Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1.

Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, an effective immunosuppressive drug. Both MPA and mycophenolate mofetil are highly specific inhibitors of guanine nucleotide synthesis and of T-cell activation. However, the mechanism by which guanine nucleotide depletion suppresses T-cell activation is unknown. Depletion of GTP inhibits ribosomal RNA synthesis in T cells by inhibiting transcription initiation factor I (TIF-IA), a GTP-binding protein that recruits RNA polymerase I to the ribosomal DNA promoter. TIF-IA-GTP binds the ErbB3-binding protein 1, and together they enhance the transcription of proliferating cell nuclear antigen (PCNA). GTP binding by TIF-IA and ErbB3-binding protein 1 phosphorylation by protein kinase C δ are both required for optimal PCNA expression. The protein kinase C inhibitor sotrastaurin markedly potentiates the inhibition of ribosomal RNA synthesis, PCNA expression, and T-cell activation induced by MPA, suggesting that the combination of the two agents are more highly effective than either alone in inducing immunosuppression.

Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells.

Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways.

The ErbB3-binding protein EBP1 modulates lapatinib sensitivity in prostate cancer cells.

Although ErbB receptors have been implicated in prostate cancer progression, ErbB-directed drugs have not proven effective for prostate cancer treatment. The ErbB3-binding protein EBP1 affects both ErbB2 and androgen receptor signaling, two components of the response to ErbB-targeted therapies. We therefore examined the effects of EBP1 expression on the response to the ErbB1/2 tyrosine kinase inhibitor lapatinib. We found a negative correlation between endogenous EBP1 levels and lapatinib sensitivity in prostate cancer cell lines. We then overexpressed or inhibited expression of EBP1. Silencing EBP1 expression increased lapatinib sensitivity and overexpression of EBP1 increased resistance in androgen-containing media. Androgen depletion resulted in an increased sensitivity of androgen-dependent EBP1 expressing cells to lapatinib, but did not affect the lapatinib sensitivity of hormone resistant cells. However, EBP1 silenced cells were still more sensitive to lapatinib than EBP1-expressing cells in the absence of androgens. The increase in sensitivity to lapatinib following EBP1 silencing was associated with increased ErbB2 levels. In addition, lapatinib treatment increased ErbB2 levels in sensitive cells that express low levels of EBP1, but decreased ErbB2 levels in resistant EBP1-expressing cells. In contrast, ErbB3 and phospho ErbB3 levels were not affected by either changes in EBP1 levels or lapatinib treatment. The production of the ErbB3/4 ligand heregulin was increased in EBP1-silenced cells. EBP1-induced changes in AR levels were not associated with changes in lapatinib sensitivity. These studies suggest that the ability of EBP1 to activate ErbB2 signaling pathways results in increased lapatinib sensitivity.

Heregulin negatively regulates transcription of ErbB2/3 receptors via an AKT-mediated pathway.

Despite the importance of the ErbB2/3 heterodimer in breast cancer progression, the negative regulation of these receptors is still poorly understood. We demonstrate here for the first time that the ErbB3/4 ligand heregulin (HRG) reduced both ErbB2 and ErbB3 mRNA and protein levels in human breast cancer cell lines. In contrast, EGFR levels were unaffected by HRG treatment. The effect was rapid with a decline in steady-state mRNA levels first noted 2 h after HRG treatment. HRG reduced the rate of transcription of ErbB2 and ErbB3 mRNA, but did not affect ErbB2 or ErbB3 mRNA stability. To test if ErbB2 kinase activity was required for the HRG-induced downregulation, we treated cells with the ErbB2/EGFR inhibitor lapatinib. Lapatinib diminished the HRG-induced decrease in ErbB2 and ErbB3 mRNA and protein, suggesting that the kinase activity of EGFR/ErbB2 is involved in the HRG-induced receptor downregulation. Further, HRG-mediated decreases in ErbB2/3 mRNA transcription are reversed by inhibiting the AKT but not MAPK pathway. To examine the functional consequences of HRG-mediated decreases in ErbB receptor levels, we performed cell-cycle analysis. HRG blocked cell-cycle progression and lapatinib reversed this block. Our findings support a role for HRG in the negative regulation of ErbB expression and suggest that inhibition of ErbB2/3 signaling by ErbB2 directed therapies may interfere with this process. J. Cell. Physiol. 229: 1831-1841, 2014. © 2014 Wiley Periodicals, Inc.

