As both perimenopausal and menopausal periods are recognized critical windows of susceptibility for breast carcinogenesis, development of a physiologically relevant model has been warranted. The traditional ovariectomy model causes instant removal of the entire hormonal repertoire produced by the ovary, which does not accurately approximate human natural menopause with gradual transition. Here, we characterized the mammary glands of 4-vinylcyclohexene diepoxide (VCD)-treated animals at different time points, revealing that the model can provide the mammary glands with both perimenopausal and menopausal states. The perimenopausal gland showed moderate regression in ductal structure with no responsiveness to external hormones, while the menopausal gland showed severe regression with hypersensitivity to hormones. Leveraging the findings on the VCD model, effects of a major endocrine disruptor (polybrominated diphenyl ethers, PBDEs) on the mammary gland were examined during and after menopausal transition, with the two exposure modes; low-dose, chronic (environmental) and high-dose, subacute (experimental). All conditions of PBDE exposure did not augment or compromise the macroscopic ductal reorganization resulting from menopausal transition and/or hormonal treatments. Single-cell RNA sequencing revealed that the experimental PBDE exposure during the post-menopausal period caused specific transcriptomic changes in the non-epithelial compartment such as Errfi1 upregulation in fibroblasts. The environmental PBDE exposure resulted in similar transcriptomic changes to a lesser extent. In summary, the VCD mouse model provides both perimenopausal and menopausal windows of susceptibility for the breast cancer research community. PBDEs, including all tested models, may affect the post-menopausal gland including impacts on the non-epithelial compartments.

BACKGROUND:Intratumour heterogeneity is a hallmark of most solid tumours, including breast cancers. We applied spatial transcriptomics and single-cell RNA-sequencing on patient-derived xenografts (PDXs) to profile spatially resolved cell populations within oestrogen receptor-positive (ER+ ) breast cancer and to elucidate their importance in oestrogen-dependent tumour growth. METHODS:Two PDXs of 'ER-high' breast cancers with opposite oestrogen-mediated growth responses were investigated: oestrogen-suppressed GS3 (80-100% ER) and oestrogen-dependent SC31 (40-90% ER) models. The observation was validated via single-cell analyses on an 'ER-low' PDX, GS1 (5% ER). The results from our spatial and single-cell analyses were further supported by a public ER+ breast cancer single-cell dataset and protein-based dual immunohistochemistry (IHC) of SC31 examining important luminal cancer markers (i.e., ER, progesterone receptor and Ki67). The translational implication of our findings was assessed by clinical outcome analyses on publicly available cohorts. RESULTS:Our space-gene-function study revealed four spatially distinct compartments within ER+ breast cancers. These compartments showed functional diversity (oestrogen-responsive, proliferative, hypoxia-induced and inflammation-related). The 'proliferative' population, rather than the 'oestrogen-responsive' compartment, was crucial for oestrogen-dependent tumour growth, leading to the acquisition of luminal B-like features. The cells expressing typical oestrogen-responsive genes like PGR were not directly linked to oestrogen-dependent proliferation. Dual IHC analyses demonstrated the distinct contribution of the Ki67+ proliferative cells toward oestrogen-mediated growth and their response to a CDK4/6 inhibitor. The gene signatures derived from the proliferative, hypoxia-induced and inflammation-related compartments were significantly correlated with worse clinical outcomes, while patients with the oestrogen-responsive signature showed better prognoses, suggesting that this compartment would not be directly associated with oestrogen-dependent tumour progression. CONCLUSIONS:Our study identified the gene signature in our 'proliferative' compartment as an important determinant of luminal cancer subtypes. This 'proliferative' cell population is a causative feature of luminal B breast cancer, contributing toward its aggressive behaviours.

Background Intratumor heterogeneity is a hallmark of most solid tumors, including breast cancers. We applied spatial transcriptomics and single-cell RNA-sequencing technologies to profile spatially resolved cell populations within estrogen receptor-positive (ER + ) metastatic breast cancers and elucidate their importance in estrogen-dependent tumor growth. Methods Spatial transcriptomics and single-cell RNA-sequencing were performed on two patient-derived xenografts (PDXs) of “ER-high” metastatic breast cancers with opposite estrogen-mediated growth responses: estrogen-suppressed GS3 (80–100% ER) and estrogen-stimulated SC31 (30–75% ER) models. The analyses included samples treated with and without 17β-estradiol. The findings were validated via scRNA-seq analyses on “ER-low” estrogen-accelerating PDX, GS1 (5% ER). The results from our spatial and single-cell analyses were further supported by the analysis of a publicly available single cell dataset and a protein-based dual immunohistochemical (IHC) evaluation using three important clinical markers [i.e., ER, progesterone receptor (PR), and Ki67]. The translational implication of these results was assessed by clinical outcome analyses on public breast cancer cohorts. Results Our novel space-gene-function study revealed a “proliferative” cell population in addition to three major spatially distinct compartments within ER + metastatic breast cancers. These compartments showed functional diversity (i.e., estrogen-responsive, proliferative, hypoxia-induced, and inflammation-related). The “proliferative ( MKI67 + )” population, not “estrogen-responsive” compartment, was crucial for estrogen-dependent tumor growth, leading to the acquisition of luminal B features. The cells with induction of typical estrogen-responsive genes such as PGR were not directly linked to estrogen-dependent proliferation. Additionally, the dual IHC analyses demonstrated the distinct contribution of the Ki67 + proliferative cells toward estrogen-mediated growth and their response to palbociclib, a CDK4/6 inhibitor. The gene signatures developed from the proliferative, hypoxia-induced, and inflammation-related compartments were significantly correlated with worse clinical outcomes, while patients with the high estrogen-responsive scores showed better prognosis, confirming that the estrogen-responsive compartment would not be directly associated with estrogen-dependent tumor progression. Conclusions For the first time, our study elucidated a “proliferative” cell population distinctly distributed in ER + metastatic breast cancers. They contribute differently toward progression of these cancers, and the gene signature in the “proliferative” compartment is an important determinant of luminal cancer subtypes.

