Drugs That Mimic Hypoxia Selectively Target EBV-Positive Gastric Cancer Cells.

Latent infection of Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cell cancers, including 10% of gastric carcinomas. We previously reported that hypoxia inducible factor-1α (HIF-1α) induces EBV's latent-to-lytic switch and identified several HIF-1α-stabilizing drugs that induce this viral reactivation. Here, we tested three classes of these drugs for preferential killing of the EBV-positive gastric cancer AGS-Akata cell line compared to its matched EBV-negative AGS control. We observed preferential killing with iron chelators [Deferoxamine (DFO); Deferasirox (DFX)] and a prolyl hydroxylase inhibitor (BAY 85-3934 (Molidustat)), but not with a neddylation inhibitor [MLN4924 (Pevonedistat)]. DFO and DFX also induced preferential killing of the EBV-positive gastric cancer AGS-BDneo and SNU-719 cell lines. Preferential killing was enhanced when low-dose DFX (10 µM) was combined with the antiviral prodrug ganciclovir. DFO and DFX induced lytic EBV reactivation in approximately 10% of SNU-719 and 20-30% of AGS-Akata and AGS-BDneo cells. However, neither DFO nor DFX significantly induced synthesis of lytic EBV proteins in xenografts grown in NSG mice from AGS-Akata cells above the level observed in control-treated mice. Therefore, these FDA-approved iron chelators are less effective than gemcitabine at promoting EBV reactivation in vivo despite their high specificity and efficiency in vitro.

Reactivation of Epstein-Barr Virus by HIF-1α Requires p53.

We previously reported that the cellular transcription factor hypoxia-inducible factor 1α (HIF-1α) binds a hypoxia response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch BZLF1 gene, Zp, inducing viral reactivation. In this study, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1α-stabilizing drugs: the iron chelator deferoxamine (Desferal [DFO]), a neddylation inhibitor (pevonedistat [MLN-4924]), and a prolyl hydroxylase inhibitor (roxadustat [FG-4592]). DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1α and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with short hairpin RNAs (shRNAs). Likewise, HIF-1α failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1α to Zp in chromatin immunoprecipitation (ChIP) assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated, thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of BZLF1 gene expression by HIF-1α requires phosphorylated, wild-type p53 as a coactivator, with HIF-1α binding recruiting p53 to Zp.IMPORTANCE EBV, a human herpesvirus, is latently present in most nasopharyngeal carcinomas, Burkitt lymphomas, and some gastric cancers. To develop a lytic-induction therapy for treating patients with EBV-associated cancers, we need a way to efficiently reactivate EBV into lytic replication. EBV's BZLF1 gene product, Zta, usually controls this reactivation switch. We previously showed that HIF-1α binds the BZLF1 gene promoter, inducing Zta synthesis, and HIF-1α-stabilizing drugs can induce EBV reactivation. In this study, we determined which EBV-positive cell lines are reactivated by classes of HIF-1α-stabilizing drugs. We found, unexpectedly, that HIF-1α-stabilizing drugs only induce reactivation when they also induce accumulation of phosphorylated, wild-type p53. Fortunately, p53 phosphorylation can also be provided by drugs such as nutlin-3, leading to synergistic reactivation of EBV. These findings indicate that some HIF-1α-stabilizing drugs may be helpful as part of a lytic-induction therapy for treating patients with EBV-positive malignancies that contain wild-type p53.

Hypoxia-inducible factor-1α plays roles in Epstein-Barr virus's natural life cycle and tumorigenesis by inducing lytic infection through direct binding to the immediate-early BZLF1 gene promoter.

When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs). We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV). Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE) located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK) and gingival epithelial (hGET) cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV's natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for development of a lytic-induction therapy for treating some EBV-associated malignancies.

The ZIIR element of the Epstein-Barr virus BZLF1 promoter plays a central role in establishment and maintenance of viral latency.

