ATF3-Induced Mammary Tumors Exhibit Molecular Features of Human Basal-Like Breast Cancer.

Basal-like breast cancer (BLBC) is an aggressive and deadly subtype of human breast cancer that is highly metastatic, displays stem-cell like features, and has limited treatment options. Therefore, developing and characterizing preclinical mouse models with tumors that resemble BLBC is important for human therapeutic development. ATF3 is a potent oncogene that is aberrantly expressed in most human breast cancers. In the BK5.ATF3 mouse model, overexpression of ATF3 in the basal epithelial cells of the mammary gland produces tumors that are characterized by activation of the Wnt/ß-catenin signaling pathway. Here, we used RNA-Seq and microRNA (miRNA) microarrays to better define the molecular features of BK5.ATF3-derived mammary tumors. These analyses showed that these tumors share many characteristics of human BLBC including reduced expression of Rb1, Esr1, and Pgr and increased expression of Erbb2, Egfr, and the genes encoding keratins 5, 6, and 17. An analysis of miRNA expression revealed reduced levels of Mir145 and Mir143, leading to the upregulation of their target genes including both the pluripotency factors Klf4 and Sox2 as well as the cancer stem-cell-related gene Kras. Finally, we show through knock-down experiments that ATF3 may directly modulate MIR145/143 expression. Taken together, our results indicate that the ATF3 mouse mammary tumor model could provide a powerful model to define the molecular mechanisms leading to BLBC, identify the factors that contribute to its aggressiveness, and, ultimately, discover specific genes and gene networks for therapeutic targeting.

Replication Study: Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Repass et al., 2016), that described how we intended to replicate an experiment from the paper 'Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma' (Castellarin et al., 2012). Here we report the results. When measuring Fusobacterium nucleatum DNA by qPCR in colorectal carcinoma (CRC), adjacent normal tissue, and separate matched control tissue, we did not detect a signal for F. nucleatum in most samples: 25% of CRCs, 15% of adjacent normal, and 0% of matched control tissue were positive based on quantitative PCR (qPCR) and confirmed by sequencing of the qPCR products. When only samples with detectable F. nucleatum in CRC and adjacent normal tissue were compared, the difference was not statistically significant, while the original study reported a statistically significant increase in F. nucleatum expression in CRC compared to adjacent normal tissue (Figure 2; Castellarin et al., 2012). Finally, we report a meta-analysis of the result, which suggests F. nucleatum expression is increased in CRC, but is confounded by the inability to detect F. nucleatum in most samples. The difference in F. nucleatum expression between CRC and adjacent normal tissues was thus smaller than the original study, and not detected in most samples.

Registered report: Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from 'Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma' by Castellarin and colleagues published in Genome Research in 2012 (Castellarin et al., 2012). The experiment to be replicated is reported in Figure 2. Here, Castellarin and colleagues performed a metagenomic analysis of colorectal carcinoma (CRC) to identify potential associations between inflammatory microorganisms and gastrointestinal cancers. They conducted quantitative real-time PCR on genomic DNA isolated from tumor and matched normal biopsies from a patient cohort and found that the overall abundance of Fusobacterium was 415 times greater in CRC versus adjacent normal tissue. These results confirmed earlier studies and provide evidence for a link between tissue-associated bacteria and tumorigenesis. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Proteomic and pathway analyses reveal a network of inflammatory genes associated with differences in skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice.

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with skin tumor promotion susceptibility, a proteomic approach was used to examine and identify proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We identified 19 differentially expressed proteins of which 5 were the calcium-binding proteins annexin A1, parvalbumin α, S100A8, S100A9, and S100A11. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially upregulated in epidermis of DBA/2 versus C57BL/6 mice following topical treatment with two other skin tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that these proteins were components of several networks that included inflammation-associated proteins known to be involved in skin tumor promotion (e.g. TNF-α, NFκB). Follow-up studies revealed that Tnf, Nfkb1, Il22, Il1b, Cxcl1, Cxcl2 and Cxcl5 mRNAs were highly expressed in epidermis of DBA/2 compared with C57BL/6 mice at 24h following treatment with TPA. Furthermore, NFκB (p65) was also highly activated at the same time point (as measured by phosphorylation at ser276) in epidermis of DBA/2 mice compared with C57BL/6 mice. Taken together, the present data suggest that differential expression of genes involved in inflammatory pathways in epidermis may play a key role in genetic differences in susceptibility to skin tumor promotion in DBA/2 and C57BL/6 mice.

Dietary energy balance modulates prostate cancer progression in Hi-Myc mice.

Male Hi-Myc mice were placed on three dietary regimens [30% calorie restriction (CR), overweight control (modified AIN76A with 10 kcal% fat), and a diet-induced obesity regimen (DIO) 60 kcal% fat]. All diet groups had approximately similar incidence of hyperplasia and low-grade prostatic intraepithelial neoplasia in the ventral prostate at 3 and 6 months of age. However, 30% CR significantly reduced the incidence of in situ adenocarcinomas at 3 months compared with the DIO group and at 6 months compared with both the overweight control and DIO groups. Furthermore, the DIO regimen significantly increased the incidence of adenocarcinoma with aggressive stromal invasion, as compared with the overweight control group (96% vs. 65%, respectively; P = 0.02) at the 6-month time point. In addition, at both 3 and 6 months, only in situ carcinomas were observed in mice maintained on the 30% CR diet. Relative to overweight control, DIO increased whereas 30% CR reduced activation of Akt, mTORC1, STAT3, and NFκB (p65) in ventral prostate. DIO also significantly increased (and 30% CR decreased) numbers of T-lymphocytes and macrophages in the ventral prostate compared with overweight control. The mRNA levels for interleukin (IL) 1α, IL1ß, IL6, IL7, IL23, IL27, NFκB1 (p50), TNFα, and VEGF family members were significantly increased in the ventral prostate of the DIO group compared with both the overweight control and 30% CR diet groups. Collectively, these findings suggest that enhanced growth factor (Akt/mTORC1 and STAT3) and inflammatory (NFκB and cytokines) signaling may play a role in dietary energy balance effects on prostate cancer progression in Hi-Myc mice.

IL7-hCD25 and IL7-Cre BAC transgenic mouse lines: new tools for analysis of IL-7 expressing cells.

IL-7 is a cytokine that is required for T-cell development and homeostasis as well as for lymph node organogenesis. Despite the importance of IL-7 in the immune system and its potential therapeutic relevance, questions remain regarding the sites of IL-7 synthesis, specific cell types involved and molecular mechanisms regulating IL-7 expression. To address these issues, we generated two bacterial artificial chromosome (BAC) transgenic mouse lines in which IL-7 regulatory elements drive expression of either Cre recombinase or a human CD25 (hCD25) cell surface reporter molecule. Expression of the IL-7.hCD25 BAC transgene, detected by reactivity with anti-hCD25 antibody, mimicked endogenous IL-7 expression. Fetal and adult tissues from crosses between IL-7.Cre transgenic mice and Rosa26R or R26-EYFP reporters demonstrated X-gal or YFP staining in tissues known to express endogenous IL-7 at some stage during development. These transgenic lines provide novel genetic tools to identify IL-7 producing cells in various tissues and to manipulate gene expression selectively in IL-7 expressing cells.