Epigenetic silencing of CREB3L1 by DNA methylation is associated with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers.

BACKGROUND: CREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the unfolded protein response, has recently been identified as a metastasis suppressor in both breast and bladder cancer. METHODS: Quantitative real time PCR (qPCR) and immunoblotting were used to determine the impact of histone deacetylation and DNA methylation inhibitors on CREB3L1 expression in breast cancer cell lines. Breast cancer cell lines and tumor samples were analyzed similarly, and CREB3L1 gene methylation was determined using sodium bisulfite conversion and DNA sequencing. Immunohistochemistry was used to determine nuclear versus cytoplasmic CREB3L1 protein. Large breast cancer database analyses were carried out to examine relationships between CREB3L1 gene methylation and mRNA expression in addition to CREB3L1 mRNA expression and prognosis. RESULTS: This study demonstrates that the low CREB3L1 expression previously seen in highly metastatic breast cancer cell lines is caused in part by epigenetic silencing. Treatment of several highly metastatic breast cancer cell lines that had low CREB3L1 expression with DNA methyltransferase and histone deacetylase inhibitors induced expression of CREB3L1, both mRNA and protein. In human breast tumors, CREB3L1 mRNA expression was upregulated in low and medium-grade tumors, most frequently of the luminal and HER2 amplified subtypes. In contrast, CREB3L1 expression was repressed in high-grade tumors, and its loss was most frequently associated with triple negative breast cancers (TNBCs). Importantly, bioinformatics analyses of tumor databases support these findings, with methylation of the CREB3L1 gene associated with TNBCs, and strongly negatively correlated with CREB3L1 mRNA expression. Decreased CREB3L1 mRNA expression was associated with increased tumor grade and reduced progression-free survival. An immunohistochemistry analysis revealed that low-grade breast tumors frequently had nuclear CREB3L1 protein, in contrast to the high-grade breast tumors in which CREB3L1 was cytoplasmic, suggesting that differential localization may also regulate CREB3L1 effectiveness in metastasis suppression. CONCLUSIONS: Our data further strengthens the role for CREB3L1 as a metastasis suppressor in breast cancer and demonstrates that epigenetic silencing is a major regulator of the loss of CREB3L1 expression. We also highlight that CREB3L1 expression is frequently altered in many cancer types suggesting that it could have a broader role in cancer progression and metastasis.

Potentially pathogenic bacteria and antimicrobial resistance in bioaerosols from cage-housed and floor-housed poultry operations.

BACKGROUND: Antibiotics are used in animal confinement buildings, such as cage-housed (CH) and floor-housed (FH) poultry operations, to lower the likeliness of disease transmission. In FH facilities, antibiotics may also be used at sub-therapeutic levels for growth promotion. Low levels of antibiotic create a selective pressure toward antimicrobial resistance (AMR) in chicken fecal bacteria. OBJECTIVE: The objective of this study was to compare bacteria and AMR genes in bioaerosols from CH and FH poultry facilities. METHODS: Bioaerosols were collected from 15 CH and 15 FH poultry operations, using stationary area samplers as well as personal sampling devices. Bacteria concentrations were determined by genus- or species-specific quantitative polymerase chain reaction (PCR) and AMR genes were detected using endpoint PCR. RESULTS: Enterococcus spp., Escherichia coli, and Staphylococcus spp. were significantly higher in bioaerosols of FH poultry operations than CH bioaerosols (P < 0.001) while Clostridium perfringens was significantly higher in area bioaerosols of CH operations than FH area bioaerosols (P < 0.05). Campylobacter spp. were detected only in bioaerosols of FH facilities. Zinc bacitracin resistance gene, bcrR, erythromycin resistance gene, ermA, and tetracycline resistance gene, tetA/C, were more prevalent in bioaerosols of FH facilities than CH bioaerosols (P < 0.01, P < 0.01, and P < 0.05, respectively). CONCLUSIONS: Most bacteria are more concentrated and most AMR genes are more prevalent in bioaerosols of FH poultry operations, where growth-promoting antibiotics may be used.