The Epstein-Barr virus (EBV) BZLF1 gene encodes the immediate-early (IE) protein Zta, which plays a central role in regulating the switch between viral latency and lytic replication. A silencing element, ZIIR, is located between the ZID and ZII positive regulatory elements in the BZLF1 promoter Zp. We report here the phenotypes of variants of EBV strain B95.8 containing base substitution mutations in this ZIIR element. HEK293 cells infected with ZIIR mutant (ZIIRmt) virus produced at least 20-fold more viral IE Zta and Rta and early (E) EAD protein than did cells infected with the parental wild-type (WT) virus, leading to viral DNA replication and production of infectious virus. However, ZIIR mutant virus was 1/10 as efficient as WT virus in establishing proliferating B-cell clones following infection of human primary blood B cells. The ZIIRmt-infected lymphoblastoid cell lines (LCLs) that did grow out exhibited a phenotype similar to the one observed in 293 cells, including marked overproduction of IE and E gene products relative to WT-infected LCLs and lytic replication of the viral genome. Incubation of the ZIIRmt-infected LCLs with the chemical inducer 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to much greater activation of Zp than did the same treatment of WT- or ZVmt-infected LCLs. Furthermore, a protein kinase C (PKC) inhibitor, bis-indolylmaleimide, eliminated this activation by TPA. Thus, we conclude that ZIIR is a potent silencing element of Zp; it plays a key role in establishment and maintenance of EBV latency by inhibiting activation of Zp through the PKC signal transduction pathway.

Phosphorylation-dependent sumoylation of estrogen-related receptor alpha1.

We previously showed that estrogen-related receptor alpha1 (ERRalpha1) can compete with estrogen receptor alpha (ERalpha) for binding to estrogen response elements (EREs), repressing transcription in the mammary carcinoma cell line MCF-7. Given that ERRalpha1 can function in the absence of ligands and exists as a phosphoprotein in vivo, we wished to determine sites of phosphorylation involved in regulating its transcriptional activity. Using a combination of electrophoretic mobility shift analysis, phospho-specific fluorescent dye staining, and site-directed mutagenesis, we identified two novel in vivo sites of phosphorylation in the A/B ligand-independent activation domain of ERRalpha1 at Ser19 and Ser22. Inhibition of phosphorylation at amino acid residue 22 did not have a significant effect on ERRalpha1's transcriptional activity. However, mutation of amino acid residue 19 from serine to alanine enhanced two-fold ERRalpha1's response to the coactivator GRIP-1. We also identified two sites of sumoylation at Lys14 and Lys403. We found that inhibition of sumoylation at Lys14 could enhance five-fold ERRalpha1's response to coactivator GRIP-1. Furthermore, phosphorylation of Ser19 enhanced the sumoylation at Lys14. Taken together, we conclude that phosphorylation at Ser19 and sumoylation at Lys14 within the A/B domain play roles in regulating ERRalpha1's transcriptional activities via affecting its response to coactivators.

ZEB1 and c-Jun levels contribute to the establishment of highly lytic Epstein-Barr virus infection in gastric AGS cells.

The induction of lytic infection has been proposed as a therapeutic strategy for treating Epstein-Barr virus (EBV)-positive malignancies. To succeed, efficient methods are needed for activating the EBV immediate-early (IE) promoters, Zp and Rp. Here we compared factors which regulate Zp and Rp in AGS gastric carcinoma cells that support a remarkably high level of persistently lytic EBV infection with HeLa cervical cells that permit only tightly latent infection. We found that the level of Zp activity assayed by transient transfection assays with reporter plasmids was high in AGS cells but low in HeLa cells. The level of Rp activity was low in both cell types. Mutational analysis indicated that sequences within Zp located between -70 and +27 relative to the transcription initiation site were sufficient to confer a high level of Zp activity in AGS cells. The Zp CRE motif was necessary for this constitutive activity, while the ZIA and ZIB MEF2D motifs were not. Consistent with these findings, immunoblot analysis indicated that phosphorylated c-Jun, which activates Zp through the CRE motif, was expressed at a much higher level in EBV-infected AGS cells than in EBV-infected HeLa cells. In contrast, ZEB1, which represses Zp via the ZV motif located near the transcription initiation site, was abundant in HeLa cells, while it was absent from AGS cells. Exogenous addition of ZEB1 led to the repression of Zp in AGS cells. We conclude that the unusually high Zp activity level in AGS cells is due to the high abundance of positively acting transcription factors such as c-Jun combined with the low abundance of negatively acting factors such as ZEB1.

Estrogen-related receptor alpha1 transcriptional activities are regulated in part via the ErbB2/HER2 signaling pathway.

