Breast Epithelial Hierarchy Research Articles

Abstract P2-26-08: Influence of genetic ancestry on breast stromal cells provides biologic basis for increased incidence of metaplastic breast cancer in women of African descent

Abstract The biologic basis of genetic ancestry-dependent variability in disease incidence and outcome is just beginning to be explored. We recently reported enrichment of a population of ZEB1-expressing cells located adjacent to the ductal epithelial cells in the normal breast of women of African Ancestry (AA) compared to European Ancestry (EA). By establishing and characterizing cell lines corresponding to these cells and validating in vitro findings with tissue microarrays of healthy breast tissue from AA, EA and Latina Ancestry (LA) women, we demonstrate that these cells have the properties of fibroadipogenic/mesenchymal stromal cells that express PROCR and PDGFR. PROCR+/ZEB1+/PDGFR+ cells, hence renamed as PZP cells, are enriched in the normal breast tissues of AA compared to EA or LA women. In vitro, PZP cells trans-differentiated into adipocytes or osteocytes. In co-culture conditions, PZP:epithelial cell communication resulted in luminal epithelial cells acquiring basal/stem cell characteristics and increased expression of IL-6 suggesting the impact of this communication on the microenvironment and breast epithelial hierarchy. Consistent with this possibility, the level of phospho-STAT3, which is a downstream target of IL-6, was higher in the normal and cancerous breast tissues of AA compared to EA women. PZP cells transformed with HRasG12V ± SV40-T/t antigens generated metaplastic carcinoma in NSG mice suggesting that these cells could be the cell-of-origin of metaplastic breast cancers. Collectively, these results identify a stromal cell component that could influence the biology of breast cancer in AA women. Citation Format: Brijesh Kumar, Katie Batic, Poornima Bhat-Nakshatri, Maggie Granatir, Rebekah Addison, Megan Szymanski, Lee Ann Baldridge, Constance Temm, George Sandusky, Sandra Althouse, Anna Maria Storniolo, Harikrishna Nakshatri. Influence of genetic ancestry on breast stromal cells provides biologic basis for increased incidence of metaplastic breast cancer in women of African descent [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 P2-26-08.

Dual TGFβ/BMP Pathway Inhibition Enables Expansion and Characterization of Multiple Epithelial Cell Types of the Normal and Cancerous Breast.

Functional modeling of normal breast epithelial hierarchy and stromal-epithelial cell interactions have been difficult due to inability to obtain sufficient stem-progenitor-mature epithelial and stromal cells. Recently reported epithelial reprogramming assay has partially overcome this limitation, but cross-contamination of cells from the feeder layer is a concern. The purpose of this study was to develop a feeder-layer-independent and inexpensive method to propagate multiple cell types from limited tissue resources. Cells obtained after enzymatic digestion of tissues collected at surgery or by core-needle biopsies were plated on tissue culture dishes precoated with laminin-5-rich-conditioned media from the rat bladder tumor cell line 804G and a defined growth media with inhibitors of ROCK, TGFβ, and BMP signaling. Cells were characterized by flow cytometry, mammosphere assay, 3D cultures, and xenograft studies. Cells from the healthy breasts included CD10+/EpCAM- basal/myoepithelial, CD49f+/EpCAM+ luminal progenitor, CD49f-/EpCAM+ mature luminal, CD73+/EpCAM+/CD90- rare endogenous pluripotent somatic stem, CD73+/CD90+/EpCAM-, estrogen receptor alpha-expressing ALCAM (CD166)+/EpCAM+, and ALDFLUOR+ stem/luminal progenitor subpopulations. Epithelial cells were luminal (KRT19+), basal (KRT14+), or dual-positive luminal/basal hybrid cells. While breast cells derived from BRCA1, BRCA2, and PALB2 mutation carriers did not display unique characteristics, cells from women with breast cancer-protective alleles showed enhanced differentiation. Cells could also be propagated from primary tumors and metastasis of breast, ovarian, and pancreatic cancer-neuroendocrine subtype. Xenograft studies confirmed tumorigenic properties of tumor-derived cells. IMPLICATIONS: Our method expands the scope of individualized studies of patient-derived cells and provides resources to model epithelial-stromal interactions under normal and pathologic conditions.