Post-transcriptional regulation of androgen receptor mRNA by an ErbB3 binding protein 1 in prostate cancer.

Androgen receptor (AR)-mediated pathways play a critical role in the development and progression of prostate cancer. However, little is known about the regulation of AR mRNA stability and translation, two central processes that control AR expression. The ErbB3 binding protein 1 (EBP1), an AR corepressor, negatively regulates crosstalk between ErbB3 ligand heregulin (HRG)-triggered signaling and the AR axis, affecting biological properties of prostate cancer cells. EBP1 protein expression is also decreased in clinical prostate cancer. We previously demonstrated that EBP1 overexpression results in decreased AR protein levels by affecting AR promoter activity. However, EBP1 has recently been demonstrated to be an RNA binding protein. We therefore examined the ability of EBP1 to regulate AR post-transcriptionally. Here we show that EBP1 promoted AR mRNA decay through physical interaction with a conserved UC-rich motif within the 3'-UTR of AR. The ability of EBP1 to accelerate AR mRNA decay was further enhanced by HRG treatment. EBP1 also bound to a CAG-formed stem-loop in the 5' coding region of AR mRNA and was able to inhibit AR translation. Thus, decreases of EBP1 in prostate cancer could be important for the post-transcriptional up-regulation of AR contributing to aberrant AR expression and disease progression.

The ErbB3 binding protein EBP1 regulates ErbB2 protein levels and tamoxifen sensitivity in breast cancer cells.

The ErbB2/3 heterodimer plays a critical role in breast cancer progression and in the development of endocrine resistance. EBP1, an ErbB3 binding protein, inhibits HRG-stimulated breast cancer growth, decreases ErbB2 protein levels and contributes to tamoxifen sensitivity. We report here that ectopic expression of EBP1 in Estrogen Receptor (ER) positive breast cancers that express ErbB2 at both high and low levels decreased ErbB2 protein levels. ErbB2 protein expression was also increased in mammary glands of Ebp1 knock out mice. To define the mechanism of ErbB2 down regulation, we examined the effects of EBP1 on ErbB2 mRNA levels, transcription of the ErbB2 gene and ErbB2 protein stability. We found that ectopic expression of EBP1 decreased steady state levels of endogenous ErbB2 mRNA in all cell lines tested. EBP1 overexpression decreased the activity of an ErbB2 promoter reporter in cells which overxpress ErbB2. However, reporter activity was unchanged or increased in cells which express low endogenous levels of ErbB2. We also found that ectopic expression of EBP1 accelerated ErbB2 protein degradation and enhanced ErbB2 ubiquitination in cells which express both low and high levels of ErbB2. Treatment with proteasome inhibitors prevented this decrease in ErbB2 protein levels. Ablation of EBP1 expression led to tamoxifen resistance that was abrogated by inhibition of ErbB2 activity. These results suggest that EBP1 inhibits expression of ErbB2 protein levels by multiple mechanisms and that EBP1's effects on tamoxifen sensitivity are mediated in part by its ability to modulate ErbB2 levels.

Metformin and Androgen Receptor-Axis-Targeted (ARAT) Agents Induce Two PARP-1-Dependent Cell Death Pathways in Androgen-Sensitive Human Prostate Cancer Cells.

We explored whether the anti-prostate cancer (PC) activity of the androgen receptor-axis-targeted agents (ARATs) abiraterone and enzalutamide is enhanced by metformin. Using complementary biological and molecular approaches, we determined the associated underlying mechanisms in pre-clinical androgen-sensitive PC models. ARATs increased androgren receptors (ARs) in LNCaP and AR/ARv7 (AR variant) in VCaP cells, inhibited cell proliferation in both, and induced poly(ADP-ribose) polymerase-1 (PARP-1) cleavage and death in VCaP but not LNCaP cells. Metformin decreased AR and ARv7 expression and induced cleaved PARP-1-associated death in both cell lines. Metformin with abiraterone or enzalutamide decreased AR and ARv7 expression showed greater inhibition of cell proliferation and greater induction of cell death than single agent treatments. Combination treatments led to increased cleaved PARP-1 and enhanced PARP-1 activity manifested by increases in poly(ADP-ribose) (PAR) and nuclear accumulation of apoptosis inducing factor (AIF). Enhanced annexin V staining occurred in LNCaP cells only with metformin/ARAT combinations, but no caspase 3 recruitment occurred in either cell line. Finally, metformin and metformin/ARAT combinations increased lysosomal permeability resulting in cathepsin G-mediated PARP-1 cleavage and cell death. In conclusion, metformin enhances the efficacy of abiraterone and enzalutamide via two PARP-1-dependent, caspase 3-independent pathways, providing a rationale to evaluate these combinations in castration-sensitive PC.

EBP1, an ErbB3-binding protein, is decreased in prostate cancer and implicated in hormone resistance.

Aberrant activation of the androgen receptor (AR) by the ErbB2/ErbB3 heterodimer contributes to the development of hormone resistance in prostate cancer. EBP1, an ErbB3-binding protein, acts as an AR corepressor. As EBP1 is decreased in preclinical models of hormone-refractory prostate cancer, we studied the expression of EBP1 in human prostate cancer. We found that the expression of the EBP1 gene was significantly decreased in prostate cancer tissues compared with benign prostate at both mRNA and protein levels. Restoration of EBP1 expression in the hormone-refractory LNCaP C81 cell line led to an amelioration of the androgen-independent phenotype based on established biological criteria and a reduction in the expression of a cohort of AR target genes. The ability of the ErbB3 ligand heregulin (HRG) to stimulate growth and AKT phosphorylation of hormone-refractory prostate cancer cells was abolished. Abrogation of EBP1 expression by short hairpin RNA in hormone-dependent LNCaP cells, which undergo apoptosis in response to HRG, resulted in HRG-stimulated cell growth. Restoration of EBP1 expression decreased the tumorigenicity of C81 xenografts in female mice, whereas elimination of EBP1 expression enhanced the ability of LNCaP cells to grow in female mice. Our data support a role for EBP1 in the development of hormone-refractory prostate cancer via inhibition of both AR- and HRG-stimulated growth and present a novel strategy for treating androgen-refractory prostate cancer.

Alterations in cell growth and signaling in ErbB3 binding protein-1 (Ebp1) deficient mice.

BACKGROUND: The ErbB3 binding protein-1 (Ebp1) belongs to a family of DNA/RNA binding proteins implicated in cell growth, apoptosis and differentiation. However, the physiological role of Ebp1 in the whole organism is not known. Therefore, we generated Ebp1-deficient mice carrying a gene trap insertion in intron 2 of the Ebp1 (pa2g4) gene. RESULTS: Ebp1-/- mice were on average 30% smaller than wild type and heterozygous sex matched littermates. Growth retardation was apparent from Day 10 until Day 30. IGF-1 production and IGBP-3 and 4 protein levels were reduced in both embryo fibroblasts and adult knock-out mice. The proliferation of fibroblasts derived from Day 12.5 knock out embryos was also decreased as compared to that of wild type cells. Microarray expression analysis revealed changes in genes important in cell growth including members of the MAPK signal transduction pathway. In addition, the expression or activation of proliferation related genes such as AKT and the androgen receptor, previously demonstrated to be affected by Ebp1 expression in vitro, was altered in adult tissues. CONCLUSION: These results indicate that Ebp1 can affect growth in an animal model, but that the expression of proliferation related genes is cell and context specific. The Ebp1-/- mouse line represents a new in vivo model to investigate Ebp1 function in the whole organism.

Inhibition of heregulin mediated MCF-7 breast cancer cell growth by the ErbB3 binding protein EBP1.

The ErbB2/3 heterodimer plays a critical role in breast cancer genesis and progression. EBP1, an ErbB3 binding protein, inhibits breast cancer growth but its effects on ErbB3 ligand mediated signal transduction or ErbB receptors is not known. We report here that ectopic expression of EBP1 in MCF-7 and AU565 breast cancer cell lines inhibited HRG-induced proliferation. ErbB2 protein levels were substantially decreased in EBP1 transfectants, while ErbB3 levels were unchanged. HRG-induced AKT activation was attenuated in EBP1 stable transfectants and transfection of a constitutively activated AKT partially restored the growth response to HRG. Down-regulation of EBP1 expression in MCF-7 cells by shRNA resulted in increased cell growth in response to HRG and increased cyclin D1 and ErbB2 expression. These results suggest that EBP1, by down-regulating ErbB signal transduction, attentuates HRG-mediated growth of breast cancer cells.

MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4.

Although microRNA (miR) 199a-3p functions as a tumor suppressor in multiple malignancies, its expression and role in esophageal cancer have not been studied. Based on our previous observation that miR-199a-3p is markedly downregulated in esophageal cancer cell lines relative to esophageal epithelial cells, we examined the function of miR-199a-3p in these cells. MiR-199a-3p is predicted to bind with high affinity to the mRNA of p21 activated kinase 4 (PAK4). This kinase has been shown to be overexpressed in several malignancies and to modulate proliferation and motility. The current study is designed to determine whether miR-199a-3p regulates the expression of PAK4 in esophageal cancer cells and to understand the functional consequences of this interaction. Herein, we demonstrate reduced expression of miR-199a-3p in human esophageal cancer specimens and cell lines compared to esophageal epithelial cells, with associated increased expression of PAK4. Forced expression of miR-199a-3p decreases expression of PAK4 in esophageal cancer cell lines. Mechanistic studies reveal that miR-199a-3p binds to the 3'UTR of PAK4 mRNA. This interaction results in reduced levels of PAK4 mRNA due to decreased mRNA stability. Downregulation of PAK4 leads to decreased cyclin D1 (CD1) transcription and protein expression, resulting in markedly impaired cellular proliferation. When PAK4 expression is rescued, both CD1 transcription and protein return to baseline levels. Our results show that miR-199a-3p functions as a tumor suppressor in esophageal cancer cells through repression of PAK4. These findings suggest that both miR-199a-3p and PAK4 may be novel therapeutic targets in the treatment of esophageal cancer.

Ebp1-mediated inhibition of cell growth requires serine 363 phosphorylation.

Ebp1 is an ErbB3 binding phosphoprotein with pleiotropic effects. Overexpression of Ebp1 represses transcription of E2F1 responsive cell cycle regulated genes and inhibits cell growth. However, the effect of phosphorylation on Ebp1-mediated transcriptional repression and cell growth inhibition is currently unknown. In this study, we show that serine 363 (S363) of Ebp1 is phosphorylated in vivo. Although total Ebp1 is located in the nucleus, organelles and the cytoplasm, Ebp1 phosphorylated at S363 (Ebp1 pS363) is localized exclusively to the nucleus. Mutation of S363 to alanine did not change the subcellular localization of Ebp1. However, the S363A mutation significantly decreased the ability of Ebp1 to repress transcription and abrogated its ability to inhibit cell growth. We have previously shown that Ebp1 can bind the E2F1 promoter in vitro and in vivo as part of a protein complex and that Ebp1-transcriptional repression is mediated via its interaction with the co-repressors HDAC2 and mSin3a present in this complex. Although Ebp1 S363A interacted with an E2F1 promoter element, it did not bind HDAC2 and mSin3a. These results indicate the importance of S363 phosphorylation in the function of Ebp1.

The N-terminal 24 amino acids of the p55 gamma regulatory subunit of phosphoinositide 3-kinase binds Rb and induces cell cycle arrest.

Although phosphoinositide 3-kinase (PI 3-kinase) is essential for cell cycle progression, the molecular mechanisms that regulate its diverse biological effects are poorly understood. We demonstrate here that Rb, a key regulator of cell cycle progression, associates with p55 kDa (p55alpha and p55gamma) regulatory subunits of PI 3-kinase in vivo and in vitro. Both confocal microscopy and biochemical analysis demonstrated the presence of p55gamma in the nucleus. The 24-amino-acid N-terminal end of p55gamma, which is unique among PI 3-kinase regulatory subunits, was sufficient to bind Rb. Addition of serum or growth factors to quiescent cells triggered the dissociation of Rb from p55. Ectopic expression of the 24-amino-acid N-terminal end of p55gamma inhibited cell cycle progression, as evidenced by induction of cell growth arrest at the G0/G1 phase, inhibition of DNA synthesis, inhibition of cyclin D and cyclin E promoter activity, and changes in the expression of cell cycle-related proteins. The inhibitory effects of the N-terminal end of p55gamma on cell cycle progression depended on the presence of functional Rb. These data demonstrate for the first time an association of p55gamma with Rb and show that modification of this association can lead to cell cycle arrest.

The ErbB3 binding protein Ebp1 interacts with Sin3A to repress E2F1 and AR-mediated transcription.

