mouse induced the development of murine prostatic intraepithelial neoplasia. Here we show that similar to EAF2, EAF1 is frequently down-regulated in advanced prostate cancer. Co-downregulation of EAF1 and EAF2 occurred in 40% of clinical specimens with Gleason score >7. We developed and characterized a murine model of prostate-epithelial specific deletion of Eaf1 in the prostate and crossed it with our previously generated mouse with conventional deletion of Eaf2. The prostates of Eaf1 deletion mice displayed murine prostatic intraepithelial neoplasia lesions with increased proliferation and inflammation. Combined deletion of Eaf1 and Eaf2 in the murine model induced an increased incidence in mPIN lesions characterized by increased proliferation and CD3+ T cells and CD19+ B cells infiltration compared to individual deletion of either Eaf1 or Eaf2 in the murine prostate. These results suggest that EAF1 may play a tumor suppressive role in the prostate. Cooperation between EAF1 and EAF2 may be important for prostate maintaining prostate epithelial homeostasis, and concurrent loss of these two tumor suppressors may promote prostate tumorigenesis and progression.

Regulation of ELL2 stability and polyubiquitination by EAF2 in prostate cancer cells.

Elongation factor for RNA polymerase 2 (ELL2) and ELL associated factor 2 (EAF2) have been reported to have tumor suppressive properties in prostate epithelial cells. We investigated ELL2 expression in human prostate cancer specimens, and ELL2 protein stability and ubiquitination in prostate cancer cells. Immunostaining analysis of human prostate cancer specimens was used to determine ELL2 expression in tumor and normal tissues. ELL2 knockdown in prostate cancer cell lines LNCaP and C4-2 was used to compare proliferation and motility. Deletion and site-directed mutagenesis was used to identify amino acid residues in ELL2 that were important for degradation. ELL2 protein was downregulated in prostate cancer specimens and was up-regulated by androgens in prostate cancer cell lines LNCaP and C4-2. ELL2 knockdown enhanced prostate cancer cell proliferation and motility. ELL2 protein has a short half-life and was stabilized by proteasome inhibitor MG132. Amino acid residues K584 and K599 in ELL2 were important for ELL2 degradation. EAF2 could stabilize ELL2 and inhibited its polyubiquitination. Our findings provide further evidence that ELL2 is a potential tumor suppressor frequently down-regulated in clinical prostate cancer specimens and provides new insights into regulation of ELL2 protein level by polyubiquitination and EAF2 binding.

Eaf2 protects human lens epithelial cells against oxidative stress‑induced apoptosis by Wnt signaling

The tumor suppressor protein ELL-associated factor 2 (Eaf2) serves an important role in lens development and maturation; however, its role in oxidative stress-induced cataract formation remains unclear. In the present study, an in vitro apoptosis model was constructed by treating HLE-B3 cells with 50 µM hydrogen peroxide (H2O2), and was confirmed by flow cytometry. Subsequently, overexpression of Eaf2 was induced in H2O2-induced HLE-B3 cells by ligating Eaf2 cDNA to a pcDNA3.0 plasmid and the role of Wnt3a in the function of Eaf2 was also assessed by inhibiting the expression of the gene in Eaf2-overexpression cells. The expression levels of glycogen synthase kinase 3β, β-catenin, Eaf2, caspase 3, Wnt3a, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein were examined using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Immunocytochemistry was used to locate Eaf2 and Wnt3 protein expression in the H2O2-induced HLE-B3 cells. The results indicated that Eaf2 was able to effectively suppress H2O2-induced apoptosis of HLE cells via inhibition of caspase 3 production and activation of Wnt3a signaling. In addition, knockdown of Wnt3a in Eaf2-overexpression cells evidently counteracted the effect of Eaf2 in antagonizing H2O2-induced apoptosis. Taken together, these findings suggested that Eaf2 may suppress oxidative stress-induced apoptosis of HLE-B3 cells exerted through the activation of Wnt3a signaling.

Combined Loss of EAF2 and p53 Induces Prostate Carcinogenesis in Male Mice.