PO-294 Role of fatty acid amide hydrolase (FAAH) in breast development and cancer

IntroductionThere are three major stages of breast development – embryonic, pubertal, and reproductive. During this complex developmental cycling, the epithelial compartment undergoes several rounds of proliferation, remodelling and cell death to eventually form an organised and functional mammary tree. The very nature of the pathways controlling these processes makes them susceptible to promote tumorigenic processes and ultimately lead to breast carcinoma.Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that hydrolyzes the endocannabinoid anandamide and related amidated signalling lipids. Here, we aimed at analysing the expression FAAH in breast physio-pathology.Material and methodsThe expression of FAAH was analysed by immunohistochemistry in a tissue microarray (TMA) containing ≈600 human breast cancer samples and in public microarrays data bases. Cell lines derived from different breast cancer subtypes have been analysed for FAAH expression and the effect of a Knock-out (CRISPR) and overexpression of FAAH examined. FACS and immunofluorescence were also performed to further assess the identity of cell populations. FAAH effects on breast CSC activity was examinated using mammosphere formation and aldehyde dehydrogenase (ALDH) activity assays.Results and discussionsWe have found that FAAH shows remarkable variations in expression within the human breast epithelial hierarchy. Particularly, FAAH expression is restricted to a certain subset of mature cells in normal breast tissue (i.e. luminal cells), with negligible levels being detected in the progenitor/stem cell subset, In line with this idea, pharmacological inhibition of FAAH with URB597 in the mammary stem cell line HC11 delayed differentiation to mature epithelial cells. In addition, we observed significant differences in duct formation between FAAH KO and WT mice during pubertal development.Our results also correlate low FAAH expression with more undifferentiated phenotypes, high histologic grade, absence of oestrogen receptor and triple-negative phenotype in breast cancer. Consistent with this, we found low FAAH mRNA levels associated with metastasis and poor prognosis gene signatures. Finally, pharmacoinhibition and knocking-out of FAAH in breast cancer lines, enriched the cultures in cancer stem cells.ConclusionTogether, our data suggest that FAAH plays a role in breast cell differentiation, both in normal development and in oncologic contexts.

Abstract P6-07-08: Dual TGFβ/BMP inhibition allows in vitro expansion of multiple cell types from normal and cancerous breast

Abstract Functional modeling of breast epithelial hierarchy and stromal-epithelial cell interactions has been difficult due to inability to obtain sufficient stem-progenitor-mature epithelial cells and stromal cells. The recently developed epithelial reprogramming assay has partially overcome this limitation, allowing propagation of epithelial cells with stem, luminal progenitor and mature cell features. However, characterizing stromal cells using this assay is difficult because irradiated fibroblasts which can be difficult to distinguish from stromal cells are needed as feeder layer. A recent study demonstrated expansion of airway basal stem cells without a feeder layer through pharmacologic inhibition of TGFβ/BMP/SMAD signaling. We sought to develop this method for culture and expansion of cells from normal and cancerous breast samples. With appropriate modifications to growth media, we were able to obtain normal and stromal cells from breast biopsies of healthy women. The expanded cell population included CD10+/EpCAM- basal/myoepithelial cells, CD49f+/EpCAM+ luminal progenitor cells, CD49f-/EpCAM+ mature luminal cells, CD73+/EpCAM+/CD90- rare endogenous pluripotent somatic stem cells, CD73+/CD90+/EpCAM- mesenchymal stem cells, ALCAM (CD166)+/EpCAM+ cells, CD44+/CD24- cells, CD44+/CD24+ cells and ALDFLUOR+ stem/luminal progenitor cells. Epithelial cells were KRT14+, KRT19+ or both further documenting heterogeneity within epithelial cell population. We have extended this technique to grow breast epithelial cells from high-risk patients including BRCA1 mutant-carriers, tumor-adjacent normal and tumor cells from the same patient, pleural effusions and liver metastasis from breast cancer patients. Phenotypic characterization showed differences in the differentiation state of adjacent-normal and tumor cells. Tumor cells from pleural effusions showed remarkable phenotypic heterogeneity with a fraction of these cells expressing estrogen receptor. The assay described here, therefore, is versatile and provides resources to model epithelial-stromal interactions under normal and cancerous conditions as well as for genomics and screening of drugs to target metastasis on an individual level. Susan G. Komen for the Cure and Department of Defense supported this work. Citation Format: Nakshatri H, Ananappa M, Prasad MS, Kumar B, Liu Y, Storniolo AM, Miller KD, Bhat-Nakshatri P. Dual TGFβ/BMP inhibition allows in vitro expansion of multiple cell types from normal and cancerous breast [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-08.

