Mature Luminal Cells Research Articles

R-loop functions in Brca1-associated mammary tumorigenesis

Deleterious accumulation of R-loops, a DNA-RNA hybrid structure, contributes to genome instability. They are associated with BRCA1 mutation-related breast cancer, an estrogen receptor α negative (ERα-) tumor type originating from luminal progenitor cells. However, a presumed causality of R-loops in tumorigenesis has not been established in vivo. Here, we overexpress mouse Rnaseh1 (Rh1-OE) in vivo to remove accumulated R-loops in Brca1-deficient mouse mammary epithelium (BKO). R-loop removal exacerbates DNA replication stress in proliferating BKO mammary epithelial cells, with little effect on homology-directed repair of double-strand breaks following ionizing radiation. Compared to their BKO counterparts, BKO-Rh1-OE mammary glands contain fewer luminal progenitor cells but more mature luminal cells. Despite a similar incidence of spontaneous mammary tumors in BKO and BKO-Rh1-OE mice, a significant percentage of BKO-Rh1-OE tumors express ERα and progesterone receptor. Our results suggest that rather than directly elevating the overall tumor incidence, R-loops influence the mammary tumor subtype by shaping the cell of origin for Brca1 tumors.

Closing classification gaps in luminal breast cancer with single-cell RNA-seq insights from normal breast lineages.

e13168 Background: Almost two decades ago, invasive breast cancer was classified into five subgroups based on expression patterns, each with distinct biological and prognostic characteristics. Advances in single-cell RNA sequencing offers insights into the expression profiles of normal luminal epithelial cells. Here, we investigate the connection between breast cancer (BC) classification and luminal expression profiles. Methods: We applied single-cell data from recent studies (Table) to compile gene signatures corresponding to expression profiles of luminal epithelial cell clusters in normal breast tissue. Normalized enrichment scores (ESs) of these signatures were calculated for bulk RNA-seq gene expression data from open source cohorts (TCGA-BRCA, SCAN-B, METABRIC). The BC molecular subtype of each tumor sample was predicted based on a modified PAM50 classification using gene expression profiling (1); LumA and LumB subtypes were combined into Luminal. ESs of signatures were compared between the BC subtypes (Table). Results: The ESs of all signatures differed significantly across BC subtypes (p < 0.001, Kruskal-Wallis test, Table 1). The ESs of signatures for LumSec-major and Luminal progenitor clusters are the highest in subtypes HER2-low and Basal-like (p < 0.001, Dwass-Steel-Critchlow-Fligner test [DSCF]). Basal-like also had the highest enrichment of the ductal signature (p < 0.001, DSCF). Luminal subtype showed the highest ESs of signatures corresponding to HR+ clusters of mature luminal cells and of the TDLU signature (p < 0.001, DSCF). HER2-low subtype showed the highest ES of the LumSec-lac signature (p < 0.001, DSCF). Conclusions: Our analyses challenge traditional classifications by showing that luminal breast cancers are less similar to most luminal epithelial clusters in normal breast tissue than other breast cancer subtypes. These findings suggest traditional expression classification may not fully capture the complexity of this largest subset of luminal-type breast cancers. 1. Turova et al., SABCS, 2023. [Table: see text]

Abstract 265: Reconstructing the early steps of breast tumor initiation

Abstract Breast cancer screening programs, detect not only the early stages of the disease but also various preneoplastic lesions which are very difficult to predict if and when they may develop into cancer. This uncertainty can lead to over-diagnosis, over-treatment and stress for millions of screened women. As consequence, discovering reliable biomarkers of this evolution could make it possible to monitor these patients appropriately and propose an effective prevention strategy. To meet this challenge, we urgently need to better understand the mechanisms that regulate human mammary homeostasis during early steps of tumor initiation. The hierarchy and the composition of human mammary epithelium, composed of stem, myoepithelial, luminal progenitors and mature luminal cells, are complex. This complexity raises the question of whether a given mammary epithelial cell is equally likely to be the cell-of-origin of breast cancer. Interestingly, increasing evidences show that disruption of lineage integrity can be a precursor of breast tumor initiation, and the basal and the luminal lineages respond differently to a given oncogenic stress and may each follow their own rule towards malignant transformation. These results strongly suggest that each lineage may have its own pliancy to be transformed by a given oncogene, hijacking the inter-tumoral heterogeneity observed in breast cancers. The objectives of this study are to develop a model to reconstruct the early steps of breast tumor initiation and understand the interplay between the initiating oncogenic event and the cell-of-origin. To answer these questions, we used a human mammary epithelial cell lines (HME) that maintains a cellular hierarchy with mammary stem cells (MaSC) and luminal cells (LC). Upon oncogenic expression of HRASG12V or c-Myc, we observed different modifications of cell homeostasis, with altered proportions of the lineages populations in the cell line. Depending on the oncogenic stress induced, we also demonstrated a modification in the morphology and the cellular composition of HME organoids, as well as in the capacity to form tumors. HRASG12V induces increase of MaSC compartment, formation of more branched organoids and generate aggressive basal-like tumors. Conversely, c-MYC leads to an almost loss of MaSC and formation of more proliferative unbranched organoid and generate tumors with limited growth. To address more precisely how cell-of-origin and lineage plasticity influence malignant transformation, we are developing for the first time a lineage tracing model in human mammary epithelium with HME. Moreover, a single cell RNA-seq was performed at different early time points during HME cellular differentiation in homeostasis and upon oncogenic stress to decipher the molecular mechanisms that underpin the very earliest steps of breast tumor initiation. Citation Format: Anaïs Grandon, Shuheng Lin, Martin Castagné, Julien Wicinski, Olivier Rosnet, Christophe Ginestier, Emmanuelle Charafe-Jauffret. Reconstructing the early steps of breast tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 265.