Mutations in the p53 tumor suppressor are frequent in patients with castration-resistant prostate cancer but less so in patients with localized disease, and patients who have Li-Fraumeni with germline p53 mutations do not have an increased incidence of prostate cancer, suggesting that additional molecular and/or genetic changes are required for p53 to promote prostate carcinogenesis. ELL-associated factor 2 (EAF2) is a tumor suppressor that is frequently downregulated in advanced prostate cancer. Previous studies have suggested that p53 binds to EAF2, providing a potential mechanism for their functional interactions. In this study, we tested whether p53 and EAF2 could functionally interact in prostate cancer cells and whether concurrent inactivation of p53 and EAF2 could promote prostate carcinogenesis in a murine knockout model. Endogenous p53 coprecipitated with EAF2 in prostate cancer cells, and deletion mutagenesis indicated that this interaction was mediated through the C terminus of EAF2 and the DNA binding domain of p53. Concurrent knockdown of p53 and EAF2 induced an increase in proliferation and migration in cultured prostate cancer cells, and conventional p53 and EAF2 knockout mice developed prostate cancer. In human prostate cancer specimens, concurrent p53 nuclear staining and EAF2 downregulation was associated with high Gleason score. These findings suggest that EAF2 and p53 functionally interact in prostate tumor suppression and that simultaneous inactivation of EAF2 and p53 can drive prostate carcinogenesis.

Bone marrow stromal antigen 2 , a novel ELL-associated factor 2 target gene, regulates proliferation and migration abilities of prostate cancer cells

Objective To investigate whether bone marrow stromal antigen 2 (BST2) which was regulated by ELL-associated factor 2 (EAF2) can promote prostate cancer cells proliferation and migration. Methods After inhibiting the expression of EAF2 by 100 pmol interfering RNA (RNAi) in C4-2 cells using lipofectamine 2000, the mRNA and protein expression levels were assessed by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting respectively. After transfecting MYC-EAF2 into 293T cells according to the manufacturer’s instruction, the expression level of BST2 was detected by Western blotting. After knockdown of EAF2 by RNAi in C4-2 cells, proliferation was tested by bromodeoxyuridine incorporation (BrdU) assay, and migration was tested by Transwell assay. Results The mRNA and protein levels were significantly increased after transfection of small interfering RNA (siRNA) against EAF2. Moreover, proliferation rate was further decreased from (22.09±1.08)% to (12.25±1.32)% (P=0.001). At the meantime, the number of migrating cells was decreased from (50.50±3.80)% to (24.25±1.43)% (P=0.001). Conclusion Our findings suggest that EAF2 regulates the mRNA and protein levels of BST2, and BST2 could regulate proliferation and migration abilities of C4-2 cells. Key words: Prostate cancer; ELL-associated factor 2; Bone marrow stromal antigen 2

Physical and Functional Interactions between ELL2 and RB in the Suppression of Prostate Cancer Cell Proliferation, Migration, and Invasion

Elongation factor, RNA polymerase II, 2 (ELL2) is expressed and regulated by androgens in the prostate. ELL2 and ELL-associated factor 2 (EAF2) form a stable complex, and their orthologs in Caenorhabditis elegans appear to be functionally similar. In C. elegans, the EAF2 ortholog eaf-1 was reported to interact with the retinoblastoma (RB) pathway to control development and fertility in worms. Because RB loss is frequent in prostate cancer, ELL2 interaction with RB might be important for prostate homeostasis. The present study explored physical and functional interaction of ELL2 with RB in prostate cancer. ELL2 expression in human prostate cancer specimens was detected using quantitative polymerase chain reaction coupled with laser capture microdissection. Co-immunoprecipitation coupled with deletion mutagenesis was used to determine ELL2 association with RB. Functional interaction between ELL2 and RB was tested using siRNA knockdown, BrdU incorporation, Transwell, and/or invasion assays in LNCaP, C4-2, and 22Rv1 prostate cancer cells. ELL2 expression was downregulated in high–Gleason score prostate cancer specimens. ELL2 could be bound and stabilized by RB, and this interaction was mediated through the N-terminus of ELL2 and the C-terminus of RB. Concurrent siRNA knockdown of ELL2 and RB enhanced cell proliferation, migration, and invasion as compared to knockdown of ELL2 or RB alone in prostate cancer cells. ELL2 and RB can interact physically and functionally to suppress prostate cancer progression.

