Search

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/β-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.

Since disease susceptibility of the intestine exhibits an anatomical bias, we propose that the chromatin landscape, especially the site-specific epigenetic differences in histone modification patterns throughout the colonic longitudinal axis, contributes to the differential incidence of site-specific pathology. To test this hypothesis, we assessed the chromatin structure associated with gene expression profiles in the rat proximal and distal colon by globally correlating chromatin immunoprecipitation next-generation sequencing analysis (ChIP-Seq) with mRNA transcription (RNA-Seq) data. Crypts were isolated from the proximal and distal colonic regions from rats maintained on a semipurified diet, and mRNA gene expression profiles were generated by RNA-Seq. The remaining isolated crypts were formaldehyde-cross-linked and chromatin immunoprecipitated with antibodies against H3K4me3, H3K9me3, and RNA polymerase II. Globally, RNA-Seq results indicate that 9,866 genes were actively expressed, of which 540 genes were differentially expressed between the proximal and distal colon. Gene ontology analysis indicates that crypt location significantly affected both chromatin and transcriptional regulation of genes involved in enterocyte movement, lipid metabolism, lymphatic development, and immune cell trafficking. Gene function analysis indicates that the PI3-kinase signaling pathway was regulated in a site-specific manner, e.g., proto-oncogenes, JUN, FOS, and ATF, were upregulated in the distal colon. Middle and long noncoding RNAs (lncRNAs) were also detected in the colon, including select lncRNAs formerly only detected in the rat nervous system. In summary, distinct combinatorial patterns of histone modifications exist in the proximal versus distal colon. These site-specific differences may explain the differential effects of chemoprotective agents on cell transformation in the ascending (proximal) and descending (distal) colon.

DNA can adopt a variety of alternative secondary (i.e., non-B DNA) conformations that play important roles in cellular metabolism, including genetic instability, disease etiology, and evolution. While we still have much to learn, research in this field has expanded dramatically in the past decade. We have summarized in our previous Methods review (Wang et al., Methods, 2009) some commonly used techniques to determine non-B DNA structural conformations and non-B DNA-induced genetic instability in prokaryotes and eukaryotes. Since that time, we and others have further characterized mechanisms involved in DNA structure-induced mutagenesis and have proposed both replication-dependent and replication-independent models. Thus, in this review, we highlight some current methodologies to identify DNA replication-related and replication-independent mutations occurring at non-B DNA regions to allow for a better understanding of the mechanisms underlying DNA structure-induced genetic instability. We also describe a new web-based search engine to identify potential intramolecular triplex (H-DNA) and left-handed Z-DNA-forming motifs in entire genomes or at selected sequences of interest.

Genetically engineered mouse cancer models are among the most useful tools for testing the in vivo effectiveness of the various chemopreventive approaches. The p53-null mouse model of mammary carcinogenesis was previously characterized by us at the cellular, molecular, and pathologic levels. In a companion article, Medina et al. analyzed the efficacy of bexarotene, gefitinib, and celecoxib as chemopreventive agents in the same model. Here we report the global gene expression effects on mammary epithelium of such compounds, analyzing the data in light of their effectiveness as chemopreventive agents. SAGE was used to profile the transcriptome of p53-null mammary epithelium obtained from mice treated with each compound versus controls. This information was also compared with SAGE data from p53-null mouse mammary tumors. Gene expression changes induced by the chemopreventive treatments revealed a common core of 87 affected genes across treatments (P < 0.05). The effective compounds, bexarotene and gefitinib, may exert their chemopreventive activity, at least in part, by affecting a set of 34 genes related to specific cellular pathways. The gene expression signature revealed various genes previously described to be associated with breast cancer, such as the activator protein-1 complex member Fos-like antigen 2 (Fosl2), early growth response 1 (Egr1), gelsolin (Gsn), and tumor protein translationally controlled 1 (Tpt1), among others. The concerted modulation of many of these transcripts before malignant transformation seems to be conducive to predominantly decrease cell proliferation. This study has revealed candidate key pathways that can be experimentally tested in the same model system and may constitute novel targets for future translational research.