Abstract 3019: Dissecting in vivo functions of R-Loops in Brca1-associated DNA replication stress and mammary tumorigenesis

Abstract Germ-line BRCA1 mutations are associated with a significantly increased incidence of breast cancer in women. Although BRCA1-associated breast tumors are basal-like and estrogen receptor α negative (ERα−), they originate from luminal progenitor cells. At the molecular level, BRCA1 is best known for its roles in resolution of DNA replication stress and promotion of homologous recombination (HR)-based double strand break (DSB) repair. In addition, BRCA1 is implicated in reduction of R-loops, a DNA-RNA hybrid structure and transcriptional byproduct associated with genome instability and transcriptional regulation. Our published findings suggest that BRCA1-dependent R-loop mitigation contributes to luminal cell-specific transcription and differentiation (Zhang et al, 2017, Nature Communications, 8:15908), which could in turn suppress Brca1-associated tumorigenesis. Using cell type-specific mouse genetics, we investigate a direct impact of R-loop removal on Brca1-associated mammary epithelial tumor formation. R-loop formation in mammary epithelial cells is efficiently reduced by over-expression of RNase H1, an R-loop-specific ribonuclease. R-loop removal does not affect mammary duct development or function. However, it drastically increases ɣH2AX foci in proliferating BRCA1-deficient epithelial cells without external DNA damage. This suggests a role of R-loops in attenuating elevated DNA replication stress caused by the loss of Brca1. While R-loop removal does not appear to affect the overall Brca1-associated mammary tumor incidence, it promotes cell differentiation from ERα− luminal progenitor cells to ERα+ mature luminal cells. Accordingly, unlike their counterparts, a significant percentage of Brca1-associated mammary tumors with RNase H1 overexpression tend to be ERα+. Thus, our findings provide in vivo evidence for a previously unappreciated role of R-loop dynamics on Brca1-associated DNA replication stress and tumor development in mammary epithelium. Citation Format: Huai-Chin Chiang, Leilei Qi, Yanfen Hu, Rong Li. Dissecting in vivo functions of R-Loops in Brca1-associated DNA replication stress and mammary tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3019.

Mechanosensitive TRPV4 channel guides maturation and organization of the bilayered mammary epithelium

Biophysical cues from the cell microenvironment are detected by mechanosensitive components at the cell surface. Such machineries convert physical information into biochemical signaling cascades within cells, subsequently leading to various cellular responses in a stimulus-dependent manner. At the surface of extracellular environment and cell cytoplasm exist several ion channel families that are activated by mechanical signals to direct intracellular events. One of such channel is formed by transient receptor potential cation channel subfamily V member, TRPV4 that is known to act as a mechanosensor in wide variaty of tissues and control ion-influx in a spatio-temporal way. Here we report that TRPV4 is prominently expressed in the stem/progenitor cell populations of the mammary epithelium and seems important for the lineage-specific differentiation, consequently affecting mechanical features of the mature mammary epithelium. This was evident by the lack of several markers for mature myoepithelial and luminal epithelial cells in TRPV4-depleted cell lines. Interestingly, TRPV4 expression is controlled in a tension-dependent manner and it also impacts differentation process dependently on the stiffness of the microenvironment. Furthermore, such cells in a 3D compartment were disabled to maintain normal mammosphere structures and displayed abnormal lumen formation, size of the structures and disrupted cellular junctions. Mechanosensitive TRPV4 channel therefore act as critical player in the homeostasis of normal mammary epithelium through sensing the physical environment and guiding accordingly differentiation and structural organization of the bilayered mammary epithelium.

Signaling Pathway Alterations Driven by BRCA1 and BRCA2 Germline Mutations are Sufficient to Initiate Breast Tumorigenesis by the PIK3CAH1047R Oncogene.

This study provides a single-cell atlas of breast tissues of BRCA1/2 mutation carriers and demonstrates that aberrant signaling due to BRCA1/2 mutations is sufficient to initiate breast cancer by mutant PIK3CA.

Expression of type VI collagen α3 chain in canine mammary carcinomas

This study aimed to investigate the expression of type VI collagen α3 chain (COL6a3) in neoplastic cells of canine mammary gland carcinomas (CMGCs) using immunohistochemistry (IHC) and to evaluate the association between COL6a3 expression and tumor histological features, histological grades, and the differentiation status of neoplastic epithelial cells. COL6a3 expression in carcinoma cells was significantly associated with histologically low malignancy and low mitotic indices. In addition, COL6a3+ carcinoma cells were more frequently detected in simple carcinomas (tubular and tubulopapillary types) than in solid carcinomas. These findings indicate that reduced expression of COL6a3 in carcinoma cells contributes to the malignant phenotype in CMGCs. We also showed that COL6a3 expression in the carcinoma cells was more frequently detected in CK19+/CD49f + and/or CK19+/CK5+ tumors. In addition, COL6a3+/CK19+/CD49f + and COL6a3+/CK19+/CK5+ tumors consisted of CK19+/CD49f + and CK19+/CD49f- cells, and CK19+/CK5+ and CK19+/CK5- cells, respectively. Most of these tumors more frequently expressed GATA3, but not Notch1. These results indicate that COL6a3 is expressed in CMGCs containing both luminal progenitor-like and mature luminal-like cells and showing differentiation ability into mature luminal cells. It is possible that COL6 may be involved in the differentiation of luminal progenitor-like carcinoma cells into mature luminal-like carcinoma cells in CMGCs, which may suppresses the development of malignant phenotypes in CMGCs.

Abstract PD4-08: PD4-08 A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors

Abstract A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors Austin Whitman, Hyunsoo Kim, Kamila Wisniewska, Rasha Kakati, Susana Garcia Recio, Hector Franco, Charles Perou, Philip Spanheimer Background: Resistance to endocrine therapy is a primary cause of treatment failure and death in patients with estrogen receptor (ER)-positive breast cancer. Intratumor heterogeneity is associated with resistance to therapy across tumors, and specifically in ER+/HER2- breast cancer, heterogeneity in ER and PR expression is associated with a worse response to endocrine therapy. We hypothesize that subpopulations within and across ER+/HER2- human breast tumors have distinct responses to tamoxifen and that discerning heterogeneity in response will improve understanding of inherent and emerging resistance to endocrine therapy. Methods: We developed an operating room-to-laboratory pipeline immediately after surgical resection for studies using alive tissue. Tissue samples were obtained and single cell suspensions created using physical and enzymatic dissociation. Cells were treated with tamoxifen (10 M) or control media for 12 hours in suspension and single cell RNA libraries generated using the 10X Genomics droplet-based kit and sequenced using the Illumina NextSeq2000. Results: We obtained normal breast tissue from 2 women undergoing reduction mammoplasty and tumor tissue from 10 women with ER+/HER2- invasive breast carcinoma. In tamoxifen treated and control matched pairs, a total of 22,195 cells from normal breast and 94,558 cells from tumor samples were sequenced. Computational analysis using consensus clustering was performed and cell types assigned using canonical correlation. Both tumor and normal samples identified clustering by cell type and not by patient revealing significant variability in cell type abundance between samples. In the normal breast samples, we performed differentially expressed genes (DEG) analysis comparing tamoxifen treatment to control for each cell type (Immune cells, fibroblasts, basal epithelial cells, luminal progenitor cells, and mature luminal cells) and enrichment analysis of up- and down-regulated genes performed. Strong depletion of estrogen induced genes was observed in tamoxifen-treated normal luminal progenitor and mature luminal cells, but not in basal epithelial cells or fibroblasts, demonstrating distinct, subpopulation-specific response to tamoxifen. In the 10 tumor matched pairs, 4 had a high epithelial proportion and tumor cells identified using inferred copy number variation. Tumor cells in 3 of these 4 samples showed significant down regulation of estrogen response genes with tamoxifen treatment. Using scBCSubtype to assign PAM50 subtype to individual tumor cells, the 3 responsive tumors were comprised primarily of LumA cells while the unresponsive tumor was predominantly LumB. Finally, we developed a novel score to quantify responsiveness at the single cell level based on downregulation of estrogen response genes with tamoxifen treatment relative to matched cluster-specific untreated expression. This analysis demonstrated heterogeneity in response to tamoxifen in tumor cells and identified distinct subpopulations of responsive and unresponsive tumor cells to tamoxifen treatment. Conclusion: We developed a novel ex vivo model to determine heterogeneity in therapeutic response to tamoxifen in normal human breast tissue and primary human breast tumors. We demonstrate differences in tamoxifen response by cell type and identify distinctly responsive and resistant subpopulations within human tumors. This provides a foundation to define features of responsive and resistant populations on the individual cell and specimen basis, and should allow us to develop precise, single cell-based predictors of response to endocrine therapy, and to identify genes and pathways driving resistance to therapy. Citation Format: Hyunsoo Kim, Austin Whitman, Kamila Wisniewska, Susana Garcia-Recio, Rasha Kakati, Hector L. Franco, Charles M. Perou, Philip Spanheimer. PD4-08 A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors [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 PD4-08.

Abstract IA024: Single nuclei chromatin accessibility and transcriptomic map of breast tissues of women of diverse genetic ancestry

Abstract Single nuclei analysis is allowing robust classification of cell types in an organ that helps to establish relationships between cell-type specific gene expression and chromatin accessibility status of gene regulatory regions. Using the institutional resource of breast tissues of healthy donors of various genetic ancestry, we have developed a comprehensive chromatin accessibility (snATAC-seq) and gene expression (snRNA-seq) atlas of human breast tissues. Our analyses included 51,367 nuclei with average sequence coverage of 1195 genes per nuclei. These nuclei were derived from 22 donors of Ashkenazi descent, 20 of European non-Ashkenazi ancestry, 10 of Asian ancestry, 10 Hispanic/Latina, 10 Native American, 6 from BRCA1 mutation carriers, and 5 from BRCA2 mutation carriers. Although tissues from 20 women of African Ancestry were included in sequencing, poor quality sequencing reads prevented inclusion of data from those samples in the final analysis. Integrated analysis revealed 10 distinct cell types in the healthy breast, which included three major epithelial cell subtypes (mature luminal, luminal progenitor, basal), two endothelial subtypes, two adipocyte subtypes, fibroblasts, T-cells, and macrophages. Mature luminal cells could be further divided into two distinct hormone sensitive subtypes, HSa and HSb, with HSa subtype expressing higher levels of Estrogen Receptor alpha (ESR1) and NEK10, a tyrosine kinase that controls p53 activity and limits cell proliferation. The luminal progenitors could be broadly classified into alveolar progenitors (AP) and basal-luminal hybrid progenitors (BL). AP cells expressed higher levels of ELF5, a known marker of alveolar cells, compared to BL cells. Basal cells could be classified into two subtypes with Basal-BAa cells being the most dominant and expressing higher levels of TP63 and NFIB compared to Basal-BAb cells. ESR1 expression pattern was distinctly different in tissues from Native Americans compared to the rest, with a high level of ESR1 expression extending to AP cells. In fact, overall AP cell numbers were ~3-fold higher in Native Americans compared to others (18.9% versus 1.5-8.8%). Furthermore, Ingenuity pathway analysis of differentially expressed genes revealed elevated Estrogen Receptor signaling in ML and AP cell types of Native Americans compared to those of other genetic ancestry. Despite significant differences in expression and activity in Native Americans compared to others, the chromatin accessibility map of the ESR1 gene regulatory regions in ML and LP cells did not show any genetic ancestry dependent variability. In general, cell subtype-specific gene expression did not correlate with chromatin accessibility differences, suggesting that transcriptional regulation independent of chromatin accessibility governs cell type-specific gene expression in the breast. Collectively, these results reveal complexities in gene expression in different cell types of the breast Citation Format: Poornima Bhat-Nakshatri, Duojiao Chen, Aditi Khatpe, Hongyu Gao, Yunlong Liu, Anna Maria Storniolo, Harikrishna Nakshatri. Single nuclei chromatin accessibility and transcriptomic map of breast tissues of women of diverse genetic ancestry [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA024.

Inactivation of LATS1/2 drives luminal-basal plasticity to initiate basal-like mammary carcinomas

Basal-like breast cancers, an aggressive breast cancer subtype that has poor treatment options, are thought to arise from luminal mammary epithelial cells that undergo basal plasticity through poorly understood mechanisms. Using genetic mouse models and ex vivo primary organoid cultures, we show that conditional co-deletion of the LATS1 and LATS2 kinases, key effectors of Hippo pathway signaling, in mature mammary luminal epithelial cells promotes the development of Krt14 and Sox9-expressing basal-like carcinomas that metastasize over time. Genetic co-deletion experiments revealed that phenotypes resulting from the loss of LATS1/2 activity are dependent on the transcriptional regulators YAP/TAZ. Gene expression analyses of LATS1/2-deleted mammary epithelial cells notably revealed a transcriptional program that associates with human basal-like breast cancers. Our study demonstrates in vivo roles for the LATS1/2 kinases in mammary epithelial homeostasis and luminal-basal fate control and implicates signaling networks induced upon the loss of LATS1/2 activity in the development of basal-like breast cancer.

Abstract 1458: Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues

Abstract Single cell mRNA atlases of multiple organs have been reported using scRNA-seq. We had previously reported single cell mRNA atlas of the breast. In this study, we performed scATAC-seq and scRNA-seq of breast tissues from 22 women of Ashkenazi (A) ancestry (19,935 nuclei), 20 women of European (non-A) ancestry (19,090 nuclei), six BRCA1 (6,261 nuclei) and five BRCA2 (3,400 nuclei) mutation carriers to correlate cell type-specific gene expression with chromatin accessibility. An integrated analysis revealed 17 cell clusters including basal, luminal progenitor, and mature luminal epithelial cells, two endothelial subtypes and two fibroblast/mesenchymal stem-like cell types. Basal cells could be further subdivided into two subclusters that differentially expressed genes such as TSHZ2, POSTN and ETV6. Similarly, there were four luminal progenitor subclusters that differentially expressed genes such as MALAT1, SOX13, and MDM4. Although ESR1 expression was observed predominantly in mature luminal cells, binding sites for ESR1 were accessible in multiple epithelial and non-epithelial cells with the exception of basal cells. FOXA1 expression and binding site enrichment were observed mostly in mature luminal cells. GATA3 expression was observed in all epithelial cell types with highest expression in mature luminal cells but binding sites for GATA3 were accessible in multiple cell types. Expression and binding site enrichment of alveolar differentiation-associated EHF were predominant in luminal progenitor cells. TP63 expression and binding site enrichment were restricted to basal cells, which is consistent with the proposed role of TP63 in maintaining stem cells among the basal cell populations. EMT-associated ZEB1 showed an intriguing pattern. Similar to our published immunohistochemistry-based analysis of breast tissues, ZEB1 expression was detected in fibroblasts/mesenchymal cells, endothelial cells and adipocytes but not in epithelial cells. However, binding sites for this transcription factor were enriched in epithelial cell types and in T-cells. ZEB1 mRNA stability is tightly controlled by the epithelial cell-enriched miR-200 family of microRNAs, and it is possible that ZEB1 transcribed in epithelial cells is immediately targeted for degradation through miR-200. To gain insight into this possibility, we examined the chromatin accessibility status of ZEB1 regulatory regions in fibroblasts/mesenchymal cells and in epithelial cells. ZEB1 regulatory regions displayed open chromatin status in both epithelial cells and fibroblasts. With the exception of a few genes such as SIGLEC1 in macrophages and IL7R in T cells, cell type-specific expression of many genes is not controlled at the level of chromatin accessibility of their regulatory regions. Collectively, these results reveal complexities in gene expression in different cell types of the breast. Citation Format: Poornima Bhat-Nakshatri, Duojiao Chen, Hongyu Gao, Yunlong Liu, Anna Maria Storniolo, Harikrishna Nakshatri. Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues [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 1458.

Abstract 2936: Multi-omic characterization of transitional cell populations in breast cancer

Abstract Breast cancer is a heterogeneous collection of diseases grouped by hormone receptor status or by expression of key subtype-determining genes. Breast cancer subtypes, in particular basal-like breast cancer and the luminal breast cancer subtypes, differ by hormonal receptor status, proliferation, genomic instability and mutational signatures, treatment response, and prognosis. The highly distinct signatures of basal-like and luminal breast cancer suggest that they may have different cells of origin within the breast duct. As part of the Washington University Human Tumor Atlas Network (WU-HTAN) program, we generated multi-omic data for 53 samples from 37 breast cancer tumors and 4 normal adjacent tissues. Genomic subtyping was applied to both bulk and single-nucleus RNA sequencing. Analysis of single-nucleus gene expression and chromatin accessibility in epithelial cells underscores similarities between basal-like breast cancer and luminal progenitor cells within the breast duct, and between luminal breast cancer and mature luminal ductal cells. This study links distinct breast cancer subtypes to normal breast cell populations and suggests distinct cells of origin for these cancer types. Citation Format: Michael D. Iglesia, Reyka G. Jayasinghe, Daniel Cui Zhou, Nadezhda V. Terekhanova, John Herndon, Alla Karpova, Siqi Chen, Nataly Naser Al Deen, Kazuhito Sato, Feng Chen, Deborah J. Veis, Ryan C. Fields, William E. Gillanders, Li Ding. Multi-omic characterization of transitional cell populations in breast cancer [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 2936.

Abstract 11: Prophylactic use of tamoxifen could reduce the risk of breast cancer in women who do not breast feed postpartum

Abstract Background: Triple negative breast cancer (TNBC) is associated with poor survival, particularly affecting African American women (AAW). Epidemiological studies indicate prolonged breast feeding reduces breast cancer (BC) risk, including TNBC. AAW have significantly lower rates of breast feeding compared to Caucasian women. To understand this link we developed a mouse model mimicking abrupt (AI) and gradual involution (GI). AI led to increased estrogen signaling, cell proliferation and chronic inflammation, which was followed by hyperplasia and squamous metaplasia in mammary glands1. There was an increase in the luminal progenitor (LP) cell population, the cells of origin of TNBC, and a decrease in mature luminal (ML) cells in AI glands. In this study, we sought to determine if blocking estrogen signaling with tamoxifen (TAM) could revert the negative effects of AI, and if so, could be a prophylactic option to reduce BC risk in women who do not breast feed. Methods: Uniparous FVB/N mice (~8 weeks) were allowed to nurse six pups per dam at partum. To induce AI, all pups were removed on postpartum (PP) day 7 (d7). For TAM treatment, 5mg sustained release TAM citrate pellet or placebo was implanted in the subscapular region on PP d8. Mammary glands were harvested on PP d28 and d120. FFPE sections were used for histology and immunohistochemistry. Single cell suspensions were analyzed for mammary epithelial subpopulations using Fluorescence Activated Cell Sorting. Affymetrix and qPCR were used for gene expression analysis. Mass cytometry was performed on mammary glands harvested at PP d120. Results: TAM treatment for 21 days completely abrogated hyperplastic and metaplastic changes in AI glands harvested on d120. Treatment initiation on PP d8, d15 and d35 had the same effect. TAM treatment reduced the cell proliferation and collagen deposition in AI glands. De-enrichment of estrogen signaling pathways and decrease in Elf5 expression, a luminal progenitor marker, were observed upon TAM treatment in d28 glands. Mass cytometry revealed a marked reduction in LP population and a significant increase in ML population in TAM treated AI glands on d120, restoring to the levels in age matched virgin mice. Significant increases in progenitor-like markers TSPAN8, Ly6D, CD200 and decreases in CD49f and CD47 expression in LP cells were observed, indicating return to a normal uniparous LP state. Expression of Ly-6D in ML cells, a ML cell marker, was also rescued upon TAM treatment. Conclusion: Using our mouse model of AI and GI, we show that suppression of estrogen signaling after initiation of AI offered marked protection against precancerous changes. TAM restored the balance of epithelial lineages and normalized the LP and basal cells in AI glands to the post-involution phenotype. Our data provide a rationale for considering short-term TAM treatment for women who do not breastfeed to reduce risk of BC. 1. Basree et.al. PMID 31315645 Citation Format: Bhuvaneswari Ramaswamy, Neelam Shinde, Gary K. Gray, Resham Mawalkar, Allen Zhang, Mustafa Basree, Xiaoli Zhang, Ramesh Ganju, Gina M. Sizemore, Joan S. Brugge, Sarmila Majumder. Prophylactic use of tamoxifen could reduce the risk of breast cancer in women who do not breast feed postpartum [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 11.

Pathogenic BRCA1 variants disrupt PLK1-regulation of mitotic spindle orientation

Preneoplastic mammary tissues from human female BRCA1 mutation carriers, or Brca1-mutant mice, display unexplained abnormalities in luminal differentiation. We now study the division characteristics of human mammary cells purified from female BRCA1 mutation carriers or non-carrier donors. We show primary BRCA1 mutant/+ cells exhibit defective BRCA1 localization, high radiosensitivity and an accelerated entry into cell division, but fail to orient their cell division axis. We also analyse 15 genetically-edited BRCA1 mutant/+ human mammary cell-lines and find that cells carrying pathogenic BRCA1 mutations acquire an analogous defect in their division axis accompanied by deficient expression of features of mature luminal cells. Importantly, these alterations are independent of accumulated DNA damage, and specifically dependent on elevated PLK1 activity induced by reduced BRCA1 function. This essential PLK1-mediated role of BRCA1 in controlling the cell division axis provides insight into the phenotypes expressed during BRCA1 tumorigenesis.

Abstract P3-07-09: Single cell transcriptomic analysis reveals the effects of BRCA1 and BRCA2 mutations on distinct signaling networks and cancer susceptibility

Abstract Background: Inheritance of BRCA1 and BRCA2 mutations is associated with increased risk of breast and ovarian cancers. Previous studies with low-throughput flow cytometry-based assays suggested elevated number of luminal progenitor cells in the breast tissues of BRCA1 mutation carriers compared to breast tissues of non-carriers. However, breast epithelial cell-specific transcriptome differences between BRCA1, BRCA2 mutation carriers, and non-carriers and how these differences alter susceptibility to transformation are yet to be elucidated. Methods: We generated a single cell transcriptome atlas of breast tissues from BRCA1 (six samples, 17,220 cells), BRCA2 (four samples, 25,046 cells) mutation carriers and non-carriers (11 samples, >50,000 cells). Using previously described markers, epithelial cells were sub-clustered into basal, luminal progenitor, and mature luminal cells. Genes differentially expressed in epithelial cells of BRCA1 and BRCA2 mutation carriers compared to those in non-carrier donors were subjected to Ingenuity Pathway Analysis to determine signaling pathways uniquely active in BRCA1 and BRCA2 mutant epithelial cells. Breast epithelial cells derived from three donor types were immortalized using hTERT and then transformed with PIK3CAH1047R mutant or H-RasG12V ± SV40-T/t antigens, and tumorigenicity was determined in vivo. Results: BRCA1 but not BRCA2 mutations altered the ratio between basal, luminal progenitor and mature luminal cells in breast tissues compared to breast tissues in non-carriers. A unique cluster of cells within luminal progenitors was underrepresented in case of BRCA2 mutation carriers compared to non-carriers or BRCA1 mutation carriers. BRCA1 or BRCA2 mutations specifically altered transcriptomes which are an integral part of mTOR and MYCN signaling, and the translational machinery. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included YAP1, BRD4, SMARCA4, and TGFβ1 signaling. BRCA2 mutations were associated with upregulation of IL-6, FOXO3, and TNFSF11 signaling. Breast epithelial cells from BRCA2 mutation carriers but not BRCA1 mutation carriers or non-carriers modified to overexpress hTERT + PIK3CAH1047R generated tumors in NSG mice. These tumors displayed large cystic structures with basilar epithelial cells lining the rim of cysts with focal areas of cellular hyperplasia and neoplastic cells that extended into the lumen. However, BRCA1/2 mutation status did not influence tumorigenicity by hTERT+ H-RASG12V +SV40-T/t antigens. Conclusions: Our studies provide a high resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers, which also reveal potentially targetable signaling networks uniquely deregulated in these cells. BRCA2 mutations are associated with distinct susceptibility to PIK3CA mutation-driven transformation. Since PIK3CA mutations are observed in clinically normal breast tissues, screening for such mutations in BRCA2 mutation carriers may help to detect pre-neoplastic or early stage breast cancer. Citation Format: Harikrishna Nakshatri, Poornima Bhat-Nakshatri, Duojiao Chen, Katie Chen, Henry Mang, Christopher A Herodotou, Aditi S Khatpe, Patrick C McGuire, Xiaoling Xuei, Yunlong Liu, George Sandusky, Anna Maria Storniolo. Single cell transcriptomic analysis reveals the effects of BRCA1 and BRCA2 mutations on distinct signaling networks and cancer susceptibility [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 P3-07-09.

Abstract P1-05-04: Novel breast cancer proteomic subtyping with connection to cell of origin

Abstract Introduction: In clinical practice, immunohistochemistry (IHC) is combined with clinicopathologic information to stratify patients into subtypes and guide treatment decisions. In our mass spectrometry study of 116 breast cancers (BrCAs), we identified 34 significant proteins which provide additional prognostic information. This novel proteomic subtyping, combining IHC with proteomics, separates HER2 negative (Her2-) BrCA patients into Luminal-like and TN-like subtypes. A novel subtype, luminal by IHC and TN-like by proteomics (L/T), is associated with unfavorable outcome compared with the luminal subtype (L/L) by both IHC and proteomics. These IHC-based Her2- subtypes are described as L/L, L/T, T/L and T/T subtypes. We investigated the cell-of-origin of our enhanced subtypes using gene set enrichment analysis with gene sets consisting of breast cancer cell-of-origin signatures. We further identified genes representing our 34 proteins in the significant gene sets and investigated their association with survival outcomes across The Cancer Genome Atlas (TCGA) pan-cancers. Method: The single-cell RNA-sequencing data analyses of health breast samples identified 23 breast cell-of-origin signatures (Bhat-Nakshatri et al., 2021) with 46 up and down-regulated gene sets based on the provided fold changes. Using our cohort, we performed differential analysis for each enhanced subtype (L/L, L/T, T/T) versus the other subtypes separately. Pre-ranked gene set enrichment analyses were used to identify significantly enriched gene sets. The significance of enriched gene sets was reported at FDR<0.05. We further identified genes of 34 biomarkers that overlapped in the identified significant gene sets and investigated the impact of the genes on survival across TCGA pan-cancers. Univariate overall survival (OS) and progression-free interval (PFI) analyses were performed on each cancer cohort and the significance of the association with survival outcome was reported by log-rank p-value <0.05. Results: Our signature was compared to the clusters from healthy tissues. We found up-regulated genes in L/L subtype were enriched in gene sets elevated in mature luminal annotated clusters (N8 and N12). Down-regulated genes in L/L subtype were enriched in the elevated genes in the N3 (luminal progenitor) and N9 (luminal progenitor/basal) clusters. Up-regulated genes in L/T subtype were enriched in the elevated genes in the N19 (luminal progenitor) and N11 (basal) clusters. Down-regulated genes in L/T were enriched in the elevated genes in the N8 (mature luminal) cluster and down-regulated genes in the N9 (luminal progenitor/basal) cluster. Up-regulated genes in T/T subtype were enriched in genes highly expressed in the luminal progenitor (N3) and luminal progenitor/basal (N9) clusters. Down-regulated genes in T/T subtype were enriched in the elevated genes in the mature luminal annotated clusters (N8, N12). One gene, KPNA2, was up-regulated in both the N9 (luminal progenitor/basal) cluster and the Basal-like (TN-like) subtype. The higher expression of this gene had significant association with worse OS outcomes in most of the cancers. Conclusions: T/T and L/T subtypes likely arise from progenitor and basal cells. The L/L subtype may arise from mature luminal cells. The novel L/T subtype appears to have characteristics that result in poorer survival. Disclaimers The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DOD or the Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Guisong Wang, Punit Shah, Rick Searfoss, J. Leigh Fantacone-Campbell, Jeffrey A. Hooke, Brenda Deyarmin, Rebecca N. Zingmark, Stella Somiari, Jianfang Liu, Leonid Kvecher, Lori A. Sturtz, Praveen-Kumar Raj-Kumar, Elder Granger, Linda Vahdat, Niven R. Narain, Mary L. Cutler, Rangaprasad Sarangarajan, Hai Hu, Michael A. Kiebish, Albert J. Kovatich, Craig D. Shriver. Novel breast cancer proteomic subtyping with connection to cell of origin [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 P1-05-04.

Cell-type-specific epigenomic variations associated with BRCA1 mutation in pre-cancer human breast tissues.

BRCA1 germline mutation carriers are predisposed to breast cancers. Epigenomic regulations have been known to strongly interact with genetic variations and potentially mediate biochemical cascades involved in tumorigenesis. Due to the cell-type specificity of epigenomic features, profiling of individual cell types is critical for understanding the molecular events in various cellular compartments within complex breast tissue. Here, we produced cell-type-specific profiles of genome-wide histone modifications including H3K27ac and H3K4me3 in basal, luminal progenitor, mature luminal and stromal cells extracted from a small pilot cohort of pre-cancer BRCA1 mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+), using a low-input ChIP-seq technology that we developed. We discovered that basal and stromal cells present the most extensive epigenomic differences between mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+), while luminal progenitor and mature luminal cells are relatively unchanged with the mutation. Furthermore, the epigenomic changes in basal cells due to BRCA1 mutation appear to facilitate their transformation into luminal progenitor cells. Taken together, epigenomic regulation plays an important role in the case of BRCA1 mutation for shaping the molecular landscape that facilitates tumorigenesis.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract NG05: Epigenetic mechanisms of endocrine therapy response in breast cancer

Abstract NG05: Epigenetic mechanisms of endocrine therapy response in breast cancer

Single-Cell RNA Sequencing Reveals the Cellular Origin and Evolution of Breast Cancer in BRCA1 Mutation Carriers.

The cell of origin and the development of breast cancer are not fully elucidated in BRCA1 mutation carriers, especially for estrogen receptor (ER)-positive breast cancers. Here, we performed single-cell RNA sequencing (RNA-seq) on 82,122 cells isolated from the breast cancer tissues and adjacent or prophylactic normal breast tissues from four BRCA1 mutation carriers and three noncarriers. Whole-exome sequencing was performed on breast tumors from the four BRCA1 mutation carriers; for validation, bulk RNA-seq was performed on adjacent normal breast tissues from eight additional BRCA1 mutation carriers and 14 noncarriers. Correlation analyses suggested that breast cancers in BRCA1 mutation carriers might originate from luminal cells. The aberrant luminal progenitor cells with impaired differentiation were significantly increased in normal breast tissues in BRCA1 mutation carriers compared with noncarriers. These observations were further validated by the bulk RNA-seq data from additional BRCA1 mutation carriers. These data suggest that the cell of origin of basal-like breast tumors (ERneg) in BRCA1 mutation carriers might be luminal progenitor cells. The expression of TP53 and BRCA1 was decreased in luminal progenitor cells from normal breast tissue in BRCA1 mutation carriers, which might trigger the basal/mesenchymal transition of luminal progenitors and might result in basal-like tumor development. Furthermore, ERhigh luminal tumors might originate from mature luminal cells. Our study provides in-depth evidence regarding the cells of origin of different breast cancer subtypes in BRCA1 mutation carriers. SIGNIFICANCE: Single-cell RNA-seq data indicate that basal-like breast cancer (ERneg) might originate from luminal progenitors, and ERhigh luminal breast cancer might originate from mature luminal cells in BRCA1 mutation carriers.