Background: While estrogen typically promotes the progression of hormone-dependent breast cancer via the activation of estrogen receptor (ER)-α, estrogen-induced suppression of ER+ breast cancer has been clinically observed. Our previous study demonstrated that estrogen increased the percentage of cells expressing IL24, linking to the estrogen-dependent growth inhibition of an ER+ aromatase inhibitor (AI)-resistant tumor (Mori et al. Cancers, 2021). To continue our evaluation, we investigated whether progesterone (P4) and dihydrotestosterone (DHT) would affect the growth of this estrogen-suppressive tumor. Methods: An estrogen-suppressive patient-derived xenograft (PDX) model (named GS3) was established from an AI resistant ER+/HER2– brain metastatic breast cancer. E2 (1mg), P4 (10mg), DHT (12.5mg), or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC) and RNA sequencing of PDX specimens were conducted to decipher molecular changes after each treatment. The single-cell RNAseq analysis was further performed to examine gene expression profiles in individual cells. Results: ERα, ERβ, Progesterone receptor (PR), and androgen receptor (AR) genes in GS3 are wild-type and are not amplified. Measurements of tumor volume showed that E2, E2+P4, and DHT suppressed the growth of GS3. The Tumor growth was not modulated by P4 treatment. IHC indicated that the number of Ki-67+ cells were decreased after E2, E2+P4, and DHT treatments, but were not changed after P4 treatment. PR+ cells appeared after E2 and E2+P4 treatments, and the AR+ cells increased after DHT treatment. Bulk-RNA sequencing indicated that E2 and E2+P4 treatments resulted in comparable gene expression patterns, while those of placebo and P4-treated tumors were similar. GSEA analysis showed that in E2 treatment, the hallmark estrogen response gene sets were upregulated, and the hallmark G2M checkpoint gene set was downregulated. However, in DHT treatment, the hallmark interferon alpha/gamma response gene sets and androgen response gene set were upregulated, and the hallmark TNFA signaling via NFkB gene set was downregulated. Single-cell RNA sequencing analysis of Placebo/E2/DHT samples revealed 9 clusters; cells from E2-treated and DHT-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes, although both E2 and DHT treatments resulted in tumor regression. DHT promoted cell cycle arrest, but it did not increase IL24 expression. The hallmark oxidative phosphorylation and androgen response gene sets were upregulated in all clusters from DHT-treated tumors. Trajectory analysis of single cells revealed that three major branches associated with clusters selective to the treatments of placebo, E2, and DHT were separated from a common branch which are consist of G2M phase cells. Conclusions: E2 and DHT were effective suppressor of GS3, but not P4. Based on the results from IHC, bulk-RNAseq, and Single-cell RNAseq, the mechanism of DHT-induced tumor regression is different from that by E2. Our results suggest that DHT/E2 could be treatment options for patients with relapsed ER+ AI-resistance breast cancer. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Jinhui Wang, Xiwei Wu, Noriko Kanaya, George Somlo, Shiuan Chen. Efficacy of Gonadal steroids on aromatase inhibitor-resistant ER+ breast cancer: Insights from single-cell trajectory analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-05.

Abstract Introduction Polybrominated diphenyl ethers (PBDEs) are persistent environmental contaminants and associated with breast carcinogenesis. In this study, we examined the effects of PBDEs during the menopausal transition in a 4-vinylcyclohexene diepoxide (VCD)-induced mouse menopause model, which presents a follicle-depleted and ovary-intact animal that closely approximate the gradual menopause transition in humans. The aim of this study was to compare the impacts of PBDE exposures on the mammary gland at the macroscopic gland structures and the single-cell resolution under three treatment schedules. Materials and Methods We treated nine-week-old female C57BL/6 mice with VCD (160 mg/kg) for 15 days. Then, the animals had high dose PBDE exposure for 1 week during peri- (Perimenopause/High group) or post-menopausal periods (Menopause/High group). The third group was exposed to low dose PBDE (the 1/20th of the high dose) for 20 weeks during the postmenopausal period (Menopause/Low group). In each group, the mice received various combinations of 17B-estradiol and progesterone treatments simultaneously. Our experimental design allowed us to directly compare the effects of PBDE under three potential exposure conditions. Results The mammary glands significantly regressed by the VCD treatment, more evident at the postmenopausal period. All PBDE exposures did not augment or compromise the macroscopic ductal reorganization resulting from the VCD and/or hormonal treatments. Single-cell sequencing revealed that the PBDE exposure in the Menopause/High group caused specific transcriptomic changes in the non-epithelial compartment such as Errfi1 upregulation in fibroblasts and AY036118 downregulation in multiple cell types. The PBDE exposure in the Menopause/Low group resulted in similar transcriptomic changes to a lesser extent. Conclusion PBDEs may affect the postmenopausal gland through impacts on the non-epithelial compartments. Low dose PBDE exposure, which is presumably equivalent to the maximum environmental exposure in humans, can exert the influences. Presentation: No date and time listed

Fibroblasts have been shown to be one of the essential players for mammary gland organization. Here, we identify two major types of mouse mammary gland fibroblasts through single-cell RNA sequencing analysis: Dpp4+ fibroblasts and Dpp4- fibroblasts. Each population exhibits unique functional characteristics as well as discrete localization in normal mouse mammary glands. Remarkably, estrogen, a crucial mediator of mammary gland organization, alters the gene expression profiles of fibroblasts in a population-specific manner, without distinct activation of estrogen receptor signaling. Further integrative analysis with the inclusion of five other publicly available datasets reveals a directional differentiation among the mammary gland fibroblast populations. Moreover, the combination with the mouse mammary epithelium atlas allows us to infer multiple potential interactions between epithelial cells and fibroblasts in mammary glands. This study provides a comprehensive view of mouse mammary gland fibroblasts at the single-cell level.

Background: Estrogen typically promotes the progression of hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1. While estrogen-induced tumor suppression in ER+ breast cancer has been clinically observed as an unexpected outcome of aromatase inhibitor (AI)-resistance, the molecular mechanisms have not yet been fully defined. Methods: Characterization of estrogen regulation in the two ER+ breast cancer patient-derived xenograft (PDX) models with opposite responses to estrogen offered us an unprecedented opportunity to assess how 17β-estradiol (E2) modulates ER+ cancer. We succeeded in establishing estrogen-stimulating (SC31) and estrogen-suppressing (GS3) PDX models. In vivo tumor promotion or suppression by estrogen was confirmed through experiments by implanting E2 pellets in mice carrying SC31/GS3, and then single cell analysis was performed using SC31/GS3 tumors. To investigate whether GS3 would change its response to E2, we performed intermittent E2 treatment every 28 days (E2 pellet on/off every 28 days) and whole-genome RNA sequencing using GS3 tumors. Results: SC31 and GS3 behaved oppositely regarding estrogen-mediated tumor growth. Immunohistochemistry indicated that the number of ERα+ cells and Ki-67+ cells were increased in SC31 and decreased in GS3 after E2 treatment, but progesterone receptor+ cells appeared in both SC31 and GS3 after E2 treatment at the protein level. The E2-induced suppression of GS3 involves ERα, not ERβ, which was wild-type and not amplified. Single cell RNA sequencing analysis of these PDXs had revealed that E2 upregulated the expression of estrogen-regulated genes (e.g., PGR and AREG) in both SC31 and GS3. However, E2 treatment induced cell cycle promotion in SC31, while E2 induced cell cycle arrest in GS3. These gene-expression changes occurred in both ESR1+ cells and ESR1- cells, demonstrating for the first time the influence of estrogen on ESR1- cells in ER+breast tumors. This result suggests that 100% ER positivity is not essential for endocrine response. E2 also upregulated a tumor suppressor gene, IL24, only in GS3. More IL24+ cells were ESR1+ and in G1 phase than IL24- cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24+ cells after E2 treatment. After three rounds of intermittent E2 treatment on GS3, an E2 independent growth developed. Approximately 60% of genes in the intermittent E2-treated sample had the same trend as E2-treated samples (mainly ER target genes), in which 40% of the genes behaved similarly to the placebo-treated sample (mainly cell cycle progression genes). Furthermore, lower levels of IL24 were linked to estrogen independence. Conclusions: Estrogen/ERα signaling increases the expression of estrogen-regulated genes, but it can modulate HR+ tumor growth in different manners. While E2 should activate ERα and regulate ESR1+ cells, our findings regarding ESR1- cells were new and suggested crosstalk between ESR1+ cells and ESR1- cells in both estrogen-stimulating and -suppressing ER+ tumors. Furthermore, our studies demonstrate the potential roles of tumor suppressor gene, IL24 in HR+ cancer. Our findings point to the need to identify biomarkers for patients with estrogen-suppressing tumors who can benefit from E2 treatment after AI resistance; measurements of ER and PR expression are insufficient. Expression of IL24 in AI-resistant tumors may be one such indicator for favorable response to E2 therapy. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Jinhui Wang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Masafumi Nakamura, Andrea Bild, Shiuan Chen. Estrogen-mediated mechanisms in estrogen receptor-positive breast cancer at the single cell level [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-04.

Abstract Mammary gland is an essential organ for milk production. It continues to develop after birth under the influence of ovarian hormones, especially estrogen. Among its cellular components, fibroblasts are recognized as an important player for mammary gland organization. Although human mammary gland fibroblasts have been classified into two subtypes based on histological localizations and different marker expressions (i. e., interlobular and intralobular fibroblasts), their detailed characteristics and functions are still limited. Moreover, despite the importance of experimental mouse models for studying mammary gland biology, heterogeneity of mouse mammary gland fibroblasts has not been fully defined. Here, we performed single-cell RNA sequencing (scRNA-seq) to determine cellular populations and their potential contributions to mammary gland organization. Based on the comprehensive gene expression profiles at a single cell level, we identified two major clusters of the mouse mammary gland fibroblasts. One of the major clusters showed the highly specific expression of Dpp4 gene, which is a known marker for human interlobular fibroblasts, and the upregulation of the inflammatory response gene signature. The other major cluster was consisted of three subclusters without Dpp4 expression, which suggested the distinct contributions to adipogenesis and/or ECM modeling in mammary gland stroma. Through the histological validation, we defined these two major subsets as "DPP4+" and "DPP4-" fibroblasts. Since our scRNA-seq was performed on two different ovarian hormone-depleted mouse models followed with hormone treatments, we evaluated the effect of the hormones, especially estrogen. The results demonstrated that the estrogen treatment altered the gene expression profiles of the DPP4+ and a subcluster of the DPP4- fibroblasts in a population-specific manner. Estrogen upregulated interferon-regulated genes and ECM genes in these fibroblast populations, respectively, but not typical estrogen-regulated genes, even in the ERα+ DPP4+ fibroblasts. To define these mammary gland fibroblasts better, we integrated our datasets with a recently established "steady-state mouse fibroblasts atlas" (1) as well as four other scRNA-seq datasets containing normal mouse mammary gland fibroblasts. Using this integrated dataset, we revealed the directional differentiation among the mammary gland fibroblasts, going from the fibroblasts and branching toward the DPP4- fibroblasts with more specialized functions. Moreover, the cell-cell interaction analysis combined with a mammary epithelium dataset from our previous publication (2) indicated possible cell-type-specific communications between mammary gland fibroblasts and epithelial cells. In conclusion, this study provided a comprehensive view of mouse mammary gland fibroblast biology at the single-cell level. Our findings would provide fundamental insights for further investigations on the roles of different fibroblast populations in mammary gland organization and pathological conditions, such as breast cancer. Reference: (1) Buechler et al., Nature. 2021 May;593(7860): 575–579. (2) Saeki et al., Commun Biol. 2021 Jun;4(1): 1–16. Presentation: No date and time listed

Identification of chemicals that affect hormone-regulated systems will help to predict endocrine disruption. In our previous study, a 46 gene biomarker was found to be an accurate predictor of estrogen receptor (ER) α modulation in chemically treated MCF-7 cells. Here, potential ERα modulators were identified using the biomarker by screening a microarray compendium consisting of ∼1600 gene expression comparisons representing exposure to ∼1200 chemicals. A total of ∼170 chemicals were identified as potential ERα modulators. In the Connectivity Map 2.0 collection, 75 and 39 chemicals were predicted to activate or suppress ERα, and they included 12 and six known ERα agonists and antagonists/selective ERα modulators, respectively. Nineteen and eight of the total number were also identified as active in an ERα transactivation assay carried out in an MCF-7-derived cell line used to screen the Tox21 10K chemical library in agonist or antagonist modes, respectively. Chemicals predicted to modulate ERα in MCF-7 cells were examined further using global and targeted gene expression in wild-type and ERα-null cells, transactivation assays, and cell-free ERα coregulator interaction assays. Environmental chemicals classified as weak and very weak agonists were confirmed to activate ERα including apigenin, kaempferol, and oxybenzone. Novel activators included digoxin, nabumetone, ivermectin, and six progestins. Novel suppressors included emetine, mifepristone, niclosamide, and proscillaridin. Our strategy will be useful to identify environmentally relevant ERα modulators in future high-throughput transcriptomic screens.

Background: Estrogen is known to promotes hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1 . However, several clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)-resistant cases of ER + breast cancer. Considering potential impact of such clinical observation, we decided to determine the mechanisms of estrogen-induced tumor regression. Methods: A unique estrogen-inhibitory patient-derived xenograft (PDX) tumor, GS3, was established from an AI resistant ER + /HER2 – brain metastatic breast cancer. In vivo estrogen suppression was confirmed through experiments by implanting 17β-estradiol (E2) pellets in mice carrying GS3, and then the single-cell analysis was performed using GS3 tumors. In vitro E2 suppression analysis was carried out using organoids from GS3. Results: The E2-induced suppression of GS3 involves ERα, which was wild-type and not amplified. Single cell RNA sequencing analysis of this PDX has revealed that E2 treatment (for 1 week) induces cell cycle arrest in both ESR1 + cells and ESR1 – cells, demonstrating the unexpected influence of estrogen on ESR1 – cells in ER + breast cancer. E2 upregulated the expression of estrogen-regulated genes, including a tumor suppressor gene, IL24 , and lower levels of IL24 were linked to estrogen independence, after three rounds of intermittent E2 treatment. IL24 + cells included more G1 phase cells of cell cycle compared to IL24 – cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24 + cells after E2 treatment. The number of apoptotic cells was significantly increased after long term (for 4 weeks) E2 treatment. Western blotting analysis demonstrated that long term E2 treatment induced expression of apoptosis-associated protein cleaved-PARP and reduction of the pro-survival protein Bcl-xl level. Conclusions: There is the need of markers for patients who can benefit from E2 treatment after AI resistance, and measurements of ER and PR expression are not enough. Analysis of GS3 PDX has revealed that estrogen induces cell cycle arrest and apoptosis. Our study has revealed the cross-talk between ESR1 + and ESR1 – cells as well as potential roles of IL24 in estrogen-suppressive tumors.

The female mammary epithelium undergoes reorganization during development, pregnancy, and menopause, linking higher risk with breast cancer development. To characterize these periods of complex remodeling, here we report integrated 50K mouse and 24K human mammary epithelial cell atlases obtained by single-cell RNA sequencing, which covers most lifetime stages. Our results indicate a putative trajectory that originates from embryonic mammary stem cells which differentiates into three epithelial lineages (basal, luminal hormone-sensing, and luminal alveolar), presumably arising from unipotent progenitors in postnatal glands. The lineage-specific genes infer cells of origin of breast cancer using The Cancer Genome Atlas data and single-cell RNA sequencing of human breast cancer, as well as the association of gland reorganization to different breast cancer subtypes. This comprehensive mammary cell gene expression atlas (https://mouse-mammary-epithelium-integrated.cells.ucsc.edu) presents insights into the impact of the internal and external stimuli on the mammary epithelium at an advanced resolution. Kohei Saeki et al. present a mammary cell atlas derived from mouse and human single-cell RNA-sequence data. They characterize key life stages in which the mammary gland undergoes complex remodeling and, using breast cancer expression data, infer cells of origin for carcinogenesis of various breast cancer subtypes.

Abstract Extensive efforts, through cell line-based models, have been made to characterize the androgen receptor (AR) signaling pathway in triple-negative breast cancer (TNBC). However, these efforts have not yet reached a consensus with regards to the mechanism of AR in TNBC. On the other hand, patient-derived xenografts (PDXs) are generally considered more appropriate than cell line-based models for recapitulating the structural and molecular features of a patient’s tumor, but only a few have been reported to be AR-positive TNBC. In our study, we identified and molecularly characterized two new, AR-positive TNBC PDX models and assessed the impacts of AR agonist (DHT) and antagonist (enzalutamide) on tumor growth and gene expression profiles by utilizing immunohistochemistry (IHC), western blots, and RNA-Seq and TNBC subtyping analyses. Two PDX models, termed TN1 and TN2, were derived from two grade 3 TNBC tumors, each containing 1~5% of AR positive tumor cells. DHT activated AR in both PDX tumors by increasing AR nuclear localization and protein levels. However, the endpoint tumor volume of DHT-treated TN1 was 3-folds smaller than that of non-treated TN1 tumors. Conversely, the endpoint tumor volume of DHT-treated TN2 was 2-folds larger than that of non-treated TN2. Moreover, enzalutamide failed to antagonize DHT-induced tumor growth in TN2. The RNA-Seq analyses revealed that DHT suppressed gene expression in TN1 (961 down-regulated genes versus 149 up-regulated genes), while the DHT promoted gene expression in TN2 (673 up-regulated genes versus192 down-regulated genes). TNBC subtyping analyses based on RNA-Seq data predicted distinct molecular subtypes of TN1 and TN2: TN1 correlated to a basal-like 1 (BL1) subtype, and TN2 correlated to a basal-like 2 (BL2) subtype. These analyses suggest that TN1 and TN2, which both express functional AR, are two molecularly distinct PDX models that expand our current knowledge of AR-positive TNBC. Our results do not support that AR is a suitable therapeutic target in TNBC. To our best knowledge, the molecular mechanisms of AR in TNBC are equivocal and should be evaluated using clinically relevant models, considering both the heterogeneous expression of AR in TNBC and the general complexities of AR signaling.

Background: Estrogen typically promotes the progression of hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1 . While estrogen-induced tumor suppression in ER + breast cancer has been clinically observed as an unexpected outcome of aromatase inhibitor (AI)-resistance, the molecular mechanisms have not yet been fully defined. Characterization of estrogen regulation in two ER + breast cancer patient-derived xenograft (PDX) models with opposite responses to estrogen offered us an unprecedented opportunity to assess how 17β-estradiol (E2) modulates ER + cancer. Methods: We established two PDX breast cancer models in mice using ER + tumors from patients that responded (SC31) or were suppressed (GS3) by exogenous estrogen. In vivo tumor promotion or suppression by estrogen were confirmed through experiments by implanting E2 pellets in mice carrying SC31 or GS3, and then single-cell analysis was performed. Results: E2 promoted SC31 tumor growth but suppressed growth of GS3 in vivo . The E2-mediated suppression of GS3 involves ERα, which was wild-type and not amplified. Single-cell RNA sequencing analysis showed that E2 treatment induced cell cycle promotion in SC31, while E2 induced cell cycle arrest in GS3. However, E2 treatment upregulated the expression of estrogen-regulated genes in both tumors. These gene-expression changes by E2 occurred in both ESR1 + cells and ESR1 – cells within the same tumor, demonstrating for the first time the influence of estrogen on ESR1 – cells in ER + breast tumors. E2 also upregulated a tumor suppressor gene, IL24 , only in GS3, and lower levels of IL24 were linked to estrogen independence, after three rounds of intermittent E2 treatment. More IL24 + cells were ESR1 + and in G1 phase than IL24 – cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24 + cells after E2 treatment. Conclusions: Our study has revealed the effects of estrogen treatment on both ESR1 + and ESR1 – cells in ER + tumors, but not all ER + cancers respond the same manner to estrogen. SC31 is a tumor that is stimulated by E2, while GS3 is suppressed by E2 via cell cycle arrest. Our results indicate a potential role of IL24 in estrogen-suppressive tumors.

Abstract Background: Estrogen such as estradiol (E2) is known to promote ER+ breast cancer. However, several clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)-resistant cases of ER+ postmenopausal breast cancer. The objective of this study is to uncover the mechanisms of E2-induced tumor regression, leading to an unconventional treatment of AI resistance. Methods: An E2-suppressive patient-derived xenograft model (named GS3) was established from an AI resistant ER+/PR-/HER2- brain metastatic breast cancer. Placebo or E2 pellets were implanted in mice carrying GS3 for evaluating the effects of E2. Immunohistochemistry (IHC) and RNA sequencing of GS3 were conducted to decipher molecular changes after E2 treatments. Since the cancer tissue has a heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist, not by ERβ antagonist. IHC showed that ER, Ki-67 and CEA expressions decreased and PR expression appeared after E2 treatment. Gene set enrichment analysis (GSEA) using RNAseq results showed that the E2 response gene sets were significantly up-regulated after E2 treatment. However, the cell cycle gene sets and the TNFA/NFKB gene set were down-regulated. GS3 gained an E2 independence after three cycles of intermittent E2 treatment (E2 pellet on/off every 4 weeks; Int-E2). Interestingly, the cell cycle and TNFA/NFKB gene sets were up-regulated after Int-E2 treatment. Single-cell RNAseq analysis revealed that cells from one-week E2-treated and Placebo-treated GS3 were placed in different clusters based on principle component analysis of Highly Variable Genes. Although E2 response genes were up-regulated, the percent of ESR1+ cells decreased after E2 treatment (41.3% vs. 31.5%). The number of cells arrested at the G1 phase increased (+12.5%) after E2 treatment. GSEA using genes expressed in only ESR1+ cells showed that the TNFA/NFKB gene set was significantly down-regulated after E2 treatment. Meanwhile, GSEA using genes expressed in only ESR1– cells showed that cell cycle gene sets were significantly down-regulated. Single-cell trajectory analysis disclosed three major branches; 1) common E2 and placebo, 2) E2, and 3) placebo. In the E2 only branch, the cell cycle arrested at the G1 phase, the E2 response gene sets were up-regulated, and the NFKB gene set was down-regulated in ESR1+ cells. Significantly, E2 response gene sets were also up-regulated and cell cycle genes were down-regulated even in ESR1– cells. In the placebo branch, E2 response gene sets were not up-regulated and cell cycle genes were not down regulated. A group of MKI67+ cells (at G2M phase), including some ESR1+ cells, were present in both E2-treated and placebo-treated tumors. Conclusions: E2-induced suppression is an unexpected outcome of AI resistance. In these cases, elimination of estrogen by AI results in maintaining tumor growth. Analysis of GS3 PDX has revealed that estrogen can induce cell cycle arrest and the expression of estrogen-regulated genes. Our results also suggest the cross-talk between ESR1+ and ESR1- cells as well as potential roles of the TNFA/NFKB pathway. Our findings point out the need of markers for such patients who can benefit from E2 treatment after AI resistance, and measurements of ER and PR expression are not sufficient. An intermittent treatment strategy does not sustain the effect of estrogen-mediated suppression. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest as an unexpected outcome of aromatase inhibitor-resistance: Insights from single-cell trajectory analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-02.

A 100% ER positivity is not required for an endocrine therapy response. Furthermore, while estrogen typically promotes the progression of hormone-dependent breast cancer via the activation of estrogen receptor (ER)-α, estrogen-induced tumor suppression in ER+ breast cancer has been clinically observed. With the success in establishing estrogen-stimulated (SC31) and estrogen-suppressed (GS3) patient-derived xenograft (PDX) models, single-cell RNA sequencing analysis was performed to determine the impact of estrogen on ESR1+ and ESR1– tumor cells. We found that 17β-estradiol (E2)-induced suppression of GS3 transpired through wild-type and unamplified ERα. E2 upregulated the expression of estrogen-dependent genes in both SC31 and GS3; however, E2 induced cell cycle advance in SC31, while it resulted in cell cycle arrest in GS3. Importantly, these gene expression changes occurred in both ESR1+ and ESR1– cells within the same breast tumors, demonstrating for the first time a differential effect of estrogen on ESR1– cells. E2 also upregulated a tumor-suppressor gene, IL-24, in GS3. The apoptosis gene set was upregulated and the G2M checkpoint gene set was downregulated in most IL-24+ cells after E2 treatment. In summary, estrogen affected pathologically defined ER+ tumors differently, influencing both ESR1+ and ESR1– cells. Our results also suggest IL-24 to be a potential marker of estrogen-suppressed tumors.

Abstract Background: Estradiol (E2) is known to promote breast cancer. However, several small-scale prospective clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)- resistant cases of hormone receptor-positive postmenopausal breast cancer. Considering that the practicality and safety of E2 treatments are still undefined, the objective of this study is to uncover the mechanisms of E2-induced tumor regression, which will lead to a better design of E2 therapies. Methods: While most of our ER+ breast cancer patient-derived xenograft (PDX) models are positively regulated by E2, an estrogen-suppressive model (named GS3) was established from an AI resistant ER+/PR−/HER2− brain metastatic breast cancer. E2 pellets (1mg each) or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC), RNA sequencing, and reverse phase protein array (RPPA) analyses of PDX specimens were conducted to decipher molecular changes after E2 treatments. Importantly, since the cancer tissue has a very heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist (MPP), not by ERβ antagonist (PHTPP). IHC showed that PR expression appeared, Ki-67 and CEACAM5 (CEA) expressions decreased, and the number of apoptotic cells significantly increased after E2 treatment. RNA sequencing indicated that estrogen-regulated genes, e.g., PGR, EGR3, PDZK1, and GREB1, were up-regulated, while tumor growth was repressed by E2. Single cell RNAseq analysis demonstrated that cells from E2-treated and Placebo-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes (HVGs). Importantly, after one-week, many cells of E2-treated PDXs were arrested within the G1 phase of the cell cycle (G1, 60%; S, 19%; G2M, 21%), whereas, cells of placebo-treated samples advanced to the S and G2M phases (G1, 47%; S, 27%; G2M, 26%). Gene Set Variation Analysis of cells from E2-treated PDXs revealed enrichment of genes associated with reactive oxygen species (ROS) mechanism. E2 increased cells expressing pro-apoptotic genes, such as TP53, BOK,IL24, and PGLYRP2. Gene expressions including mitogen-activated protein kinase (MAPK) signaling pathway, such as GADD45A, MAP3K5 (ASK1), MAPK9 (JNK2), and especially MAPKAPK2 were also increased in E2 treated PDXs. Conclusions: E2/ERα-mediated suppression of GS3 tumor growth was accompanying the inhibition of the cell cycle progression and increased expression of pro-apoptotic genes. It is known that classical MAPK pathway induces cell proliferation, meanwhile, JNK MAPK pathway induces p53 signaling pathway and apoptosis. We hypothesize that E2 induces ROS and promotes apoptosis of GS3 due to JNK MAPK pathway activation. This could be an unexpected outcome of AI resistance. Using this valuable estrogen-down-regulated ER+ breast cancer PDX and informative integrative omics analyses, critical molecular mechanisms on E2-mediated apoptosis are being revealed in our laboratory. The ongoing research using E2-sensitive PDXs will identify key markers to select AI resistant patients who are expected to respond effectively to estrogen-induced apoptosis treatments as well as to evaluate the efficacy of drug combination with estrogen, including CDK 4/6 inhibitors. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest and apoptosis in aromatase inhibitor-resistant breast cancer: Insights from single cells analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-08-01.