Ectopic expression of ebp1, a member of the PA2G4 family, inhibits the proliferation and induces the differentiation of human breast and prostate cancer cell lines. Ebp1 inhibits transcription of E2F1 and androgen receptor regulated genes such as prostate specific antigen (PSA) through its interactions with histone deacetylases (HDACs). To further understand Ebp1's interactions with other components of the transcriptional repression machinery, we examined the association of Ebp1 with the corepressor Sin3A. Ebp1 interacted with Sin3A both in vitro and in vivo as demonstrated by glutathione S-transferase (GST) pull-down and coimmunoprecipitation analysis. The C-terminal domain of Ebp1, responsible for its ability to repress transcription and arrest cell growth, was necessary and sufficient for binding Sin3A. The C-terminal domain of Sin3A, containing the paired amphipathic domain 4 and the HDAC interacting domain, bound Ebp1. Recombinant Sin3A bound Ebp1 directly, but recombinant HDAC2 failed to bind Ebp1. Chromatin immunoprecipitation (ChIP) and DNA affinity precipitation analysis demonstrated that Ebp1 and Sin3A associate at the PSA and E2F1 promoters. Functionally, Sin3A enhanced the ability of Ebp1 to repress transcription of androgen receptor (AR) and E2F1 regulated genes. These results demonstrate that Ebp1 participates in transcriptional regulation via its interaction with the Sin3-HDAC.

Cancer stem cells and individualized therapy.

The concept of individualized cancer chemotherapy emerged three decades ago from the observation that a small fraction of cells in primary tumors can form colonies in soft agar similar to stem cells of the hematopoietic system. In a series of retrospective and prospective clinical studies, clonogenic tumor growth and effects of anticancer agents on the putative cancer stem cells were assessed as predictive factors. The results of these trials showed that clonogenic growth is associated with poor outcome and drug resistance. Recent breakthroughs enabling isolation and the molecular classification of cancer stem cells have renewed interest in cancer stem cells as a therapeutic target. Here, we provide a current overview of cancer stem cell biology and highlight possibilities for targeted intervention with existing and novel experimental anticancer agents.

Repression of E2F1-mediated transcription by the ErbB3 binding protein Ebp1 involves histone deacetylases.

Ebp1, an ErbB3 binding protein that is a member of the proliferation-associated PA2G4 family, inhibits the proliferation and induces the differentiation of human ErbB positive breast and prostate cancer cell lines. Ebp1 binds the tumor suppressor retinoblastoma protein (Rb) both in vivo and in vitro, and Rb and Ebp1 cooperate to inhibit the transcription of the E2F1-regulated cyclin E promoter. We show here that Ebp1 can inhibit the transcription of other E2F-regulated reporter genes and of several endogenous E2F-regulated genes important in cell cycle progression in both Rb positive and Rb null cells. The Ebp1-mediated transcriptional repression depended on the presence of an E2F1 consensus element in the promoters. A fusion of Ebp1 with the GAL4 DNA binding domain protein had independent transcriptional repression activity that mapped to the C-terminal region of Ebp1. This C-terminal region of Ebp1 bound functional histone deacetylase (HDAC) activity and inhibitors of HDAC significantly reduced Ebp1-mediated repression. Ebp1 bound HDAC2, but not HDAC1, in vitro. An Ebp1 mutant lacking the HDAC binding domain failed to inhibit transcription. Our results suggest that Ebp1 can repress transcription of some E2F-regulated promoters and that one mechanism of Ebp1- mediated transcriptional repression is via its ability to recruit HDAC activity.

The nonsteroidal anti-inflammatory drug sulindac causes down-regulation of signal transducer and activator of transcription 3 in human oral squamous cell carcinoma cells.

