Mouse Mammary Tumor Virus Promoter Research Articles

Abstract A084: DEK overexpression in the murine mammary gland promotes epithelial hyperplasia

Abstract Introduction: Breast cancer (BC) remains a leading cause of cancer-associated mortality for women in the USA, which makes it critically important to identify and study molecular drivers of pathogenesis. We have previously identified the chromatin modifying DEK protein to have oncogenic properties. DEK is a histone H3 chaperone that is over-expressed in over 60% of breast cancers, particularly triple negative breast cancers, and is associated with poor survival. To date, all studies regarding the role of DEK in BC have manipulated DEK expression in cultured immortalized MCF10A human mammary epithelial cells and BC cell lines. However, the oncogenic functions of DEK in vivo and in normal mammary epithelium have remained unexplored. Methods: We utilized a tetracycline-off regulated, DEK overexpressing (DEK-OE) transgenic mouse model that targets mammary epithelium through the mouse mammary tumor virus (MMTV) promoter. We subsequently performed bulk RNA-Seq transcriptomic analyses, western blotting, and both histological and morphological assessments of control vs. DEK-OE mammary gland tissue to identify the oncogenic effects of DEK over-expression in vivo. Results: Bulk RNA sequencing and gene ontology analyses identified cell cycle regulation as a significantly upregulated biological process in DEK-OE versus control glands. Subsequent studies confirmed upregulation of proteins that promote the G1-S cell cycle transition including cyclins A and D and CDKs 2, 4, and 6 as well as increased numbers of Ki67+ cells. We also observed down-regulation of genes that inhibit entry the G1-S transition, such as p21 and p27. This pro-proliferative molecular status correlated with the development of mammary gland hyperplasia. Importantly, in silico analyses demonstrated gene correlations between DEK and cell cycle regulators that were also evident in human breast cancers. One potential mechanism for this transcriptional deregulation is that we identified increased histone H3K27me3 epigenetic modifications in DEK-OE cells, in both mouse and human models, which may occur through EZH2 activity. Conclusions: Combined, our work indicates that DEK is not a tumor initiator, but its oncogenic activities are likely due to its ability to promote proliferation of mammary epithelial cells. Furthermore, we report a novel function for DEK in the epigenetic regulation of histone H3 K27 trimethylation, which is mediated by the well-known EZH2 oncogene in triple negative breast cancers. Citation Format: Megan Johnstone, Ashley Leck, Taylor Lange, Lisa Privette Vinnedge. DEK overexpression in the murine mammary gland promotes epithelial hyperplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A084.

Comparison of Tumor Control between FLASH and CONV in an Orthotopic Breast Cancer Model

Post-lumpectomy radiotherapy (RT) reduces in-breast tumor recurrence by eradicating residual, occult breast cancer (BC) that may be in the mm size scale. The ability of FLASH-RT to eradicate BC relative to conventional dose rate (CONV) RT is unknown. ∼ 20Gy RT is currently used clinically for single-fraction breast IORT. Determine the effectiveness of FLASH compared to CONV in eradicating small tumors in an orthotopic, syngeneic model of BC using single-fraction 20 or 30Gy RT. Radiation sensitive, mammary tumor cell line Py117 from the transgenic model of the mouse mammary tumor virus promoter driving the polyoma middle T antigen (MMTV- PyMT) efficiently forms non-metastatic, orthotopic tumors in C57BL/6 mice. 106 Py117 cells were injected orthotopically into the left 4th mammary fat pad of C57Bl/6J mice. Radiotherapy was performed with a custom jig that allows for fixed positioning of the target volume (2x2cm radiation field) with 5mm of margin into surrounding tissue. Tumors were irradiated at ∼30mm3 volume or, for comparison, at a range of greater volumes (200-800mm3) with 20 or 30Gy FLASH or CONV with 16-17 MeV electrons. Small 30mm3 tumors regressed until ∼ day 15 after 20Gy single fraction RT then regrew for both FLASH and CONV. 30mm3 tumors were eradicated with both FLASH and CONV at 30Gy with no regrowth up to day 35 post-RT. Larger tumors irradiated with 30Gy regressed until ∼ day 12 post-RT then regrew for both FLASH and CONV. There was no significant difference in growth delay or tumor eradication between FLASH and CONV in any cohort. FLASH was as effective as CONV in controlling growth and eradicating murine BC. Based on other preclinical studies, single-fraction doses between 20 and 30Gy, as well as hypofractioned RT schedules, may identify FLASH doses that achieve comparable tumor control with less toxicity than CONV. Such findings would encourage clinical trials of FLASH in human BC.

Characterization of Mammary Tumors Arising from MMTV-PyVT Transgenic Mice.

Among genetically engineered mouse models of breast cancer, MMTV-PyVT is a mouse strain in which the oncogenic polyoma virus middle T antigen is driven by the mouse mammary tumor virus promoter. The aim of the present study was to perform morphologic and genetic analyses of mammary tumors arising from MMTV-PyVT mice. To this end, mammary tumors were obtained at 6, 9, 12, and 16 weeks of age for histology and whole-mount analyses. We conducted whole-exome sequencing to identify constitutional and tumor-specific mutations, and genetic variants were identified using the GRCm38/mm10 mouse reference genome. Using hematoxylin and eosin analysis and whole-mount carmine alum staining, we demonstrated the progressive proliferation and invasion of mammary tumors. Frameshift insertions/deletions (indels) were noted in the Muc4. Mammary tumors showed small indels and nonsynonymous single-nucleotide variants but no somatic structural alterations or copy number variations. In summary, we validated MMTV-PyVT transgenic mice as a multistage model for mammary carcinoma development and progression. Our characterization may be used as a reference for guidance in future research.

Ectopic expression of OCT4B1 Decreases Fertility Rate and Changes Sperm Parameters in Transgenic Mice.

The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic "master switch" in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.

Haploinsufficiency Interactions between RALBP1 and p53 in ERBB2 and PyVT Models of Mouse Mammary Carcinogenesis.

Simple SummaryRlip knockout has been reported to prevent cancer in highly cancer-susceptible mice lacking p53, and Rlip knockdown kills many types of cancer cells. In humans, breast cancer shows diverse characteristics, including HER2-driven subtypes and viral-driven subtypes. HER2 can be targeted; however, escape of the cancer from targeted therapies remains a problem. In this work we evaluated the capacity of Rlip knockout to prevent breast cancer in genetically engineered mouse models of HER2-driven breast cancer (Erbb2 model) and polyomavirus-driven breast cancer (PyVT model). We found that in Erbb2 mice, Rlip knockout significantly delayed oncogenesis and reduced the expression of genes associated with poor prognosis in patients. In PyVT mice, Rlip knockout did not delay oncogenesis or tumor growth, but Rlip knockdown reduced tumor metastasis to the lung. We conclude that Rlip inhibitors may significantly improve survival in HER2-positive patients, but are unlikely to offer benefits to patients with polyomavirus-associated tumors.We recently reported that loss of one or both alleles of Ralbp1, which encodes the stress-protective protein RLIP76 (Rlip), exerts a strong dominant negative effect on both the inherent cancer susceptibility and the chemically inducible cancer susceptibility of mice lacking one or both alleles of the tumor suppressor p53. In this paper, we examined whether congenital Rlip deficiency could prevent genetically-driven breast cancer in two transgenic mouse models: the MMTV-PyVT model, which expresses the polyomavirus middle T antigen (PyVT) under control of the mouse mammary tumor virus promoter (MMTV) and the MMTV-Erbb2 model which expresses MMTV-driven erythroblastic leukemia viral oncogene homolog 2 (Erbb2, HER2/Neu) and frequently acquires p53 mutations. We found that loss of either one or two Rlip alleles had a suppressive effect on carcinogenesis in Erbb2 over-expressing mice. Interestingly, Rlip deficiency did not affect tumor growth but significantly reduced the lung metastatic burden of breast cancer in the viral PyVT model, which does not depend on either Ras or loss of p53. Furthermore, spontaneous tumors of MMTV-PyVT/Rlip+/+ mice showed no regression following Rlip knockdown. Finally, mice lacking one or both Rlip alleles differentially expressed markers for apoptotic signaling, proliferation, angiogenesis, and cell cycling in PyVT and Erbb2 breast tumors. Our results support the efficacy of Rlip depletion in suppressing p53 inactivated cancers, and our findings may yield novel methods for prevention or treatment of cancer in patients with HER2 mutations or tumor HER2 expression.