The aromatase inhibitors (AIs), letrozole (Femar®/Femara®) and exemestane (Aromasin®), are widely used to treat estrogen receptor (ER) positive breast cancer in postmenopausal patients. In the setting of metastatic breast cancer, these drugs may be used after another causing new responses in selected patients after progressing on the first choice. The precise explanation for this "lack of cross resistance" is still missing. NEOLETEXE is a neoadjuvant, randomized, open-label, cross-over trial. Postmenopausal patients with ER-positive, HER-2 negative, locally advanced breast cancer were enrolled. All patients were randomized to treatment starting with either letrozole or exemestane for at least 2 months followed by another 2 months on the alternative AI. The total estrogenic activities in blood samples were determined using the AroER tri-screen assay developed in the Chen laboratory. Using this highly sensitive assay, estrogenic activity was detected at three time points for all patients. Importantly, a significantly higher total estrogenic activity was found during therapy with exemestane compared to letrozole in 21 out of 26 patients. When letrozole was included in the AroER tri-screen assay, the estrogenic activities in most samples collected during exemestane treatment were further reduced, suggesting that low levels of androgens remained in specimens obtained after exemestane treatment. Our results suggest the AroER tri-screen to be a very sensitive method to estimate the overall estrogen-mediated activity in human samples even during therapy with highly potent aromatase inhibitors. In the present study, serum estrogen activity was significantly higher during exemestane therapy when compared to letrozole therapy.

Abstract Menopause is considered a critical window of susceptibility (WOS) for its sensitivity to endocrine-disrupting chemicals due to decline of endogenous estrogen. Using a surgical menopausal (ovariectomized) mouse model, we assessed how mammary gland tissue was affected by both 17β-estradiol (E2) and polybrominated diphenyl ethers (PBDEs). These endocrine-disrupting chemicals have been used as flame retardants in common household products since the 1970s and are vastly detected in human serum. During physiologically relevant exposure to E2, PBDEs enhanced E2-mediated regrowth of mammary glands with terminal end budlike structures. To better elucidate the effects of E2 and PBDEs, single-cell RNA sequencing (scRNAseq) analysis was performed. In a definitive manner, E2 was found to induce Pgr expression in both Esr1+ and Esr1- cells. We identified PBDE-impacted cell populations, Esr1+/Pgr+ and Esr1-/Pgr+ cells, in both mature luminal epithelial and progenitor cells, as well as the mammary gland population of M2 macrophages. Significantly, scRNAseq data show gene expression differences among cells with differential expression of Esr1 and Pgr. The results help clarify how exposure to both E2 and endocrine-disrupting chemicals like PBDEs after menopause impacts mammary gland structure. Ultimately, the findings advance understanding of how such exposure can increase the risk of developing breast cancer through the expansion of estrogen-responsive cells. (Supported by NIH U01ES026137.) Citation Format: Shiuan Chen, Noriko Kanaya, Gregory Chang, Xiwei Wu, Kohei Saeki, Lauren Bernal, Timothy Dynold, Susan L. Neuhausen. Single-cell RNA-sequencing analysis of estrogen and the endocrine-disrupting chemical induced mouse mammary gland reorganization after surgical menopause [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A30.

Extensive efforts, through cell line-based models, have been made to characterize the androgen receptor (AR) signaling pathway in triple-negative breast cancer (TNBC). However, these efforts have not yet reached a consensus with regards to the mechanism of AR in TNBC. Considering that patient-derived xenografts (PDXs) are more appropriate than cell line-based models for recapitulating the structural and molecular features of a patient's tumor, we have identified and molecularly characterized two new AR-positive TNBC PDX models and assessed the impacts of AR agonist [dihydrotestosterone (DHT)] and antagonist (enzalutamide) on tumor growth and gene expression profiles by utilizing immunohistochemistry, western blots, and RNA-Seq analyses. Two PDX models, termed TN1 and TN2, were derived from two grade-3 TNBC tumors, each harboring 1∼5% of AR nuclear positive cancer cells. DHT activated AR in both PDX tumors by increasing nuclear localization and AR protein levels. However, the endpoint tumor volume of DHT-treated TN1 was 3-folds smaller than that of non-treated TN1 tumors. Conversely, the endpoint tumor volume of DHT-treated TN2 was 2-folds larger than that of non-treated TN2. Moreover, enzalutamide failed to antagonize DHT-induced tumor growth in TN2. The RNA-Seq analyses revealed that DHT mainly suppressed gene expression in TN1 (961 down-regulated genes versus 149 up-regulated genes), while DHT promoted gene expression in TN2 (673 up-regulated genes versus 192 down-regulated genes). RNA-Seq data predicted distinct TNBC molecular subtypes for TN1 and TN2. TN1 correlated to a basal-like 1 (BL1) subtype, and TN2 correlated to a basal-like 2 (BL2) subtype. These analyses suggest that TN1 and TN2, which both express functional AR, are two molecularly distinct PDX models. The molecular characterization of these PDX models expands our current knowledge on AR-positive TNBC. Our results do not support that AR is a suitable therapeutic target in TNBC. To our best knowledge, the molecular mechanisms of AR in TNBC are equivocal and should be evaluated using clinically relevant models, considering both the heterogeneous expression of AR in TNBC and the general complexities of AR signaling.