Global gene expression measured by DNA microarray platforms have been extensively used to classify breast carcinomas correlating with clinical characteristics, including outcome. We generated a breast cancer Serial Analysis of Gene Expression (SAGE) high-resolution database of ∼2.7 million tags to perform unsupervised statistical analyses to obtain the molecular classification of breast-invasive ductal carcinomas in correlation with clinicopathologic features. Unsupervised statistical analysis by means of a random forest approach identified two main clusters of breast carcinomas, which differed in their lymph node status (P = 0.01); this suggested that lymph node status leads to globally distinct expression profiles. A total of 245 (55 up-modulated and 190 down-modulated) transcripts were differentially expressed between lymph node (+) and lymph node (−) primary breast tumors (fold change, ≥2; P < 0.05). Various lymph node (+) up-modulated transcripts were validated in independent sets of human breast tumors by means of real-time reverse transcription-PCR (RT-PCR). We validated significant overexpression of transcripts for HOXC10 (P = 0.001), TPD52L1 (P = 0.007), ZFP36L1 (P = 0.011), PLINP1 (P = 0.013), DCTN3 (P = 0.025), DEK (P = 0.031), and CSNK1D (P = 0.04) in lymph node (+) breast carcinomas. Moreover, the DCTN3 (P = 0.022) and RHBDD2 (P = 0.002) transcripts were confirmed to be overexpressed in tumors that recurred within 6 years of follow-up by real-time RT-PCR. In addition, meta-analysis was used to compare SAGE data associated with lymph node (+) status with publicly available breast cancer DNA microarray data sets. We have generated evidence indicating that the pattern of gene expression in primary breast cancers at the time of surgical removal could discriminate those tumors with lymph node metastatic involvement using SAGE to identify specific transcripts that behave as predictors of recurrence as well. (Mol Cancer Res 2007;5(9):881–90)

Microarray technologies have provided the ability to monitor the expression of whole genomes rapidly. However, concerns persist with regard to quantitation and reproducibility, and the detection limits for individual genes in particular arrays are generally unknown. This article describes a semiautomated PCR-based technology, Q-RAGE, which rapidly provides measurements of mRNA abundance with extremely high sensitivity using fluorescent detection of specific products separated by capillary electrophoresis. A linear relationship between template concentration and fluorescent signal can be demonstrated down to template concentrations in the low aM region, corresponding to approximately 0.04 zmol (24 molecules) per reaction. The technique is shown to be quantitative over five orders of magnitude of template concentration, and average mRNA abundances of approximately 0.01 molecule per cell can be detected. A single predefined set of 320 primers provides 90–95% coverage of all eukaryotic genomes. Analysis of a set of 19 p53-regulated genes in untreated cultures of normal human epithelial cells, derived from three different tissues, revealed a 600-fold range of apparent constitutive expression levels. For most of the genes assayed, good correlations were observed among the expression levels in normal mammary, bronchial, and epidermal epithelial cells.

Much evidence has accumulated implicating the p53 gene as of importance in breast carcinogenesis. However, much still remains to be uncovered on the specific downstream pathways influenced by this important activator/repressor of transcription. This study investigated the effects of a p53 null genotype on the transcriptome of 'normal' mouse mammary epithelium using a unique in vivo model of preneoplastic transformation. We used SAGE for the comparative analysis of p53 wild type (wt) and null mammary epithelium unexposed and exposed to hormonal stimulation. Analysis of the hormone exposed samples provided a comprehensive view of the dramatic changes in gene expression as consequence of the functional differentiation of the mammary epithelium in an in vivo system. We detected the dysregulation in p53(null) epithelium of