EAF2 regulates DNA repair through Ku70/Ku80 in the prostate

Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor signaling in prostate cancers. ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor and its intracellular localization can be modulated by ultraviolet light, suggesting a potential role for EAF2 in androgen regulation of DNA repair in prostate cancer cells. Here we show that knockdown of EAF2 or its homolog EAF1 sensitized prostate cancer cells to DNA damage and the sensitization did not require p53. EAF2 knockout mouse prostate was also sensitized to γ-irradiation. Furthermore, EAF2 knockdown blocked androgen repression of LNCaP or C4-2 cells from doxorubicin induction of γH2ax, a DNA damage marker. In human prostate cancer specimens, EAF2 expression was inversely correlated with the level of γH2ax. Further analysis showed that EAF2 and EAF1 are required for the recruitment and retention of Ku70/Ku80 to DNA damage sites and play a functional role in nonhomologous end-joining DNA repair. These findings provide evidence for EAF2 as a key factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells.

EAF2 mediates germinal centre B-cell apoptosis to suppress excessive immune responses and prevent autoimmunity.

Regulated apoptosis of germinal centre (GC) B cells is critical for normal humoral immune responses. ELL-associated factor 2 (EAF2) regulates transcription elongation and has been shown to be an androgen-responsive potential tumour suppressor in prostate by inducing apoptosis. Here we show that EAF2 is selectively upregulated in GC B cells among various immune cell types and promotes apoptosis of GC B cells both in vitro and in vivo. EAF2 deficiency results in enlarged GCs and elevated antibody production during a T-dependent immune response. After immunization with type II collagen, mice lacking EAF2 produce high levels of collagen-specific autoantibodies and rapidly develop severe arthritis. Moreover, the mutant mice spontaneously produce anti-dsDNA, rheumatoid factor and anti-nuclear antibodies as they age. These results demonstrate that EAF2-mediated apoptosis in GC B cells limits excessive humoral immune responses and is important for maintaining self-tolerance.

Expression and prognostic significance of ELL-associated factor 2 in human prostate cancer.

ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor in the prostate. The purpose of this study was to investigate the expression of EAF2 protein in human prostate cancer specimens along with BPH specimens as a control, and to evaluate potential association of EAF2 expression with clinical characteristics and overall survival of the prostate cancer patients. The expression of EAF2 was evaluated in 44 prostate cancer and 23 BPH tissue specimens using immunohistochemistry. The relationships of EAF2 expression with clinical characteristics and overall survival rates were analyzed by Chi-square test and Kaplan-Meier method. The immunostaining intensity of EAF2 in BPH specimens was significantly higher than that in prostate cancer (p < 0.05). EAF2 expression decreased significantly in high-grade and advanced-stage human prostate tumors and inversely correlated with PSA level, Gleason scores, bone metastasis and tumor stage. Importantly, loss of EAF2 expression was associated with a significant decrease in patient survival. Expression of EAF2 is decreased in prostate carcinogenesis, and EAF2 loss is associated with high-risk patients and poor survival.

Effects of ELL-associated factor 2 on ultraviolet radiation-induced cataract formation in mice.

ELL-associated factor 2 (Eaf2) has an important role in crystalline lens development and maturation; however, its role in ultraviolet radiation (UV)-induced cataract formation has remained elusive. The present study compared UV-induced cell apoptosis, activation of caspase-3 and caspase-9 and changes in protein expression levels of B-cell lymphoma 2 (bcl-2), bcl-2-associated X protein (bax) and phosphorylated extracellular signal-regulated kinase in wild-type and Eaf2-knockout mice. The results showed that Eaf2 knockout can reduce UV-induced apoptosis in crystalline lenses and mitigate the formation of cataracts. Further functional studies indicated that Eaf2 can induce the activation of caspase-3 and caspase-9, increase the protein expression of the pro-apoptotic protein bax and inhibit the expression of the anti-apoptotic protein bcl-2; thereby, Eaf2 promotes cell apoptosis and is implicated in the formation and development of cataracts. The present study laid a theoretical foundation for the development of drugs for cataract treatment.

FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation, and migration in prostate cancer cells.

ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor in the prostate. However, the mechanisms underlying tumor suppressive function of EAF2 are still largely unknown. Identification of factors capable of modulating EAF2 function will help elucidate the mechanisms underlying EAF2 tumor suppressive function. Using eaf-1(the ortholog of EAF2) mutant C. elegans model, RNAi screen was used to identify factors on the basis of their knockdown to synergistically enhance the reduced fertility phenotype of the eaf-1 mutant C. elegans. In human cells, the interaction of EAF2 with FOXA1 and the effect of EAF2 on the FOXA1 protein levels were determined by co-immunoprecipitation and protein stability assay. The effect of EAF2 and/or FOXA1 knockdown on the expression of AR-target genes was determined by real-time RT-PCR and luciferase reporter assays. The effect of EAF2 and/or FOXA1 knockdown on LNCaP human prostate cancer cell proliferation and migration was tested using BrdU assay and transwell migration assay. RNAi screen identified pha-4, the C. elegans ortholog of mammalian FOXA1, on the basis of its knockdown to synergistically enhance the reduced fertility phenotype of the eaf-1 mutant C. elegans causing sterility. EAF2 co-immunoprecipitated with FOXA1. EAF2 knockdown enhanced endogenous FOXA1 protein level, whereas transfected GFP-EAF2 down-regulated the FOXA1 protein. Also, EAF2 knockdown enhanced the expression of AR-target genes, cell proliferation, and migration in LNCaP cells. However, FOXA1 knockdown inhibited the effect of EAF2 knockdown on AR-target gene expression, cell proliferation, and migration in LNCaP cells, suggesting that FOXA1 can modulate EAF2 regulation of AR transcriptional activation, cell proliferation, and migration. These findings suggest that regulation of the AR signaling pathway, cell proliferation, and migration through FOXA1 represents an important mechanism of EAF2 suppression of prostate carcinogenesis.

Pirin down‐regulates the EAF2/U19 protein and alleviates its growth inhibition in prostate cancer cells

The tumor suppressor ELL associated factor 2 (EAF2/U19) has been reported to induce apoptosis of LNCaP cells and suppress AT6.1 xenograft prostate tumor growth. EAF2/U19 expression level is down-regulated in advanced human prostate cancer. EAF2/U19 is also a putative transcription factor with a transactivation domain and capability of sequence-specific DNA binding. Identification of binding partners and regulators of EAF2/U19 is essential to understand its function in regulating apoptosis/survival of prostate cancer cells. Through a yeast two-hybrid screening system, we identified Pirin as a binding partner of EAF2. We further determined the interaction between epitope-tagged EAF2/U19 and Pirin by co-immunoprecipitation in mammalian cells. The effect of Pirin on EAF2/U19 inhibition of LNCaP growth was assayed by colony formation. Pirin co-immunoprecipitated with EAF2/U19 and the overexpressed Pirin decreased the expression level of EAF2/U19 protein in prostate cancer cell lines LNCaP and PC3. Furthermore, overexpression of EAF2/U19 suppressed LNCaP colony formation, and co-expression of Pirin significantly blocked the growth inhibition induced by EAF2/U19 overexpression. Pirin is a newly identified binding partner of EAF2/U19 capable of down-regulating EAF2/U19 protein and alleviating its inhibition of prostate cancer cell survival/proliferation. Pirin may play an important role involved in EAF2/U19 function as an androgen-responsive gene and tumor repressor.

Development of a Reactive Stroma Associated with Prostatic Intraepithelial Neoplasia in EAF2 Deficient Mice

