Quantification of Epithelial Cell Differentiation in Mammary Glands and Carcinomas from DMBA- and MNU-Exposed Rats

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin β1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin β3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer

Epstein-Barr Virus Infection Promotes Epithelial Cell Growth by Attenuating Differentiation-Dependent Exit from the Cell Cycle

Latent infection by Epstein-Barr virus (EBV) is an early event in the development of EBV-associated carcinomas. In oral epithelial tissues, EBV establishes a lytic infection of differentiated epithelial cells to facilitate the spread of the virus to new hosts. Because of limitations in existing model systems, the effects of latent EBV infection on undifferentiated and differentiating epithelial cells are poorly understood. Here, we characterize latent infection of an hTERT-immortalized oral epithelial cell line (NOKs). We find that although EBV expresses a latency pattern similar to that seen in EBV-associated carcinomas, infection of undifferentiated NOKs results in differential expression of a small number of host genes. In differentiating NOKs, however, EBV has a more substantial effect, reducing the extent of differentiation and delaying the exit from the cell cycle. This effect may synergize with preexisting cellular abnormalities to prevent exit from the cell cycle, representing a critical step in the development of cancer.Epstein-Barr virus (EBV) is a human herpesvirus that is associated with lymphomas as well as nasopharyngeal and gastric carcinomas. Although carcinomas account for almost 90% of EBV-associated cancers, progress in examining EBV’s role in their pathogenesis has been limited by difficulty in establishing latent infection in nontransformed epithelial cells. Recently, EBV infection of human telomerase reverse transcriptase (hTERT)-immortalized normal oral keratinocytes (NOKs) has emerged as a model that recapitulates aspects of EBV infection in vivo, such as differentiation-associated viral replication. Using uninfected NOKs and NOKs infected with the Akata strain of EBV (NOKs-Akata), we examined changes in gene expression due to EBV infection and differentiation. Latent EBV infection produced very few significant gene expression changes in undifferentiated NOKs but significantly reduced the extent of differentiation-induced gene expression changes. Gene set enrichment analysis revealed that differentiation-induced downregulation of the cell cycle and metabolism pathways was markedly attenuated in NOKs-Akata relative to that in uninfected NOKs. We also observed that pathways induced by differentiation were less upregulated in NOKs-Akata. We observed decreased differentiation markers and increased suprabasal MCM7 expression in NOKs-Akata versus NOKs when both were grown in raft cultures, consistent with our transcriptome sequencing (RNA-seq) results. These effects were also observed in NOKs infected with a replication-defective EBV mutant (AkataΔRZ), implicating mechanisms other than lytic-gene-induced host shutoff. Our results help to define the mechanisms by which EBV infection alters keratinocyte differentiation and provide a basis for understanding the role of EBV in epithelial cancers

An EBNA3C-deleted Epstein-Barr virus (EBV) mutant causes B-cell lymphomas with delayed onset in a cord blood-humanized mouse model.

EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. While EBNA3C undoubtedly plays a key role in allowing EBV to successfully infect B cells, many EBV+ lymphomas do not express this protein, suggesting that cellular mutations and/or signaling pathways may obviate the need for EBNA3C in vivo under certain conditions. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (Δ3C EBV) in a cord blood-humanized mouse model (CBH). We found that the Δ3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and Δ3C viruses induced B-cell lymphomas with restricted B-cell populations and heterogeneous T-cell infiltration. In comparison to WT-infected tumors, Δ3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that Δ3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti-apoptotic proteins, BCL2 and IRF4, were expressed in Δ3C-infected tumors, likely helping cells avoid c-Myc-induced apoptosis. Unexpectedly, Δ3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo

Characterization of rat mammary epithelial cells (RMECs) based on cell surface and intracellular markers.

<p>(<b>A</b>) Representative flow cytometric histograms and dot plots showing gating for propidium iodide (PI)-negative (live) cells (left panel); exclusion of endothelial cells and leukocytes based on CD31 and CD45 expression, respectively (middle left panel); CD61 expression in CD45–CD31– RMECs (middle right panel); CD24 and CD29 expression in CD45–CD31– RMECs identifies two major populations indicated with a red or blue circle (right panel). (<b>B</b>) Dot plots of intracellular cytokeratin (CK) 14 and CK19 expression in CD45–CD31– RMECs (upper left panel); intracellular smooth muscle actin (SMA) staining with phalloidin and CD29 expression in CD45–CD31– RMECs (upper right panel); overlay of dot plots showing CD24 and CD29 expression in CK14+CK19- cells and CK19+CK14- cells (lower left panel); overlay of dot plots of phalloidin bright cells on CD24 and CD29 expression in CD45–CD31– RMECs (lower right panel). Based on CK14, CK19, and SMA expression, the luminal (red) and basal (blue) populations in CD45–CD31– RMECs are identified. (<b>C</b>) Contour plot showing binding of Peanut Lectin (PNL) or anti-Thy-1 in CD45–CD31– RMECs (left panel), overlaid histograms showing CD29 expression on PNL+Thy1-, PNL-Thy-1+ cells (middle left panel), contour plots showing anti-Thy-1 (middle right panel) or PNL binding in CD29med or CD29hi cells (right panel). For all panels, rats of 12 weeks of age were used.</p

Features of the actively dividing cells in CD45–CD31– RMECs.