Background Estrogens are known to regulate the proliferation of breast cancer cells and to modify their phenotypic properties. Identification of estrogen-regulated genes in human breast tumors is an essential step toward understanding the molecular mechanisms of estrogen action in cancer. To this end we generated and compared the Serial Analysis of Gene Expression (SAGE) profiles of 26 human breast carcinomas based on their estrogen receptor α (ER) status. Thus, producing a breast cancer SAGE database of almost 2.5 million tags, representing over 50,000 transcripts. Results We identified 520 transcripts differentially expressed between ERα-positive (+) and ERα-negative (-) primary breast tumors (Fold change ≥ 2; p < 0.05). Furthermore, we identified 220 high-affinity Estrogen Responsive Elements (EREs) distributed on the promoter regions of 163 out of the 473 up-modulated genes in ERα (+) breast tumors. In brief, we observed predominantly up-regulation of cell growth related genes, DNA binding and transcription factor activity related genes based on Gene Ontology (GO) biological functional annotation. GO terms over-representation analysis showed a statistically significant enrichment of various transcript families including: metal ion binding related transcripts (p = 0.011), calcium ion binding related transcripts (p = 0.033) and steroid hormone receptor activity related transcripts (p = 0.031). SAGE data associated with ERα status was compared with reported information from breast cancer DNA microarrays studies. A significant proportion of ERα associated gene expression changes was validated by this cross-platform comparison. However, our SAGE study also identified novel sets of genes as highly expressed in ERα (+) invasive breast tumors not previously reported. These observations were further validated in an independent set of human breast tumors by means of real time RT-PCR. Conclusion The integration of the breast cancer comparative transcriptome analysis based on ERα status coupled to the genome-wide identification of high-affinity EREs and GO over-representation analysis, provide useful information for validation and discovery of signaling networks related to estrogen response in this malignancy.

Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV-induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH-1 mice epidermis with that of UV-induced squamous cell carcinomas (SCCs) from SKH-1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase-14 (reduced 8.5-fold in SCCs); cathepsins D and S (reduced 3-fold and increased 11.3-fold, respectively, in SCCs); decorin, glutathione S-transferase omega-1, hypoxia-inducible factor 1 alpha, insulin-like growth factor binding protein-7, and matrix metalloproteinase-13 (increased 18-, 12-, 12-, 18.3-, and 11-folds, respectively, in SCCs). Chemokine (C-C motif), ligand 27 (CCL27), which was found downregulated 12.7-fold in SCCs by SAGE, was also observed to be strongly downregulated 6-24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV-irradiated versus sham-treated SKH-1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV-treated skin and SCCs included keratins 6 and 16, small proline-rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.

The responses of a line of normal human mammary epithelial cells, HME87, to treatment with the ultimate carcinogen benzo[a]pyrene diol epoxide (BPDE) were analyzed using a directed gene expression analysis technique, RAGE. Under conditions where cell number was decreased by 50% 24 or 48 h post-treatment, flow cytometry demonstrated no establishment of a G(1)/S arrest nor induction of apoptosis; cells continued to enter S phase from G(1) for at least 24 h but were blocked at G(2)/M. Using the RAGE technique, changes in gene expression were assayed for over 1000 genes, and multiple time-point data were collected for approximately 90 genes. In accord with the cell cycle studies, expression of the p21-WAF1 gene, the major mediator of p53-dependent G(1)/S arrest, did not increase until 24 h post-treatment. The expression of other target genes for transactivation by p53 was increased at early time points, including GADD45, an effector of the G(2)/M checkpoint, and WIP1. Analyses of proteins in treated cells indicated that p53 was phosphorylated at Ser15 but not at Ser20 within 30 min of treatment, and this correlated with an increase in the total amount of p53 protein. Significant expression changes were noted in a number of transcription factor genes, including ATF3 and E2A, genes that have not been previously connected to a response to DNA damage involving bulky chemical adducts. In addition, expression of the XPC gene was induced by BPDE treatment; the XPC product is thought to be involved in recognition of DNA damage by the nucleotide excision repair system.