The Hippo kinases LATS1 and 2 control human breast cell fate via crosstalk with ERα.

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.

Anatomical localization of progenitor cells in human breast tissue reveals enrichment of uncommitted cells within immature lobules.

IntroductionLineage tracing studies in mice have revealed the localization and existence of lineage-restricted mammary epithelial progenitor cells that functionally contribute to expansive growth during puberty and differentiation during pregnancy. However, extensive anatomical differences between mouse and human mammary tissues preclude the direct translation of rodent findings to the human breast. Therefore, here we characterize the mammary progenitor cell hierarchy and identify the anatomic location of progenitor cells within human breast tissues.MethodsMammary epithelial cells (MECs) were isolated from disease-free reduction mammoplasty tissues and assayed for stem/progenitor activity in vitro and in vivo. MECs were sorted and evaluated for growth on collagen and expression of lineages markers. Breast lobules were microdissected and individually characterized based on lineage markers and steroid receptor expression to identify the anatomic location of progenitor cells. Spanning-tree progression analysis of density-normalized events (SPADE) was used to identify the cellular hierarchy of MECs within lobules from high-dimensional cytometry data.ResultsIntegrating multiple assays for progenitor activity, we identified the presence of luminal alveolar and basal ductal progenitors. Further, we show that Type I lobules of the human breast were the least mature, demonstrating an unrestricted pattern of expression of luminal and basal lineage markers. Consistent with this, SPADE analysis revealed that immature lobules were enriched for basal progenitor cells, while mature lobules consisted of increased hierarchal complexity of cells within the luminal lineages.ConclusionsThese results reveal underlying differences in the human breast epithelial hierarchy and suggest that with increasing glandular maturity, the epithelial hierarchy also becomes more complex.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0453-3) contains supplementary material, which is available to authorized users.

Abstract PL04-02: The breast epithelial hierarchy and its implications for tumor heterogeneity

Abstract Breast cancer is a highly heterogeneous disease at both the molecular and pathological levels. To understand this heterogeneity and ‘cells of origin’ of breast cancer, it is important to dissect the normal mammary epithelial hierarchy. Discrete populations of mouse and human mammary epithelial cells can be isolated on the basis of cell surface marker expression. To date, subpopulations that are highly enriched for mammary stem (MaSC), luminal progenitor and mature luminal cells have been identified. Notably, these are functionally analogous across human and mouse and share highly conserved transcriptomes. Lineage tracing is an important strategy for assessing the stem cell hierarchy as it allows stem and progenitor cell fate to be studied in situ in the context of development, tissue maintenance and disease. We have combined lineage tracing with a novel three-dimensional (3D) imaging strategy to explore the relative contributions of stem and progenitor cells to post-natal mammary gland development and tissue homeostasis. Cell lineage tracing studies also provide the current gold standard for identifying ‘cells of origin’ in cancer. Towards this end, we are utilizing newly generated transgenic strains harboring lineage-specific gene regulatory regions to direct the expression of specific mammary oncogenic lesions to discrete epithelial cell types. In terms of human breast cancer, the interrogation of molecular profiles has provided insight into potential ‘cells of origin’ in preneoplastic tissue for basal-like cancers arising in BRCA1 mutation carriers. Finally, the identification of deregulated genes/pathways in these crucial target cell populations may enable earlier detection of malignancies and lead to preventative therapies for individuals at high risk of developing breast cancer. Citation Format: Jane E. Visvader, Anne Rios, Naiyang Fu, Emma Nolan, Geoffrey Lindeman. The breast epithelial hierarchy and its implications for tumor heterogeneity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr PL04-02. doi:10.1158/1538-7445.AM2014-PL04-02