Inhibition of Stress-Induced Viral Promoters by a Bovine Herpesvirus 1 Non-Coding RNA and the Cellular Transcription Factor, β-Catenin.

The ability to establish, maintain, and reactivate from latency in sensory neurons within trigeminal ganglia (TG) is crucial for bovine herpesvirus 1 (BoHV-1) transmission. In contrast to lytic infection, the only viral gene abundantly expressed during latency is the latency-related (LR) gene. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency, in part because the glucocorticoid receptor (GR) transactivates viral promoters that drive expression of key viral transcriptional regulator proteins (bICP0 and bICP4). Within hours after dexamethasone treatment of latently infected calves, LR gene products and β-catenin are not readily detected in TG neurons. Hence, we hypothesized that LR gene products and/or β-catenin restrict GR-mediated transcriptional activation. A plasmid expressing LR RNA sequences that span open reading frame 2 (ORF2-Stop) inhibited GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) and mouse mammary tumor virus (MMTV) promoter activity in mouse neuroblastoma cells (Neuro-2A). ORF2-Stop also reduced productive infection and GR steady-state protein levels in transfected Neuro-2A cells. Additional studies revealed that the constitutively active β-catenin mutant reduced the transactivation of the IEtu1 promoter by GR and dexamethasone. Collectively, these studies suggest ORF2 RNA sequences and Wnt/β-catenin signaling pathway actively promote maintenance of latency, in part, by impairing GR-mediated gene expression.

Urethane-induced Mammary Carcinogenesis Susceptibility in Transgenic Mice Expressing a Dominant-negative TGF-β Type II Receptor.

Transforming growth factor-β (TGF-β) plays dual suppressive and oncogenic roles in mammary carcinogenesis. To analyze whether TGF-β exerts suppressive or oncogenic actions on mammary carcinogenesis, transgenic mice overexpressing a dominant-negative mutant type II TGF-β receptor (TβRII-DNR) driven by the mouse mammary tumor virus (MMTV) promoter were treated with a low dose of urethane, a carcinogen present in fermented food products and alcoholic beverages. Lobular proliferative lesions, showing high β-casein expression, developed in the mammary glands of TβRII-DNR+/+ mice aged >61 weeks. Compared with wild-type mice, TβRII-DNR+/+ mice administered with urethane showed significant increases in dysplastic hyperplasias and adenocarcinomas of the mammary glands. The functional decline of TGF-β signaling in mammary glands led to a high susceptibility to urethane-induced mammary carcinogenesis. TGF-β signaling may act as a tumor suppressor during mammary tumor development.

Abstract 1604: Modulating effects of genistein in a mouse model of conditional BRCA1 deletion and triple negative breast cancer cells

Abstract Germline mutations in BRCA1 (BRCA1+/-) confer ~72% risk of breast cancer, the majority of which are triple negative breast cancer (TNBC). Hypermethylation of BRCA1 is observed at high frequency in sporadic TNBC and may play a role in tumor development in BRCA1 mutation carriers. The aryl hydrocarbon receptor (AhR) coordinates epigenetic silencing of BRCA1 and is overexpressed in some TNBC. The purpose of our study is to investigate the effect of dietary genistein on BRCA1 epigenetic regulation, the AhR pathway and mammary tumorigenesis in BRCA1+/- and BRCA1+/+ mice. We used Cre-lox recombination to derive a mouse model of conditional BRCA1+/-. We used BRCA1F22/24 (The Jackson Laboratory, stock no. 017835) mice, which harbor loxP sites flanking exons 22-24 of the BRCA1 gene. We crossed dams that were heterozygous for BRCA1F22/24 (BRCA1+/FL) and transgenic for Cre recombinase (TG) under control of the mouse mammary tumor virus (MMTV) promoter with wildtype NJ males to derive our colony. Breeding pairs were administered either control or genistein-enriched (4 ppm) diets upon set up, which were continued throughout the life of the offspring. Changes in mRNA expression and protein levels were determined, respectively, by RT-PCR and Western blot. Crewas not expressed in mammary glands of non-TG offspring and Cre expression had no effect on BRCA1 mRNA levels in BRCA1+/+ mice, which do not harbor BRCA1 loxP sites. BRCA1 expression was ~40% decreased in mammary glands of TG, BRCA1+/FL mice compared with TG, BRCA1+/+ mice. Hepatic BRCA1 expression was not changed, confirming conditional BRCA1 knockdown. Preliminary data indicate BRCA1 expression in mammary glands of genistein-fed TG, BRCA1+/+ mice was elevated (~1.3 fold) compared with control-fed mice. Moreover, the genistein diet induced a compensatory increase in BRCA1 expression in mammary glands of TG, BRCA1+/FL mice. We found genistein rescued BRCA1 levels in TNBC HCC38 cells that have constitutively active AhR and hypermethylated BRCA1. These data suggest dietary genistein may have protective effects against tumorigenesis in these mice. To that end, we are currently monitoring tumor development, BRCA1 methylation, and changes in the AhR pathway in BRCA1+/+ and BRCA1+/FL mice fed either the genistein-enriched or control diet. Citation Format: Micah G. Donovan, Ornella I. Selmin, Thomas C. Doetschman, Donato F. Romagnolo. Modulating effects of genistein in a mouse model of conditional BRCA1 deletion and triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1604.

1991-P: Mammary Tumors Increase Diet-Induced Obesity and Insulin Resistance in Female MMTV-PyMT Mice

Diabetes and cancer share multiple risk factors including obesity and insulin resistance, but the potential relationship between these two major diseases is unknown. Here we examined the metabolic effects of high-fat diet (HFD) feeding in female MMTV-PyMT mice, a breast cancer model expressing the polyoma middle T antigen driven by the Mouse Mammary Tumor Virus promoter. Female PyMT and wild type (WT) mice were fed a HFD (55% fat by calories) for 4 weeks starting at 4∼5 weeks of age (n=10-12/group). During HFD, PyMT mice became more obese than WT mice with significant increases in both fat and lean mass (Figure 1; *P&amp;lt;0.05). This was largely due to ∼5% and 30% decreases in energy expenditure and physical activity, respectively, in HFD-PyMT mice (Figure 2). Food intake did not differ between groups. Hyperinsulinemic-euglycemic clamps were performed to measure glucose metabolism in awake mice. HFD-PyMT mice showed increased insulin resistance with ∼25% decreases in glucose infusion rate and whole body glucose turnover compared to HFD-WT mice (Figure 3). Increased insulin resistance was due to significantly reduced glucose uptake in white adipose tissue (Figure 4). In conclusion, these results demonstrate that tumor-bearing female PyMT mice developed increased obesity and insulin resistance after high-fat feeding, and our findings implicate an important relationship between obesity, cancer, and type 2 diabetes. Disclosure H. Noh: None. H. Kang: None. J. Mercado-Matos: None. R.H. Friedline: None. S. Suk: None. X. Hu: None. D.A. Tran: None. L.A. Tauer: None. M. Ko: None. J. Choi: None. T. Luong: None. Y. Tsuchida: None. L.M. Shaw: None. J.K. Kim: None. Funding National Institutes of Health (5U2C-DK093000)

A Transgenic MMTV-Flippase Mouse Line for Molecular Engineering in Mammary Gland and Breast Cancer Mouse Models.

Genetically engineered mouse models have become an indispensable tool for breast cancer research. Combination of multiple site-specific recombination systems such as Cre/loxP and Flippase (Flp)/Frt allows for engineering of sophisticated, multi-layered conditional mouse models. Here, we report the generation and characterization of a novel transgenic mouse line expressing a mouse codon-optimized Flp under the control of the mouse mammary tumor virus (MMTV) promoter. These mice show robust Flp-mediated recombination in luminal mammary gland and breast cancer cells but no Flp activity in non-mammary tissues, with the exception of limited activity in salivary glands. These mice provide a unique tool for studying mammary gland biology and carcinogenesis in mice.

A cis-Acting Element Downstream of the Mouse Mammary Tumor Virus Major Splice Donor Critical for RNA Elongation and Stability

BackgroundThe mouse mammary tumor virus (MMTV) encodes a functional signal peptide, a cleavage product of envelope and Rem proteins. Signal peptide interacts with a 3′ cis-acting RNA element, the Rem-responsive element (RmRE), to facilitate expression of both unspliced genomic (gRNA) and spliced mRNAs. An additional RmRE has been proposed at the 5′ end of the genome, facilitating nuclear export of the unspliced gRNA, whereas the 3′ RmRE could facilitate translation of all other mRNAs, including gRNA. ResultsTo address this hypothesis, a series of mutations were introduced into a 24-nt region found exclusively in the unspliced gRNA. Mutant clones using MMTV or human cytomegalovirus promoters were tested in both transient and stable transfections to determine their effect on gRNA nuclear export, stability, and translation. Nuclear export of the gRNA was affected only in a small mutant subset in stably transfected Jurkat T cells. Quantitative real-time RT-PCR of actinomycin D-treated cells expressing MMTV revealed that multiple mutants were severely compromised for RNA expression and stability. Both genomic and spliced nuclear RNAs were reduced, leading to abrogation of Gag and Env protein expressed from unspliced and spliced mRNAs, respectively. RT-PCRs with multiple primer pairs indicated failure to elongate genomic MMTV transcripts beyond ~500 nt compared to the wild type in a cell line-dependent manner. ConclusionsMMTV contains a novel cis-acting element downstream of the major splice donor critical for facilitating MMTV gRNA elongation and stability. Presence of a mirror repeat within the element may represent important viral/host factor binding site(s) within MMTV gRNA.

Abstract 5114: The role of amphiregulin in mammary gland development and breast cancer

Abstract Breast cancer is the most prevalent cancer in women worldwide and afflicts more than 3.1 million women in the US alone. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast cancer and is associated with poor clinical outcome. Several therapies involving anti-EGFR agents have been developed to target breast cancer. However, clinical trials have shown mixed outcomes with these therapies. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor (EGFR), is enriched in invasive breast cancer, particularly ERα-positive breast tumors. In normal physiology, AREG mediates estrogen-induced ductal morphogenesis during mammary gland development. In the absence of AREG, the ductal tree fails to elongate and remains rudimentary throughout adulthood. The structure of a healthy mammary duct consists of luminal cells surrounded by a layer of myoepithelial cells. Studies of mammary gland transplantations suggest that a marker of myoepithelial cells (keratin 14, K14) is only expressed if AREG is present in the mammary epithelium. Multiple studies have provided evidence that myoepithelial cells are natural tumor suppressors. During tumor growth, the disappearance of myoepithelial cells indicates progression to invasive carcinoma. Therefore we hypothesize that AREG plays an important role in maintenance of the maintenance of myoepithelial cells in the mammary epithelium and loss of AREG promotes tumor progression as a result of an early loss of myoepithelial cells. In the mammary glands of AREG WT and AREG KO mice without the PyMT transgene, we have found that mammary ducts in AREG KO mice have significantly fewer myoepithelial cells. Immunofluorescent staining of K14 has revealed a thinner and discontinuous myoepithelial layer in the AREG KO mice. To investigate the role of AREG in breast cancer development and progression, we have utilized a spontaneous mammary tumor mouse model where oncogenesis is driven by expression of the polyoma middle T oncoprotein (PyMT) through the mouse mammary tumor virus (MMTV) promoter. We have crossed AREG knockout mice with the MMTV-PyMT mice to generate AREG KO mice that spontaneously form mammary tumors (AREG KO PyMT). The absence of AREG in both the mammary epithelial and stromal compartments accelerates tumor growth. By 12 weeks of age, tumors of AREG KO PyMT mice are significantly larger than that of AREG WT PyMT mice. AREG KO tumors are histologically less necrotic, apoptotic, and progressed than AREG WT tumors. Furthermore, AREG KO tumors show higher staining of PyMT by immunofluorescence. Together, these results suggest that loss of AREG stimulates tumor initiation but inhibits progression due to fewer myoepithelial cells beginning during development. Citation Format: Serena Mao, Minji Park, Ramon Cabrera, John Christin, George Karagiannis, Wenjun Guo, John Condeelis, Jeffrey Segall. The role of amphiregulin in mammary gland development and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5114.

Abstract 1844: Investigating the role of amphiregulin in breast cancer

Abstract Breast cancer is the most prevalent cancer in women worldwide and afflicts more than 3.1 million women in the US alone. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast cancer and is associated with poor clinical outcome. Several therapies involving anti-EGFR agents have been developed to target breast cancer. However, clinical trials have shown mixed outcomes with these therapies. Amphiregulin (AREG) is a ligand for EGFR that mediates estrogen-induced mammary ductal morphogenesis during development. Expression of AREG is enriched in invasive breast carcinomas, particularly ERα-positive breast tumors. Studies have shown that AREG increases the in vitro invasiveness of breast cancer cells. Furthermore, suppression of AREG expression in transformed human breast epithelial cells reduces tumor formation when injected in nude mice. These observations suggest that AREG plays an important role in breast cancer development and progression. To investigate the role of AREG in breast cancer development and progression, we utilized a spontaneous mammary tumor mouse model where oncogenesis is driven by expression of the polyoma middle T oncoprotein (PyMT) through the mouse mammary tumor virus (MMTV) promoter. We have crossed AREG knockout mice with the MMTV-PyMT mice to generate AREG KO mice that spontaneously form mammary tumors. Tumors in AREG KO PyMT mice initially grow more quickly but eventually reach 2 cm in diameter at a relatively time as compared to tumors formed in AREG WT PyMT mice. Histologically, AREG KO tumors appear more differentiated and display a greater proportion of tumor stroma. This prompted us to further hypothesize that AREG may have diverse effects in the tumoral or stromal compartment of developing tumors. To individually assess the effects of AREG knockout in the tumor cells and the stroma, we utilized a transplantation model in which we prepared tumor chunks from AREG WT and KO PyMT tumors and transplanted them into AREG WT and KO mice. Through this model, we have found that AREG expression in the tumor has a greater impact on tumor growth and progression than the tumor stroma. Regardless of the AREG status of the recipient mice, AREG KO tumors display a significant growth impairment and reduced transplant efficiency. In summary, these results demonstrate that AREG is important for mammary tumor progression. Citation Format: Serena P. Chiang, George S. Karagiannis, John S. Condeelis, Jeffrey E. Segall. Investigating the role of amphiregulin in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1844. doi:10.1158/1538-7445.AM2017-1844

Bromodomain inhibition shows antitumoral activity in mice and human luminal breast cancer.

BET bromodomain inhibitors, which have an antitumoral effect against various solid cancer tumor types, have not been studied in detail in luminal breast cancer, despite the prevalence of this subtype of mammary malignancy. Here we demonstrate that the BET bromodomain inhibitor JQ1 exerts growth-inhibitory activity in human luminal breast cancer cell lines associated with a depletion of the C-MYC oncogene, but does not alter the expression levels of the BRD4 bromodomain protein. Interestingly, expression microarray analyses indicate that, upon JQ1 administration, the antitumoral phenotype also involves downregulation of relevant breast cancer oncogenes such as the Breast Carcinoma-Amplified Sequence 1 (BCAS1) and the PDZ Domain-Containing 1 (PDZK1). We have also applied these in vitro findings in an in vivo model by studying a transgenic mouse model representing the luminal B subtype of breast cancer, the MMTV-PyMT, in which the mouse mammary tumor virus promoter is used to drive the expression of the polyoma virus middle T-antigen to the mammary gland. We have observed that the use of the BET bromodomain inhibitor for the treatment of established breast neoplasms developed in the MMTV-PyMT model shows antitumor potential. Most importantly, if JQ1 is given before the expected time of tumor detection in the MMTV-PyMT mice, it retards the onset of the disease and increases the survival of these animals. Thus, our findings indicate that the use of bromodomain inhibitors is of great potential in the treatment of luminal breast cancer and merits further investigation.

Abstract A12: The immune composition of a spontaneous mouse breast cancer

Abstract The MMTV-PyMT mouse strain is a widely used model for the study of breast cancer. In this strain, the mouse mammary tumor virus (MMTV) promoter drives expression of the polyoma middle T (PyMT) oncogene in breast epithelium resulting in widespread transformation of the tissue and formation of progressive tumors that eventually metastasize to the lungs and lymph nodes, closely recapitulating the human disease. It is know that MMTV-PyMT tumors are infiltrated by large numbers of immune cells and the role of some immune populations in cancer progression and metastasis has been described. However a detailed characterization of the myeloid and lymphoid populations infiltrating MMTV-PyMT during tumor progression is yet to be performed. In this study we used comprehensive flow cytometry panels to enumerate and quantify B cells, αβ T cells (CD4 and CD8), γδ T cells, NK T cells, NK cells, neutrophils, monocytes, dendritic cells and macrophages within MMTV-PyMT tumors in different stages of progression. Given that hypoxia is a prominent aspect of breast cancer we extended this characterization to strains with targeted deletion of hypoxia-inducible factor (HIF) 1α and HIF2α in myeloid cells. We found lymphoid cells to be the predominant immune populations within PyMT tumors particularly B cells and αβ T cells (CD4+ and CD8+) but their frequencies did not markedly change with progression or tumor mass. Surprisingly the majority of CD8 T cells expressed the NK cell marker NK1.1 (&amp;gt;80%). In addition we found dendritic cells (CD11chigh, MHC IIhigh) to be the prevalent myeloid population and that the frequency of this population had a strong positive correlation with tumor mass. Myeloid deletion of HIF1a, but not HIF2a, resulted in increased tumor mass and dendritic cell infiltration while other immune population frequencies remained unchanged. Whether dendritic cells directly affect tumor growth and to what extent HIF1α is involved in this process remains to be elucidated. We expect that this immune characterization of PyMT tumors will serve as a reference for future breast cancer immunology studies and possibly assist in the development of new immunotherapies. Citation Format: Pedro Velica, Helene Rundqvist, Milos Gojkovic, Randall S. Johnson. The immune composition of a spontaneous mouse breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A12.

X-ray structure of the MMTV-A nucleosome core

The conformation of DNA bound in nucleosomes depends on the DNA sequence. Questions such as how nucleosomes are positioned and how they potentially bind sequence-dependent nuclear factors require near-atomic resolution structures of the nucleosome core containing different DNA sequences; despite this, only the DNA for two similar α-satellite sequences and a sequence (601) selected in vitro have been visualized bound in the nucleosome core. Here we report the 2.6-Å resolution X-ray structure of a nucleosome core particle containing the DNA sequence of nucleosome A of the 3'-LTR of the mouse mammary tumor virus (147 bp MMTV-A). To our knowledge, this is the first nucleosome core particle structure containing a promoter sequence and crystallized from Mg(2+) ions. It reveals sequence-dependent DNA conformations not seen previously, including kinking into the DNA major groove.

Abstract A063: Cancer cell expression of CCR2 regulates the PD-L1/ PD-1 immune checkpoint in breast cancer

Abstract C-C chemokine receptor type 2 (CCR2) is highly expressed in human breast cancer tissue by both cancer cells and infiltrating immune cells. High expression of CCR2 is correlated with advanced tumor stage, metastasis, and early relapse. Studies in mice have suggested that CCR2 expression on infiltrating inflammatory monocytes promotes primary tumor growth and metastasis. However, the role of CCR2 expression on cancer cells has not been well studied. To determine how CCR2 directly impacts tumor growth and metastasis, we used a murine model of luminal breast cancer, the mouse mammary tumor virus (MMTV) promoter polyoma middle T antigen (PyMT) model. We crossed MMTV-PyMT mice to Ccr2-/- mice and found that loss of CCR2 did not alter tumor onset. However, primary tumors of MMTV-PyMT;Ccr2+/+ mice grew significantly faster than tumors of MMTV-PyMT;Ccr2-/- mice. Lung metastases were also larger in MMTV-PyMT;Ccr2+/+ mice than in MMTV-PyMT;Ccr2-/- mice. We determined by RNA in situ hybridization that CCR2 was indeed expressed by both host cells and cancer cells in MMTV-PyMT tumors. To differentiate between the contributions of CCR2 expression in the tumor and host, we transplanted primary cancer cells derived from MMTV-PyMT;Ccr2+/+ and MMTV-PyMT;Ccr2-/- mice to syngeneic Ccr2+/+ and Ccr2-/- hosts. Surprisingly, CCR2 expression in the host had no significant effect on tumor growth. However, deletion of CCR2 from cancer cells resulted in delayed tumor growth and increased survival. Furthermore, when Ccr2+/+ and Ccr2-/- cancer cells were transplanted to athymic nude mice (lacking T cells), the resulting tumors no longer showed a delayed growth. Consistent with this finding, flow cytometry of infiltrating leukocytes in tumors transplanted to syngeneic mice revealed increased levels of CD8+ cytotoxic T cells and antigen-presenting CD103+ dendritic cells into Ccr2-/- tumors compared to wild type tumors. These data suggest that CCR2 expression on cancer cells regulates an immune response to the tumors. Supporting this conclusion, expression of programmed cell death ligand 1 (PD-L1) on Ccr2-/- cancer cells was reduced to just 30% of the level on wild type cancer cells, indicating that CCR2 expression on cancer cells suppresses the anti-tumor immune response. Together, our results establish a novel role for cancer cell CCR2 expression in breast cancer progression by suppression of cytotoxic T cells through an immune checkpoint. Citation Format: Miriam R. Fein, Ana S. Almeida, Anaïs Eberhardt, Mikala Egeblad. Cancer cell expression of CCR2 regulates the PD-L1/ PD-1 immune checkpoint in breast cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A063.