ELL-associated factor 2 (EAF2) is an androgen-responsive tumor suppressor frequently deleted in advanced prostate cancer that functions as a transcription elongation factor of RNA Pol II through interaction with the ELL family proteins. EAF2 knockout mice on a 129P2/OLA-C57BL/6J background developed late-onset lung adenocarcinoma, hepatocellular carcinoma, B-cell lymphoma and high-grade prostatic intraepithelial neoplasia. In order to further characterize the role of EAF2 in the development of prostatic defects, the effects of EAF2 loss were compared in different murine strains. In the current study, aged EAF2−/− mice on both the C57BL/6J and FVB/NJ backgrounds exhibited mPIN lesions as previously reported on a 129P2/OLA-C57BL/6J background. In contrast to the 129P2/OLA-C57BL/6J mixed genetic background, the mPIN lesions in C57BL/6J and FVB/NJ EAF2−/− mice were associated with stromal defects characteristic of a reactive stroma and a statistically significant increase in prostate microvessel density. Stromal inflammation and increased microvessel density was evident in EAF2-deficient mice on a pure C57BL/6J background at an early age and preceded the development of the histologic epithelial hyperplasia and neoplasia found in the prostates of older EAF2−/− animals. Mice deficient in EAF2 had an increased recovery rate and a decreased overall response to the effects of androgen deprivation. EAF2 expression in human cancer was significantly down-regulated and microvessel density was significantly increased compared to matched normal prostate tissue; furthermore EAF2 expression was negatively correlated with microvessel density. These results suggest that the EAF2 knockout mouse on the C57BL/6J and FVB/NJ genetic backgrounds provides a model of PIN lesions associated with an altered prostate microvasculature and reactive stromal compartment corresponding to that reported in human prostate tumors.

Concomitant loss of EAF2/U19 and Pten synergistically promotes prostate carcinogenesis in the mouse model

Multiple genetic alterations are associated with prostate carcinogenesis. Tumor-suppressor genes phosphatase and tensin homolog deleted on chromosome 10 (Pten) and androgen upregulated gene 19 (U19), which encodes ELL-associated factor 2 (EAF2), are frequently inactivated or downregulated in advanced prostate cancers. Previous studies showed that EAF2 knockout caused tumors in multiple organs and prostatic intraepithelial neoplasia (PIN) in mice. However, EAF2-knockout mice did not develop prostate cancer even at 2 years of age. To further define the roles of EAF2 in prostate carcinogenesis, we crossed the Pten+/- and EAF2+/- mice in the C57/BL6 background to generate EAF2-/-Pten+/-, Pten+/-, EAF2-/- and wild-type mice. The prostates from virgin male mice with the above four genotypes were analyzed at 7 weeks, 19 weeks and 12 months of age. Concomitant loss of EAF2 function and inactivation of one Pten allele induced spontaneous prostate cancer in 33% of the mice. Prostatic tissues from intact EAF2-/- Pten+/- mice exhibited higher levels of phospho-Akt, -p44/42 and microvessel density. Moreover, phospho-Akt remained high after castration. Consistently, there was a synergistic increase in prostate epithelial proliferation in both intact and castrated EAF2-/-Pten+/- mice. Using laser-capture microdissection coupled with real-time reverse transcription-PCR, we confirmed that co-downregulation of EAF2 and Pten occurred in >50% clinical prostate cancer specimens with Gleason scores of 8-9 (n=11), which is associated with poor prognosis. The above findings together demonstrated synergistic functional interactions and clinical relevance of concurrent EAF2 and Pten downregulation in prostate carcinogenesis.

Eafs Control Erythroid Cell Fate by Regulating c-myb Expression through Wnt Signaling

ELL associated factor 1 and ELL associated factor 2 (EAF1/2 factors) are reported to play important roles in tumor suppression and embryogenesis. Our previous studies showed that eaf factors mediated effective convergence and extension (C&E) movements and modulated mesoderm and neural patterning by regulating both non-canonical and canonical Wnt signaling in the early embryonic process. In this study, through knockdown of both eaf1 and eaf2 in embryos, we found that differentiation of primary erythroid cells was blocked, but hematopoietic precursor cells maintained in eafs morphants. Co-injection of c-myb-MO rescued the erythroid differentiation in eafs morphants, as indicated by the restored expression of the erythroid-specific gene, βe3 globin. In addition, low dosage of c-myb effectively blocked the βe3 globin expression in embryos, and did not affect the expression of markers of hematopoietic progenitor cells and other mesoderm, which was similar to the phenotypes we observed in eafs morphants. We also revealed that knockdown Wnt signaling by transiently inducing dn-Tcf in embryos at the bud stage down-regulated the increased c-myb to normal level and also restored βe3 globin expression in eafs morphants. Our evidence points to a novel role for eaf factors in controlling erythroid cell fate by regulating c-Myb expression through canonic Wnt signaling.