TATA-binding associated factors have distinct roles during early mammalian development.

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.

Induced mammary cancer in rat models: pathogenesis, genetics, and relevance to female breast cancer.

Mammary cancer, or breast cancer in women, is a polygenic disease with a complex etiopathogenesis. While much remains elusive regarding its origin, it is well established that chemical carcinogens and endogenous estrogens contribute significantly to the initiation and progression of this disease. Rats have been useful models to study induced mammary cancer. They develop mammary tumors with comparable histopathology to humans and exhibit differences in resistance or susceptibility to mammary cancer depending on strain. While some rat strains (e.g., Sprague-Dawley) readily form mammary tumors following treatment with the chemical carcinogen, 7,12-dimethylbenz[a]-anthracene (DMBA), other strains (e.g., Copenhagen) are resistant to DMBA-induced mammary carcinogenesis. Genetic linkage in inbred strains has identified strain-specific quantitative trait loci (QTLs) affecting mammary tumors, via mechanisms that act together to promote or attenuate, and include 24 QTLs controlling the outcome of chemical induction, 10 QTLs controlling the outcome of estrogen induction, and 4 QTLs controlling the outcome of irradiation induction. Moreover, and based on shared factors affecting mammary cancer etiopathogenesis between rats and humans, including orthologous risk regions between both species, rats have served as useful models for identifying methods for breast cancer prediction and treatment. These studies in rats, combined with alternative animal models that more closely mimic advanced stages of breast cancer and/or human lifestyles, will further improve our understanding of this complex disease.

Interindividual variation contributes to differential PCB 126 induced gene expression in primary breast epithelial cells and tissues.

PCB 126 is a pervasive, dioxin-like chemical pollutant which can activate the aryl hydrocarbon receptor (AhR). Despite being banned from the market, PCB 126 can be detected in breast milk to this day. The extent to which interindividual variation impacts the adverse responses to this chemical in the breast tissue remains unclear. This study aimed to investigate the impact of 3 nM PCB 126 on gene expression in a panel of genetically diverse benign human breast epithelial cell (HBEC) cultures and patient derived breast tissues. Six patient derived HBEC cultures were treated with 3 nM PCB 126. RNAseq was used to interrogate the impact of exposure on differential gene expression. Gene expression changes from the top critical pathways were confirmed via qRT-PCR in a larger panel of benign patient derived HBEC cultures, as well as in patient-derived breast tissue explant cultures. RNAseq analysis of HBEC cultures revealed a signature of 144 genes significantly altered by 3 nM PCB 126 treatment. Confirmation of 8 targets using a panel of 12 HBEC cultures and commercially available breast cell lines demonstrated that while the induction of canonical downstream target gene, CYP1A1, was consistent across our primary HBECs, other genes including AREG, S100A8, IL1A, IL1B, MMP7, and CCL28 exhibited significant variability across individuals. The dependence on the activity of the aryl hydrocarbon receptor was confirmed using inhibitors. PCB 126 can induce significant and consistent changes in gene expression associated with xenobiotic metabolism in benign breast epithelial cells. Although the induction of most genes was reliant on the AhR, significant variability was noted between genes and individuals. These data suggest that there is a bifurcation of the pathway following AhR activation that contributes to the variation in interindividual responses.

Genetic variation in sensitivity to estrogens and breast cancer risk.

Breast cancer risk is intimately intertwined with exposure to estrogens. While more than 160 breast cancer risk loci have been identified in humans, genetic interactions with estrogen exposure remain to be established. Strains of rodents exhibit striking differences in their responses to endogenous ovarian estrogens (primarily 17ß-estradiol). Similar genetic variation has been observed for synthetic estrogen agonists (ethinyl estradiol) and environmental chemicals that mimic the actions of estrogens (xenoestrogens). This review of literature highlights the extent of variation in responses to estrogens among strains of rodents and compiles the genetic loci underlying pathogenic effects of excessive estrogen signaling. Genetic linkage studies have identified a total of the 35 quantitative trait loci (QTL) affecting responses to 17ß-estradiol or diethylstilbestrol in five different tissues. However, the QTL appear to act in a tissue-specific manner with 9 QTL affecting the incidence or latency of mammary tumors induced by 17ß-estradiol or diethylstilbestrol. Mammary gland development during puberty is also exquisitely sensitive to the actions of endogenous estrogens. Analysis of mammary ductal growth and branching in 43 strains of inbred mice identified 20 QTL. Regions in the human genome orthologous to the mammary development QTL harbor loci associated with breast cancer risk or mammographic density. The data demonstrate extensive genetic variation in regulation of estrogen signaling in rodent mammary tissues that alters susceptibility to tumors. Genetic variants in these pathways may identify a subset of women who are especially sensitive to either endogenous estrogens or environmental xenoestrogens and render them at increased risk of breast cancer.

Genetic modifiers regulating DNA replication and double-strand break repair are associated with differences in mammary tumors in mouse models of Li-Fraumeni syndrome.

Breast cancer is the most common tumor among women with inherited variants in the TP53 tumor suppressor, but onset varies widely suggesting interactions with genetic or environmental factors. Rodent models haploinsufficent for Trp53 also develop a wide variety of malignancies associated with Li-Fraumeni syndrome, but BALB/c mice are uniquely susceptible to mammary tumors and is genetically linked to the Suprmam1 locus on chromosome 7. To define mechanisms that interact with deficiencies in p53 to alter susceptibility to mammary tumors, we fine mapped the Suprmam1 locus in females from an N2 backcross of BALB/cMed and C57BL/6J mice. A major modifier was localized within a 10 cM interval on chromosome 7. The effect of the locus on DNA damage responses was examined in the parental strains and mice that are congenic for C57BL/6J alleles on the BALB/cMed background (SM1-Trp53+/-). The mammary epithelium of C57BL/6J-Trp53+/- females exhibited little radiation-induced apoptosis compared to BALB/cMed-Trp53+/- and SM1-Trp53+/- females indicating that the Suprmam1B6/B6 alleles could not rescue repair of radiation-induced DNA double-strand breaks mostly relying on non-homologous end joining. In contrast, the Suprmam1B6/B6 alleles in SM1-Trp53+/- mice were sufficient to confer the C57BL/6J-Trp53+/- phenotypes in homology-directed repair and replication fork progression. The Suprmam1B6/B6 alleles in SM1-Trp53+/- mice appear to act in trans to regulate a panel of DNA repair and replication genes which lie outside the locus.

Effects of Benzophenone-3 and Propylparaben on Estrogen Receptor-Dependent R-Loops and DNA Damage in Breast Epithelial Cells and Mice.

BACKGROUND: Endocrine-disrupting chemicals have been shown to have broad effects on development, but their mutagenic actions that can lead to cancer have been less clearly demonstrated. Physiological levels of estrogen have been shown to stimulate DNA damage in breast epithelial cells through mechanisms mediated by estrogen-receptor alpha (ERα). Benzophenone-3 (BP-3) and propylparaben (PP) are xenoestrogens found in the urine of >96% of U.S. OBJECTIVES: We investigated the effect of BP-3 and PP on estrogen receptor-dependent transactivation and DNA damage at concentrations relevant to exposures in humans. METHODS: In human breast epithelial cells, DNA damage following treatment with 17ß-estradiol (E2), BP-3, and PP was determined by immunostaining with antibodies against γ-H2AX and 53BP1. Estrogenic responses were determined using luciferase reporter assays and gene expression. Formation of R-loops was determined with DNA: RNA hybrid-specific S9.6 antibody. Short-term exposure to the chemicals was also studied in ovariectomized mice. Immunostaining of mouse mammary epithelium was performed to quantify R-loops and DNA damage in vivo. RESULTS: Concentrations of 1µM and 5µM BP-3 or PP increased DNA damage similar to that of E2 treatment in a ERα-dependent manner. However, BP-3 and PP had limited transactivation of target genes at 1µM and 5µM concentrations. BP-3 and PP exposure caused R-loop formation in a normal human breast epithelial cell line when ERα was introduced. R-loops and DNA damage were also detected in mammary epithelial cells of mice treated with BP-3 and PP. CONCLUSIONS: Acute exposure to xenoestrogens (PP and BP-3) in mice induce DNA damage mediated by formation of ERα-dependent R-loops at concentrations 10-fold lower than those required for transactivation. Exposure to these xenoestrogens may cause deleterious estrogenic responses, such as DNA damage, in susceptible individuals. https://doi.org/10.1289/EHP5221.

Mucins in lung cancer: diagnostic, prognostic, and therapeutic implications.

Aberrant expression of mucins is associated with cancer development and metastasis. An overexpression of few mucins contributes to oncogenesis by enhancing cancer cell growth and providing constitutive survival signals. This review focuses on the importance of mucins both in the normal bronchial epithelial cells and the malignant tumors of the lung and their contribution in the diagnosis and prognosis of lung cancer patients. During lung cancer progression, mucins either alone or through their interaction with many receptor tyrosine kinases mediate cell signals for growth and survival of cancer cells. Also, stage-specific expression of certain mucins, like MUC1, is associated with poor prognosis from lung cancer. Thus, mucins are emerging as attractive targets for developing novel therapeutic approaches for lung cancer. Several strategies targeting mucin expression and function are currently being investigated to control lung cancer progression.

Pathobiological implications of MUC4 in non-small-cell lung cancer.

INTRODUCTION: Altered expression of MUC4 plays an oncogenic role in various cancers, including pancreatic, ovarian, and breast. This study evaluates the expression and role of MUC4 in non-small-cell lung cancer (NSCLC). METHODS: We used a paired system of MUC4-expressing (H292) and MUC4-nonexpressing (A549) NSCLC cell lines to analyze MUC4-dependent changes in growth rate, migration, and invasion using these sublines. We also evaluated the alterations of several tumor suppressor, proliferation, and metastasis markers with altered MUC4 expression. Furthermore, the association of MUC4 expression (by immunohistochemistry) in lung cancer samples with patient survival was evaluated. RESULTS: MUC4-expressing lung cancer cells demonstrated a less proliferative and metastatic phenotype. Up-regulation of p53 in MUC4-expressing lung cancer cells led to the accumulation of cells at the G2/M phase of cell cycle progression. MUC4 expression attenuated Akt activation and decreased the expression of Cyclins D1 and E, but increased the expression of p21 and p27. MUC4 expression abrogated cancer cell migration and invasion by altering N- & E-cadherin expression and FAK phosphorylation. A decrease in MUC4 expression was observed with increasing tumor stage (mean composite score: stage I, 2.4; stage II, 1.8; stage III, 1.4; and metastatic, 1.2; p = 0.0093). Maximal MUC4 expression was associated with a better overall survival (p = 0.042). CONCLUSION: MUC4 plays a tumor-suppressor role in NSCLC by altering p53 expression in NSCLC. Decrease in MUC4 expression in advanced tumor stages also seems to confirm the novel protective function of MUC4 in NSCLC.