We previously showed that (a) estrogen-related receptor alpha1 (ERRalpha1) down-modulates estrogen receptor (ER)-stimulated transcription in low ErbB2-expressing MCF-7 mammary carcinoma cells, and (b) ERRalpha and ErbB2 mRNA levels positively correlate in clinical breast tumors. We show here that ERRalpha1 represses ERalpha-mediated activation in MCF-7 cells because it failed to recruit the coactivator glucocorticoid receptor interacting protein 1 (GRIP1) when bound to an estrogen response element. In contrast, ERRalpha1 activated estrogen response element- and ERR response element-mediated transcription in ERalpha-positive, high ErbB2-expressing BT-474 mammary carcinoma cells, activation that was enhanced by overexpression of GRIP1. Likewise, regulation of the endogenous genes pS2, progesterone receptor, and ErbB2 by ERRalpha1 reflected the cell type-specific differences observed with our reporter plasmids. Importantly, overexpression of activated ErbB2 in MCF-7 cells led to transcriptional activation, rather than repression, by ERRalpha1. Two-dimensional PAGE of radiophosphate-labeled ERRalpha1 indicated that it was hyperphosphorylated in BT-474 relative to MCF-7 cells; incubation of these cells with anti-ErbB2 antibody led to reduction in the extent of ERRalpha1 phosphorylation. Additionally, mitogen-activated protein kinases (MAPK) and Akts, components of the ErbB2 pathway, phosphorylated ERRalpha1 in vitro. ERRalpha1-activated transcription in BT-474 cells was inhibited by disruption of ErbB2/epidermal growth factor receptor signaling with trastuzumab or gefitinib or inactivation of downstream components of this signaling, MAPK kinase/MAPK, and phosphatidylinositol-3-OH kinase/Akt, with U0126 or LY294002, respectively. Thus, ERRalpha1 activities are regulated, in part, via ErbB2 signaling, with ERRalpha1 likely positively feedback-regulating ErbB2 expression. Taken together, we conclude that ERRalpha1 phosphorylation status shows potential as a biomarker of clinical course and antihormonal- and ErbB2-based treatment options, with ERRalpha1 serving as a novel target for drug development.

ZEB negatively regulates the lytic-switch BZLF1 gene promoter of Epstein-Barr virus.

Epstein-Barr virus (EBV) is a human herpesvirus capable of establishing a latent state in B lymphocytes. The product of the immediate-early BZLF1 gene, Zta, is a transcriptional transactivator essential for viral DNA amplification and virion production. Previously, we identified a negative cis-acting element within the BZLF1 promoter termed ZV. ZV contains the sequence 5'-CAGGTA-3' located at nucleotides -17 to -12 relative to the transcription initiation site. It sequence specifically binds a cellular factor, ZVR. Based on sequence binding specificity, we postulated that ZVR may be zinc finger E-box binding factor (ZEB) or a related zinc finger/homeodomain family member. We show here by immunoshift assays that ZVR and human ZEB specifically cross-react with an antibody to deltaEF1, the chicken homolog of ZEB. Competition electrophoretic mobility shift assays confirmed that ZEB binds to the ZV element with the same binding specificity as ZVR. Overexpression of ZEB in either B-lymphocytic DG75 cells or mammary epithelial MCF-7 cells repressed Zta-induced activation of the BZLF1 promoter four- to fivefold via the ZV site. Thus, we conclude that the previously identified cellular repressor ZVR is, in fact, ZEB. We also present evidence that other cellular factors likely affect the transcriptional activity of ZEB. Lastly, we identify a ZEB-binding site within the promoter of the lytic BRLF1 gene of EBV. We postulate that ZEB likely plays an important role in regulating the life cycle of EBV.

Estrogen-related receptor alpha 1 actively antagonizes estrogen receptor-regulated transcription in MCF-7 mammary cells.

The estrogen-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily. We show that the major isoform of the human ERRalpha gene, ERRalpha1, can sequence-specifically bind a consensus palindromic estrogen response element (ERE) and directly compete with estrogen receptor alpha (ERalpha) for binding. ERRalpha1 activates or represses ERE-regulated transcription in a cell type-dependent manner, repressing in ER-positive MCF-7 cells while activating in ER-negative HeLa cells. Thus, ERRalpha1 can function both as a modulator of estrogen responsiveness and as an estrogen-independent activator. Repression likely occurs in the absence of exogenous ligand since charcoal treatment of the serum had no effect on silencing activity. Mutational analysis revealed that repression is not simply the result of competition between ERalpha and ERRalpha1 for binding to the DNA. Rather, it also requires the presence of sequences within the carboxyl-terminal E/F domain of ERRalpha1. Thus, ERRalpha1 can function as either an active repressor or a constitutive activator of ERE-dependent transcription. We hypothesize that ERRalpha1 can play a critical role in the etiology of some breast cancers, thereby providing a novel therapeutic target in their treatment.