The nonsteroidal anti-inflammatory drug sulindac exerts a significant antineoplastic effect on several types of human cancers including oral squamous cell carcinoma (SCCa). Because constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been linked to carcinogenesis of various tumors including head and neck SCCa, we studied whether sulindac treatment affects the Stat3 signaling pathway in oral SCCa cells. Western blot experiments showed that short-term treatment of cells with sulindac resulted in a large reduction of phosphorylated Stat3, without significantly affecting Stat3 protein levels. In contrast, 3 days of sulindac treatment eliminated both phosphorylated and unphosphorylated Stat3 protein levels. Also, sulindac treatment exerted a significant time-dependent cell growth-inhibitory effect on oral SCCa cells under the same conditions shown to induce Stat3 down-modulation. The sulfone metabolite of sulindac, which lacks cyclooxygenase-inhibitory activity, did not affect either Stat3 expression or Stat3 phosphorylation. Antisense oligonucleotide treatment against peroxisome proliferator-activated receptor gamma did not attenuate the ability of sulindac to down-regulate Stat3. Our results suggest that down-modulation of Stat3 can be induced by sulindac treatment, thus possibly contributing to the antineoplastic effect of this drug.

Repression of androgen receptor mediated transcription by the ErbB-3 binding protein, Ebp1.

Members of the ErbB family of receptors have been implicated in regulation of androgen receptor (AR) activity. Ebp1, an ErbB-3 binding protein recently cloned in our laboratory, possesses an LXXLL motif important in mediating interactions with nuclear hormone receptors. Therefore, we sought to determine if Ebp1 could bind AR and influence AR transcriptional activation potential. We demonstrate in this study that Ebp1 bound to AR in vitro and in vivo, and that this binding was increased by androgen treatment. The C terminal 79 amino acids of Ebp1 were sufficient to bind AR. The N terminal domain of AR was responsible for binding Ebp1. Ligand-mediated transcriptional activation of both artificial and natural AR regulated promoters was inhibited by ectopic expression of ebp1 in transient transfection systems. Ebp1 deletion mutants that either lacked the C terminal AR binding region or had a mutated LXXLL motif failed to inhibit AR activated transcription. PSA expression from its endogenous promoter was also decreased in LNCaP prostate cancer cells overexpressing Ebp1. The growth of AR positive LNCaP cells was inhibited by ectopic expression of ebp1, but mutants that failed to repress transcription did not inhibit cell growth. These studies suggest that Ebp1 may play a role in the function of the AR and provide a link between ErbB receptors and the AR.

ErbB3-binding protein EBP1 decreases ErbB2 levels via a transcriptional mechanism.

Ectopic expression of EBP1, an ErbB3-interacting protein, reduces the expression of the ErbB2 protein and mRNA. However, the mechanism of EBP1-induced decrease in ErbB2 mRNA levels has not yet been determined. Since EBP1 affects both transcriptional and post-transcriptional processes, we evaluated the ability of EBP1 to regulate ErbB2 transcription and RNA stability. We discovered that while wild-type EBP1 decreased the activity of a proximal ErbB2 promoter, EBP1 mutants unable to interact with the Sin3A transcriptional repressor inhibited activity to a lesser extent. EBP1 also decreased the activity of distal ErbB2 promoters. Chromatin immunoprecipitation analysis indicated that EBP1 bound both distal and proximal endogenous ErbB2 promoters in serum-starved conditions. The ErbB3 ligand heregulin (HRG) at growth-promoting concentrations reduced EBP1 binding to the ErbB2 promoter. Although endogenous EBP1 bound ErbB2 mRNA, EBP1 overexpression or ablation of EBP1 protein by shRNA failed to alter ErbB2 mRNA stability. These results suggest that the major effect of EBP1 on ErbB2 mRNA levels is at the transcriptional level.

EBP1 inhibits translation of androgen receptor mRNA in castration resistant prostate cancer cells.

BACKGROUND: Therapies that inhibit androgen receptor (AR) are needed for treatment of castration-resistant prostate cancer (CRPC). The ErbB3 binding protein 1 (EBP1) reduces protein expression of both AR and its target genes in CRPC. Although EBP1 regulates AR in hormone-sensitive prostate cancer cells, by both destabilizing AR mRNA and inhibiting protein translation, the mechanism of EBP1 down regulation of AR in CRPC is unknown. MATERIALS AND METHODS: Western blot and quantitative PCR analysis of cell lysates and polysomes were used to assess AR mRNA, protein expression and translation. RESULTS: In contrast to hormone- dependent cells, EBP1 did not change steady state levels of AR mRNA or AR mRNA stability in hormone refractory cells. EBP1 did slow protein translation of AR mRNA. The ErbB3/4 ligand heregulin further diminished AR translation in EBP1 -transfected cells, but not in control cells. CONCLUSION: These studies suggest that one pathway of EBP1 down-regulation of AR levels may be lost in CRPC.