Abstract 2040: Adenomatous polyposis coli mediated signaling, self renewal and differentiation of breast epithelial cells

Abstract Breast cancer is the leading cause of cancer-related death in non-smoking women in the United States. Despite tremendous amounts of information about the etiology of breast cancer, many questions remain unanswered. It has been demonstrated that specific subtypes of breast cancer differ in their sensitivity to chemotherapeutic and targeted therapies. The oncogenic events and signaling pathways driving these tumor subtypes are distinct. This indicates that increased knowledge of the molecular events defining the unique subtypes will provide opportunities for novel therapeutic approaches tailored to fit the specific subtypes and will therefore lead to improvement in patient outcomes. The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in 18-70% of sporadic breast cancers depending on subtype. Oncomine database analysis revealed reduced expression of APC in aggressive estrogen receptor (ER) negative breast cancers compared to ER-positive breast cancer or normal breast. Using the ApcMin/+ mouse model, we identified pre-neoplastic lesions in the breast and enhanced breast tumorigenesis in the presence of the Polyoma middle T antigen (PyMT) oncogene. Apc mutation changed the tumor histopathology from solid to squamous adenocarcinomas, resembling the highly aggressive human metaplastic breast cancer. In this model of mouse mammary tumorigenesis, these changes occurred independent of Wnt/b-catenin signaling and required activation of focal adhesion kinase (FAK)/Src signaling, with a subsequent increase in cyclooxygenase-2 (COX-2). The applicability of these signaling pathways to human breast cancer remains unclear. Using the epithelial cell reprogramming assay for growing normal breast epithelial cells, we have begun to characterize expression of cell surface markers to delineate subtypes of epithelial cells. We have also assessed APC expression and correlated APC expression with risk and well-established clinical markers, such as ErbB2. Future studies will modulate APC expression in specific compartments of breast epithelial hierarchy to accomplish our objectives of dissecting the molecular alterations specifically downstream of APC loss and its role in the self-renewal and differentiation of breast epithelial cells. Citation Format: Alyssa Lesko, Harikrishna Nakshatri, Manjushree Anjanappa, Poornima Bhat-Nakshatri, Jenifer Prosperi. Adenomatous polyposis coli mediated signaling, self renewal and differentiation of breast epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2040. doi:10.1158/1538-7445.AM2014-2040

Invited Commentary on: Can CD44+/CD24- Tumor Stem Cells Be Used to Determine the Extent of Breast Cancer Invasion Following Neoadjuvant Chemotherapy?

