Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

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Breast cancer is one of the leading causes of cancer deaths among females. Many challenges exist in the current management of advanced stage breast cancer as there are fewer recognized therapeutic strategies, often due to therapy resistance. How breast cancer cells evade chemotherapy and the underlying mechanism remains unclear. We and others have observed that malignant cells that survive initial chemo-and radiation therapy express higher levels of CXCR2 ligands which might provide a survival benefit leading to therapy resistance. In this report, we test the hypothesis that CXCR2-dependent signaling in malignant cells might be critical for chemotherapy resistance and targeting this signaling axis may enhance the antitumor and antimetastatic activity of chemotherapeutic drugs and limit their toxicity. We used Cl66-wt, 4T1-wt, Cl66sh-CXCR2 and 4T1sh-CXCR2 cells expressing differential levels of the CXCR2 receptor to evaluate the role of targeting CXCR2 on chemotherapeutic responses. Knockdown of CXCR2 enhances paclitaxel and doxorubicin mediated toxicity at suboptimal doses. Moreover, we observed an increase in the expression of CXCL1, a CXCR2 ligand in paclitaxel and doxorubicin treated mammary tumor cells which were inhibited following CXCR2 knockdown. Knockdown of CXCR2 enhanced antitumor activity of paclitaxel in an in vivo mammary tumor model. We observed significant inhibition of spontaneous lung metastases in animals bearing CXCR2 knockdown tumors and treated with paclitaxel as compared to the control group. Our data suggest the novel role of CXCR2 and its ligands in maintaining chemotherapy resistance and provide evidence that targeting CXCR2-signaling in an adjuvant setting will help circumvent chemotherapy resistance.

Epstein-Barr virus (EBV) maintains a lifelong latent infection within a subset of its host IMPORTANCEThis study is the first one to show that the cellular transcription factor BLIMP1, a key player in both epithelial and B-cell differentiation, induces reactivation of the oncogenic herpesvirus Epstein-Barr virus (EBV) out of latency into lytic replication in a variety of cancerous epithelial cell types as well as in some, but not all, B-cell types that contain this virus in a dormant state. The mechanism by which BLIMP1 does so involves strongly turning on expression of both of the immediate early genes of the virus, probably by directly acting upon the promoters as part of protein complexes or indirectly by altering the expression or activities of some cellular transcription factors and signaling pathways. The fact that EBV ؉ cancers usually contain mostly undifferentiated cells may be due in part to these cells dying from lytic EBV infection when they differentiate and express wild-type BLIMP1.

Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors that have latent infection, such as nasopharyngeal carcinoma (NPC), and oral hairy leukoplakia (OHL) lesions that have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically infected cells, LMP1 expression is activated by the BRLF1 (R) immediate early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors KLF4 and BLIMP1 induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we found that differentiation of NOKs-Akata cells by either methylcellulose suspension or organotypic culture induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic infection-inducing effects of epithelial cell differentiation, as well as 12--tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression and then cooperate with LMP1 to activate Z and R expression. The EBV protein LMP1 is expressed in EBV-associated epithelial cell diseases, regardless of whether these diseases are due to lytic infection (such as oral hairy leukoplakia) or latent infection (such as nasopharyngeal carcinoma). However, surprisingly little is known about how LMP1 expression is regulated in epithelial cells, and there are conflicting reports about whether it plays any role in regulating viral lytic reactivation. In this study, we show that epithelial cell differentiation induces LMP1 expression by increasing expression of two cellular transcription factors (KLF4 and BLIMP1) which cooperatively activate the two LMP1 promoters. We also demonstrate that LMP1 promotes efficient lytic reactivation in EBV-infected epithelial cells by enhancing expression of the Z and R proteins. Thus, in EBV-infected epithelial cells, LMP1 expression is promoted by differentiation and positively regulates lytic viral reactivation.