<p>(<b>A</b>) Flow cytometric histogram showing gating for actively dividing cells (cells in S/G2+M phase of the cell cycle) by having >2n cellular DNA content (left panel); representative dot plot showing the actively dividing cells overlaid on CD24 and CD29 expression in the RMECs (right panel). (<b>B</b>) Representative dot plot showing gating for RMECs expressing high levels of both CD24 and CD29 (CD24hiCD29hi gate; left panel); bar graph (right panel) showing mean ± sem percentage of cells in S/G2+M phase of the cell cycle in total RMECs or CD24hiCD29hi-gated cells (n = 24 each). A significant enrichment of actively dividing cells was detected in the CD24hiCD29hi-gated cells (p<0.05; indicated with an asterisk). (<b>C</b>) Overlaid histogram showing CD49f expression in the total RMECs and actively dividing cells (left panel); bar graph (right panel) showing mean fluorescence intensity (MFI) in artificial units (a.u.) ± sem of CD49f in the total RMECs and actively dividing cells (n = 14 each). Significantly different MFI is indicated with an asterisk (p<0.05). (<b>D</b>) Representative dot plot showing CD49f expressing cells (CD49f+) overlaid on CD24 and CD29 expression of the RMECs (left panel); bar graph showing mean ± sem percentage of CD24hiCD29hi-gated cells in total RMECs or the CD49f expressing population (right panel). A significant enrichment of CD24hiCD29hi-gated cells was detected in the CD49f expressing population (p<0.05; indicated with an asterisk). For all panels, rats of 12 weeks of age were used.</p

Epithelial cell differentiation in mammary glands and carcinomas from 7,12-dimethylbenz(a)anthracene (DMBA)-or <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU)-exposed rats.

<p>(<b>A</b>) Schematic representation of the modulation of RMEC differentiation 1 week after exposure of rats to the mammary carcinogens DMBA or MNU. DMBA exposure increases CD49f expression and proliferation (not shown here). MNU exposure disrupts the luminal and basal homeostasis. (<b>B</b>) Schematic representation of the changes of RMEC differentiation in carcinomas as compared to mammary gland from untreated age-matched (22 weeks of age) control rats. Note that the RMECs from animals of 22 weeks of age have a higher percentage of luminal cells as compared to younger animals (comparing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g001" target="_blank">Fig. 1A</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g004" target="_blank">Fig. 4A</a>). In both panels cell surface expression of CD24, CD29, CD49f and CD61 and intracellular expression of focal adhesion kinase (FAK) and Y397-phosphorylated FAK (pFAK) in basal, luminal and CD24hiCD29hi-gated cells are shown.</p

Differences between the RMECs from mammary glands of untreated control rats and mammary carcinomas from rats exposed to 7,12-dimethylbenz(a)anthracene (DMBA) or <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU).

<p>(<b>A</b>) Representative pseudo-color dot plots showing CD24 and CD29 expression in the RMECs from the mammary gland of an age-matched (22 weeks of age) untreated control rat (upper left panel) and a DMBA- (upper middle panel) or MNU-induced (upper right panel) carcinoma; bar graphs (lower panel) quantifying mean ± sem percentage cells in the CD24hiD29hi gate within the total (CD45–CD31–) RMECs. A significantly different percentage comparing carcinomas to mammary glands is indicated with an asterisk (p<0.05). (<b>B</b>) Bar graphs showing the mean ± sem percentage of RMECs containing >2n cellular DNA (actively dividing cells in S/G2+M phase of cell cycle). Significantly different percentage comparing RMECs from carcinomas to control mammary glands is indicated with an asterisk (p<0.05). (<b>C</b>) Representative overlaid histograms showing upregulation of CD29 expression (upper left panel), upregulation of CD49f expression (upper middle panel) and downregulation of CD61 expression (upper right panel) in RMECs of a DMBA-induced or MNU-induced carcinoma as compared to a control mammary gland; bar graphs quantifying the mean fluorescence intensity (MFI) in artificial units (a.u.) ± sem of CD29 (lower left panel), CD49f (lower middle panel) and CD61 (lower right panel) on RMECs from control mammary glands and carcinomas. Significantly different MFI is indicated with an asterisk (p<0.05). (<b>D</b>) Representative pseudo-color dot plot showing gating for CD29 and focal adhesion kinase (FAK) in RMECs from a control mammary gland (upper left panel), a DMBA-induced (upper middle panel) and MNU-induced (upper right panel) carcinoma; bar graph (lower panel) quantifying mean ± sem percentage of CD29hiFAK+ cells. A significantly different percentage comparing carcinomas to control mammary glands is indicated with an asterisk (p<0.05). (<b>E</b>) Representative pseudo-color dot plot showing gating for CD29 and Y397-phosphorylated focal adhesion kinase (pFAK) in RMECs from a control mammary gland (upper left panel), a DMBA-induced (upper middle panel) and MNU-induced (upper right panel) carcinoma; bar graph (lower panel) quantifying mean ± sem percentage of CD29hi pFAK+ cells. A significantly different percentage comparing carcinomas to control mammary glands is indicated with an asterisk (p<0.05). In the entire <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g004" target="_blank">figure 4</a>, age-matched untreated control mammary glands: n = 16, DMBA-induced mammary carcinomas: n = 10 and MNU-induced mammary carcinomas: n = 10.</p

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells.

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV