ABSTRACT In utero exposure to estrogens is known to increase breast cancer risk in adulthood. The plasticizer bisphenol A (BPA) increases tumor susceptibility in rodent models of in utero exposure, but the mechanism of this increase is unclear. Our lab has previously shown that in utero BPA exposure causes alterations to the tissue microenvironment that are conducive to tumor initiation. We thus sought to understand when these alterations arise and how they are regulated using bulk and single-nuclei epigenomics in adult mammary glands following in utero BPA exposure. Our results indicate that in utero BPA exposure causes developmental reprogramming of mammary fibroblasts that is initiated during puberty and persists into adulthood. Fibroblast-specific changes in chromatin accessibility were identified at enhancers and annotated to genes involved in fibroblast differentiation, suggesting that BPA may exert its effects by reprogramming fibroblast heterogeneity. Single-nuclei multiomics confirmed changes in fibroblast heterogeneity and uncovered estrogen signaling as a putative regulator of the fibroblast cell state that is altered by BPA exposure. Together, these studies support a model whereby BPA acts on mesenchymal ERα to reprogram the chromatin landscape of mammary fibroblasts, leading to their altered differentiation.

Female sex is associated with lower overall melanoma incidence and mortality, with estrogen signaling thought to protect against disease progression. Genomic estrogen signaling is mediated by estrogen receptors alpha (ERα) and beta (ERβ), which function as ligand-activated transcription factors. Notably, ERβ expression shows an inverse correlation with tumor thickness in the skin and is often lost in advanced melanomas, suggesting that ERβ activity may repress melanoma development. Here, we demonstrate that germline ERβ knockout leads to reduced melanoma-free survival in a murine model of NRASQ61R spontaneous melanomas. While this implicates ERβ as a tumor suppressor, little is understood about the molecular activities of ERβ in melanomas and in melanocytes prior to transformation. To elucidate the genomic actions of the receptor, we performed ChIP-Seq in immortalized human melanocytes. We found that ERβ binding regions significantly overlap with those of MITF, a transcription factor that acts as a master regulator of melanocyte development. Changes in melanoma cellular phenotypes, such as increased proliferation, dedifferentiation, or invasiveness, are thought to be driven by differences in MITF activity. Therefore, we hypothesize that ERβ modulates MITF transcriptional activity in a way that suppresses melanoma progression. Using a reporter construct with activity driven by the tyrosinase (TYR) promoter, we showed that ERβ activation coincided with altered MITF regulation of reporter activity. As we have characterized overlapping ERβ and MITF recruitment in the TYR promoter, this result supports the hypothesis that ERβ co-binding can influence MITF transcriptional programs. Additionally, transcriptome analysis revealed that estrogen-responsive genes in melanocytes are enriched for migration and differentiation, biological processes that are directed by MITF and dysregulated in melanomas. Taken together, these findings indicate that ERβ functions as a tumor suppressor in melanoma by reprogramming MITF activity, identifying a new avenue through which melanoma may be better understood at the molecular level. Jocelyn Treneff, Amanda McCrary, Craig Burd. Estrogen receptor beta suppresses melanoma progression through modulation of melanocyte master regulator activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4055.

In utero exposures to estrogenic endocrine disrupting compounds (EDCs) can increase breast cancer risk in adulthood. It has previously been shown that the estrogenic plasticizer bisphenol A (BPA) alters development of the mammary gland and increases both mammary gland stiffness and tumor susceptibility in rodent models following in utero exposure. Because of its endocrine disrupting properties, BPA has been substituted with structural analogues with varying abilities to activate estrogen receptor alpha (ERα). However, the impact of in utero exposure to many of these analogues is unknown. In the present study, we aimed to analyze the impact of bisphenol analogues on collagen deposition and mammary gland stiffness and characterize mammary epithelial development following exposure to these compounds. With the exception of bisphenol S, all analogues significantly increased mammary gland stiffness at a 25 µg/kg body weight dose in a manner that correlated to estrogenic activity. In contrast, significant effects on epithelial development endpoints were limited and did not follow a clear pattern. These results add to the growing literature on the hazard of bisphenol analogues and support the idea that these compounds promote abnormal mammary gland development. Furthermore, these data support a model that bisphenol compounds induce a microenvironment that may promote breast cancer development in an estrogen-dependent manner.

An increasing number of cancer subtypes are treated with front-line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Preclinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of 13 C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University-Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte-specific, and clinically relevant Nras driver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non-burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune-based therapies.

Supplementary Data from Cyclin D1 Splice Variants: Polymorphism, Risk, and Isoform-Specific Regulation in Prostate Cancer

Development of the mammary gland requires both proper hormone signaling and cross talk between the stroma and epithelium. While estrogen receptor (ERα) expression in the epithelium is essential for normal gland development, the role of this receptor in the stroma is less clear. Moreover, several lines of evidence suggest that mouse phenotypes of in utero exposure to endocrine disruption act through mesenchymal ERα in the developing fetus. We utilized a Twist2-cre mouse line to knock out mesenchymal ERα. Herein, we assessed mammary gland development in the context of mesenchymal ERα deletion. We also tested the effect of in utero bisphenol A (BPA) exposure to alter the tumor susceptibility in the mouse mammary tumor virus-neu (MMTV-neu) breast cancer mouse model. Mesenchymal ERα deletion resulted in altered reproductive tract development and atypical cytology associated with estrous cycling. The mammary gland demonstrated mature epithelial extension unlike complete ERα-knockout mice, but ductal extension was delayed and reduced compared to ERα-competent mice. Using the MMTV-Neu cancer susceptibility model, ERα-intact mice exposed to BPA had reduced tumor-free survival and overall survival compared to BPA-exposed mice having mesenchymal ERα deletion. This difference is specific for BPA exposure as vehicle-treated animals had no difference in tumor development between mice expressing and not expressing mesenchymal ERα. These data demonstrate that mesenchymal ERα expression is not required for ductal extension, nor does it influence cancer risk in this mouse model but does influence the cancer incidence associated with in utero BPA exposure.

Melanin is a free-radical scavenger, antioxidant, and broadband absorber of ultraviolet (UV) radiation which protects the skin from environmental carcinogenesis. However, melanin synthesis and UV-induced reactive melanin species are also implicated in melanocyte genotoxicity. Here, we attempted to reconcile these disparate functions of melanin using a UVB-sensitive, NRAS-mutant mouse model, TpN. We crossed TpN mice heterozygous for an inactivating mutation in Tyrosinase to produce albino and black littermates on a C57BL/6J background. These animals were then exposed to a single UVB dose on postnatal day three when keratinocytes in the skin have yet to be melanized. Approximately one-third (35%) of black mice were protected from UVB-accelerated tumor formation. However, melanoma growth rates, tumor mutational burdens, and gene expression profiles were similar in melanomas from black and albino mice. Skin from albino mice contained more cyclobutane pyrimidine dimer (CPD) positive cells than black mice 1-h post-irradiation. However, this trend gradually reversed over time with CPDs becoming more prominent in black than albino melanocytes at 48 h. These results show that in the absence of epidermal pigmentation, melanocytic melanin limits the tumorigenic effects of acute UV exposure but fails to protect melanocytes from UVB-induced mutagenesis.

In utero exposure to estrogenic endocrine disrupting compounds (EDCs) increases a woman’s lifetime risk of breast cancer. Similarly, mice exposed in utero to the estrogenic EDC bisphenol A (BPA) have increased susceptibility to mammary gland tumors. It is unclear which BPA-induced alterations predispose the mammary gland to cancer transformation. There is a critical need to understand the mechanisms that drive increased cancer risk in order to assess the impact of BPA and BPA alternatives that retain estrogenic activity. We have utilized in utero BPA exposure as a model system for in utero estrogenic endocrine disruption to study the long-term consequences to the mouse mammary stroma. We found that BPA exposed fibroblasts showed significant transcriptional deregulation, with the extracellular matrix being the most altered cellular component and multiple collagen genes being more highly expressed. The fibroblasts from the BPA exposed mice decreased fluid permeability of the extracellular matrix, indicative of an increased density in the extracellular matrix. Also, in utero BPA exposure increased mammary gland stiffness. Changes to breast density, stiffness, and collagen deposition are all associated with breast cancer risk. Further, we test BPA alternative compounds with varying affinities for the estrogen receptor in our in utero model to assess the phenotypes in the mouse mammary stroma which are associated with breast cancer risk. Additionally, we use a mesenchymal estrogen receptor alpha (ERα) knockout mouse model to dissect the in utero cellular target of EDCs. Citation Format: Clarissa Wormsbaecher, Andrea R. Hindman, Alex Avendano, Marcos Cortes-Medina, Jonathan W. Song, Craig J. Burd. The estrogenic activities of endocrine disruptors alter the extracellular matrix and tissue stiffness in the mammary gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2687.

A distinct profile of NRAS mutants is observed in each tumor type. It is unclear whether these profiles are determined by mutagenic events or functional differences between NRAS oncoproteins. Here, we establish functional hallmarks of NRAS mutants enriched in human melanoma. We generate eight conditional, knock-in mouse models and show that rare melanoma mutants (NRAS G12D, G13D, G13R, Q61H, and Q61P) are poor drivers of spontaneous melanoma formation, whereas common melanoma mutants (NRAS Q61R, Q61K, or Q61L) induce rapid tumor onset with high penetrance. Molecular dynamics simulations, combined with cell-based protein–protein interaction studies, reveal that melanomagenic NRAS mutants form intramolecular contacts that enhance BRAF binding affinity, BRAF-CRAF heterodimer formation, and MAPK > ERK signaling. Along with the allelic series of conditional mouse models we describe, these results establish a mechanistic basis for the enrichment of specific NRAS mutants in human melanoma.

ABSTRACT Distinct NRAS mutants are enriched in various tumor types. Here, we generated a suite of fully congenic, conditional, Nras knock-in mouse models ( LSL-Nras Q61R, -K, -L, -H, -P, -Q; G12D and G13D, -R ) to test the hypothesis that melanocyte transformation requires functions specific to the NRAS mutants enriched in human melanoma (Q61R and Q61K). Consistent with the rarity of NRAS codon 12 and 13 mutants in human melanoma, spontaneous melanomas were rare or absent in mice expressing NRAS G12D, G13D or G13R. Mice expressing less common codon 61 alleles (Q61H, Q61P) also developed few or no tumors. NRAS Q61R, Q61K, or Q61L expression, by contrast, induced rapid melanoma onset with high penetrance. Cohorts of heterozygous mice containing one LSL-Nras Q61R and one LSL-Nras Q61K, -L, -H, -P , or -Q allele were generated to assess potential interactions between NRAS mutants. The ability of each Nras variant to substitute for an Nras Q61R allele was consistent with its own ability to drive spontaneous melanoma formation. However, LSL-Nras Q61Q / Q61R mice rarely developed tumors. In vitro experiments in mouse embryonic fibroblasts (MEFs) highlighted activation of the MAPK pathway as a defining difference between tumorigenic and non-tumorigenic NRAS mutants. Enhanced MAPK activation was associated with the promotion of BRAF-BRAF and BRAF-CRAF dimers. These results support the development of cancer preventative strategies specific to the properties of the commonly observed RAS mutants in each tumor type.

BRAF-mutant melanomas are more likely than NRAS-mutant melanomas to arise in anatomical locations protected from chronic sun damage. We hypothesized that this discrepancy in tumor location is a consequence of the differential sensitivity of BRAF and NRAS-mutant melanocytes to ultraviolet light (UV)-mediated carcinogenesis. We tested this hypothesis by comparing the mutagenic consequences of a single neonatal, ultraviolet-AI (UVA; 340-400 nm) or ultraviolet-B (UVB; 280-390 nm) exposure in mouse models heterozygous for mutant Braf or homozygous for mutant Nras Tumor onset was accelerated by UVB, but not UVA, and the resulting melanomas contained recurrent mutations affecting the RING domain of MAP3K1 and Actin-binding domain of Filamin A. Melanomas from UVB-irradiated, Braf-mutant mice averaged twice as many single-nucleotide variants and five times as many dipyrimidine variants than tumors from similarly irradiated Nras-mutant mice. A mutational signature discovered in UVB-accelerated tumors mirrored COSMIC signatures associated with human skin cancer and was more prominent in Braf- than Nras-mutant murine melanomas. These data show that a single UVB exposure yields a greater burden of mutations in murine tumors driven by oncogenic Braf.

Background In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility. Methods We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands. Results We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not. Conclusions As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood.

Over 70% of breast cancers express the estrogen receptor (ER) and depend on ER activity for survival and proliferation. While hormone therapies that target receptor activity are initially effective, patients invariably develop resistance which is often associated with activation of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. While the mechanism by which estrogen regulates proliferation is not fully understood, one gene target of ER, growth regulation by estrogen in breast cancer 1 (GREB1), is required for hormone-dependent proliferation. However, the molecular function by which GREB1 regulates proliferation is unknown. Herein, we validate that knockdown of GREB1 results in growth arrest and that exogenous GREB1 expression initiates senescence, suggesting that an optimal level of GREB1 expression is necessary for proliferation of breast cancer cells. Under both of these conditions, GREB1 is able to regulate signaling through the PI3K/Akt/mTOR pathway. GREB1 acts intrinsically through PI3K to regulate phosphatidylinositol (3,4,5)-triphosphate levels and Akt activity. Critically, growth suppression of estrogen-dependent breast cancer cells by GREB1 knockdown is rescued by expression of constitutively activated Akt. Together, these data identify a novel molecular function by which GREB1 regulates breast cancer proliferation through Akt activation and provides a mechanistic link between estrogen signaling and the PI3K pathway.

Over 70% of breast cancers express the estrogen receptor (ER) and depend on ER activity for survival and proliferation. While hormone therapies that target receptor activity are initially effective, patients invariably develop resistance which is often associated with activation of the PI3K/Akt/mTOR pathway. While the mechanism by which estrogen regulates proliferation is not fully understood, one gene target of ER, growth regulation by estrogen in breast cancer 1 (GREB1), is required for hormone-dependent proliferation. However, the molecular function by which GREB1 regulates proliferation is unknown. Herein, we validate that knockdown of GREB1 results in growth arrest and that exogenous GREB1 expression initiates oncogene-induced senescence, suggesting that an optimal level of GREB1 expression is necessary for proliferation of breast cancer cells. Under both of these conditions, GREB1 is able to regulate signaling through the PI3K/Akt/mTOR pathway. GREB1 acts intrinsically through PI3K to regulate PIP levels and Akt activity. Critically, growth suppression of estrogen-dependent breast cancer cells by GREB1 knockdown is rescued by expression of constitutively activated Akt. Together, these data identify a novel molecular function by which GREB1 regulates breast cancer proliferation through Akt activation and provides a mechanistic link between estrogen signaling and the PI3K pathway.

ABSTRACT It is difficult to discern the relative contributions of ultraviolet-A (UVA; 320-400nm) and ultraviolet-B (UVB; 280-320nm) radiation to human melanoma development. Here, we compared the tumorigenic consequences of a single UVA or UVB exposure in mouse models predisposed to Braf- or Nras-mutant melanoma. Exposures approximated the amount of UVA or UVB energy contained in ∼40 minutes of summer sunlight. While UVA accelerated melanoma onset in a subset of mice, UVB universally reduced tumor latency and induced gene mutations relevant to the human disease. Genomic analyses uncovered distinct mutational signatures specific to each UV spectrum. The UVB-specific signature was biased for mutations on the untranscribed DNA strand and closely mirrored mutational signatures enriched in human cutaneous melanoma. The UVA-specific signature mimicked SBS51, a mutational signature found in human uveal melanoma. Distinctions in the trinucleotide patterns of the UVA and UVB signatures suggest that cytosine deamination plays a key role in UVB-mediated melanomagenesis.

Activation of the transcription factor estrogen receptor α (ERα) and the subsequent regulation of estrogen-responsive genes play a crucial role in the development and progression of the majority of breast cancers. One gene target of ERα, growth regulation by estrogen in breast cancer 1 (GREB1), is associated with proliferation and regulation of ERα activity in estrogen-responsive breast cancer cells. The GREB1 gene encodes three distinct isoforms: GREB1a, GREB1b and GREB1c, whose molecular functions are largely unknown. Here, we investigate the role of these isoforms in regulation of ERα activity and proliferation. Interaction between GREB1 and ERα was mapped to the amino terminus shared by all GREB1 variants. Analysis of isoform-specific regulation of ERα activity suggests none of the GREB1 isoforms possess potent co-regulator activity. Exogenous expression of GREB1a resulted in elevated expression of some ER-target genes, independent of ERα activity. Despite this slight specificity of GREB1a for gene regulation, exogenous expression of either GREB1a or GREB1b resulted in decreased proliferation in both ER-positive and ER-negative breast carcinoma cell lines, demonstrating an ER-independent function of GREB1. Interestingly, we show an increase in the expression of GREB1b and GREB1c mRNA in malignant breast tissue compared to normal patient samples, suggesting a selective preference for these isoforms during malignant transformation. Together, these data suggest GREB1a has an isoform-specific function as a transcriptional regulator while all isoforms share an ER-independent activity that regulates proliferation.

In utero exposure to the endocrine disrupting compound bisphenol A (BPA) is known to disrupt mammary gland development and increase tumor susceptibility in rodents. It is unclear whether different periods of in utero development might be more susceptible to BPA exposure. We exposed pregnant CD-1 mice to BPA at different times during gestation that correspond to specific milestones of in utero mammary gland development. The mammary glands of early-life and adult female mice, exposed in utero to BPA, were morphologically and molecularly (estrogen receptor-α and Ki67) evaluated for developmental abnormalities. We found that BPA treatment occurring before mammary bud invasion into the mesenchyme [embryonic day (E)12.5] incompletely resulted in the measured phenotypes of mammary gland defects. Exposing mice up to the point at which the epithelium extends into the precursor fat pad (E16.5) resulted in a nearly complete BPA phenotype and exposure during epithelial extension (E15.5 to E18.5) resulted in a partial phenotype. Furthermore, the relative differences in phenotypes between exposure windows highlight the substantial correlations between early-life molecular changes (estrogen receptor-α and Ki67) in the stroma and the epithelial elongation defects in mammary development. These data further implicate BPA action in the stroma as a critical mediator of epithelial phenotypes.

Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.

Recent studies implicate innate immunity to systemic lupus erythematosus (SLE) pathogenesis. Toll-like receptor (TLR)8 is estrogen-regulated and binds viral ssRNA to stimulate innate immune responses, but recent work indicates that microRNA (miR)-21 within extracellular vesicles (EVs) can also trigger this receptor. Our objective was to examine TLR8 expression/activation to better understand sex-biased responses involving TLR8 in SLE. Our data identify an estrogen response element that promotes STAT1 expression and demonstrate STAT1-dependent transcriptional activation of TLR8 with estrogen stimulation. In lieu of viral ssRNA activation, we explored EV-encapsulated miR-21 as an endogenous ligand and observed induction of both TLR8 and cytokine expression in vitro. Moreover, extracellular miR detection was found predominantly within EVs. Thus, just as a cytokine or chemokine, EV-encapsulated miR-21 can act as an inflammatory signaling molecule, or miRokine, by virtue of being an endogenous ligand of TLR8. Collectively, our data elucidates a novel innate inflammatory pathway in SLE.

The pervasive nature of estrogenic industrial and dietary compounds is a growing health concern linked to cancer, obesity, and neurological disorders. Prior analyses of endocrine disruptor action have focused primarily on the short-term consequences of exposure. However, these studies are unlikely to reflect the consequences of constant exposures common to industrialized countries. Here we examined the global effects of long-term endocrine disruption on gene transcription and estrogen signaling. Estrogen-dependent breast cancer cell lines were chronically treated with physiologically relevant levels of bisphenol A or genistein for more than 70 passages. Microarray analysis demonstrated global reprogramming of the transcriptome when compared with a similarly cultured control cell line. Estrogen-responsive targets showed diminished expression in both the presence and absence of estrogen. Estrogen receptor recruitment, H3K4 monomethylation, and deoxyribonuclease accessibility were reduced at nearby response elements. Based on these observations, we investigated the potential of long-term endocrine disruptor exposure to initiate persistent transcriptional reprogramming. Culture of chronically exposed cell lines in the absence of the endocrine disruptors did not reverse many of the signaling defects that accumulated during treatment. Taken together, these data demonstrate that chronic exposure to endocrine disrupting compounds can permanently alter physiological hormone signaling.