Heregulin Co-opts PR Transcriptional Action Via Stat3 Role As a Coregulator to Drive Cancer Growth.

Accumulated findings have demonstrated the presence of bidirectional interactions between progesterone receptor (PR) and the ErbB family of receptor tyrosine kinases signaling pathways in breast cancer. We previously revealed signal transducer and activator of transcription 3 (Stat3) as a nodal convergence point between said signaling pathways proving that Stat3 is activated by one of the ErbBs' ligands, heregulin (HRG)β1 via ErbB2 and through the co-option of PR as a signaling molecule. Here, we found that HRGβ1 induced Stat3 recruitment to the promoters of the progestin-regulated cell cycle modulators Bcl-XL and p21(CIP1) and also stimulated Stat3 binding to the mouse mammary tumor virus promoter, which carries consensus progesterone response elements. Interestingly, HRGβ1-activated Stat3 displayed differential functions on PR activity depending on the promoter bound. Indeed, Stat3 was required for PR binding in bcl-X, p21(CIP1), and c-myc promoters while exerting a PR coactivator function on the mouse mammary tumor virus promoter. Stat3 also proved to be necessary for HRGβ1-induced in vivo tumor growth. Our results endow Stat3 a novel function as a coregulator of HRGβ1-activated PR to promote breast cancer growth. These findings underscore the importance of understanding the complex interactions between PR and other regulatory factors, such as Stat3, that contribute to determine the context-dependent transcriptional actions of PR.

The polyglutamine-expanded androgen receptor has increased DNA binding and reduced transcriptional activity

Expansion of a polyglutamine-encoding trinucleotide CAG repeat in the androgen receptor (AR) to more than 37 repeats is responsible for the X-linked neuromuscular disease spinal and bulbar muscular atrophy (SBMA). Here we evaluated the effect of polyglutamine length on AR function in Xenopus oocytes. This allowed us to correlate the nuclear AR concentration to its capacity for specific DNA binding and transcription activation in vivo. AR variants with polyglutamine tracts containing either 25 or 64 residues were expressed in Xenopus oocytes by cytoplasmic injection of the corresponding mRNAs. The intranuclear AR concentration was monitored in isolated nuclei and related to specific DNA binding as well as transcriptional induction from the hormone response element in the mouse mammary tumor virus (MMTV) promoter. The expanded AR with 64 glutamines had increased capacity for specific DNA binding and a reduced capacity for transcriptional induction as related to its DNA binding activity. The possible mechanism behind these polyglutamine-induced alterations in AR function is discussed.

Glucocorticoid Receptor-DNA Dissociation Kinetics Measured in vitro Reveal Exchange on the Second Timescale

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. Recent live cell imaging studies have revealed that GR interactions with chromatin are highly dynamic, with receptor residence times on the order of seconds. These findings were surprising since early in vitro studies suggested that GR-DNA interactions were more static, with receptor-DNA exchange occurring on the order of minutes to hours. However, these latter analyses were carried out without the modern-day advantages of recombinant receptor expression, established purification protocols or rigorous characterization methods. Here we examine the DNA dissociation kinetics of full-length human GR, using highly purified and homogenous receptor preparations. We find that in vitro dissociation kinetics are not slow as previously reported, but are quite fast, with calculated receptor-DNA residence times of seconds to tens of seconds. These findings are observed at both isolated response elements and the multisite mouse mammary tumor virus (MMTV) promoter used in live cell imaging. However, we also find that dissociation kinetics are identical for all response elements and are biphasic in character. These results indicate that differences in GR affinity toward these sequences are not due to differences in off-rate but on-rate, and further suggest that GR exists in multiple and interconverting states when bound to DNA. Since we previously established that GR-DNA binding affinity is a primary determinant of transcriptional activity at these sequences, the current findings implicate DNA sequence in controlling the extent of receptor interconversion and hence extent of transcriptional activation.