The article published by Wu et al. [1] in Journal of Breast Cancer proposed that the presence of so called 'tumor stem cells' may be an additional parameter to determine the extent of breast cancer invasion in the setting of surgery following neoadjuvant chemotherapy. However, this article has some noteworthy concerns. The first one is regarding the concept of 'tumor stem cells'. The second is about the markers the author used in the article. The third is their way of determining the tumor margin. It seems that the authors are arguing CD44(+)CD24(-) cells as human breast 'tumor stem cells.' However, their argument is not so well accepted since the tumor stem cell is not a synonym of a tumor-initiating cell. While several studies provided indirect evidence for the existence of mammary stem cells in human and in mice, these stem cells have not yet been prospectively isolated at a single cell level in human breast cancer. According to the cancer stem cell model, the tumorigenic stem cells are maintained through self-renewal and have the ability to differentiate into non-tumorigenic cancer cells [2]. It has been postulated that the cancer stem cells are from the normal stem/progenitor cells as a result of accumulated mutation during the self renewal process. However, a recent study suggests that this counterpart-relationship of the human breast epithelial hierarchy is not necessary to develop a breast cancer; an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors [3]. Because a single tumor stem cell that can repopulate the heterogeneity of human breast cancer has not been identified yet, it has not been clear whether there is a certain specific 'tumor stem cell' or 'tumor initiating cells' which have tumorigenic capability. While the monumental report proposed that the CD44(+)CD24(-/low) might be the characteristics of tumorigenic breast cancer cells, there still has been little consensus on the suitable markers for the breast cancer stem cells [4]. It is still not clear whether CD44(+)CD24(-) itself is true and definite characteristics of human breast tumor stem cells or not. For example, aldehyde dehydrogenase (ALDH) was suggested as a useful marker for enriching tumor-initiating cells in immunodeficient mice [5,6]. CD44(+)CD24(-/low) subtype can be subdivided by ALDH1 biomarker, and interestingly, the overlap between CD44(+)CD24(-/low) and ALDH1 phenotypes in breast cancer seems to be very small [5,7]. Regarding the intrinsic molecular subtypes, basal-like tumors contain a higher percentage of cells with CD44(+)CD24(-/low) and ALDH1-positive cells [7]. CD44(+)CD24(-/low) might be considered as the markers for basal subtypes rather than those of breast tumor-initiating cells, and CD44(+)CD24(-/low) alone cannot be considered as a definitive marker for stem cell characteristics. In this article, the authors also suggest that six categories can be classified from the pattern of cancer cells and stem cells, which are identified in the immunofluorescence studies performed on tissue sections obtained from surgical resection of tumors following neoadjuvant chemotherapy. According to the presented methods, 0.5×0.5×0.5 cm3 sized sections were obtained at 1 cm intervals from the tumor diameter up to 3 cm outside the maximum diameter of the tumor. We are seriously concerned whether it is a sound way to identify the border of breast cancer and remove a piece of tissue at 1 cm intervals in removed fresh breast specimen after neoadjuvant chemotherapy with the naked eye. After neoadjuvant chemotherapy, the identification of patients with a pathologic complete response before surgery often becomes difficult by physical examination. Moreover, imaging studies such as mammography, ultrasonography or magnetic resonance imaging cannot reliably assess degree of response. Neoadjuvant chemotherapy-induced tumor regression is usually patchy, remaining islands of viable tumor cells, and it is not concentric [8]. In case of the remained tumor nests after neoadjuvant chemotherapy, it can be hardly feasible to identify tumor margins with the naked eye. We are deeply apprehensive that this method may not be always possible and repeatedly performed in a standardized way. A breast cancer resistant to the chemotherapy or radiation may contain higher levels of CD44(+)CD24(-/low) cells [9]. However, this does not necessarily mean that 'tumor stem cells' are resistant to the chemotherapy or radiation because of the recent evidences that CD44(+)CD24(-/low) is not an absolute marker of breast cancer stem cells. This study is very interesting in trying to find a pattern between the distribution of CD44(+) CD24(-/low) cells from the tumor margin. However, in our humble opinion, careful consideration should be given from the above viewpoints.

Tarheel Footprints in Health Care

The biologic basis of genetic ancestry-dependent variability in disease incidence and outcome is just beginning to be explored. We recently reported enrichment of a population of ZEB1-expressing cells located adjacent to the ductal epithelial cells in the normal breast of women of African Ancestry (AA) compared to European Ancestry (EA). By establishing and characterizing cell lines corresponding to these cells and validating *in vitro* findings with tissue microarrays of healthy breast tissue from AA, EA and Latina Ancestry (LA) women, we demonstrate that these cells have the properties of fibroadipogenic/mesenchymal stromal cells that express PROCR and PDGFRα. PROCR+/ZEB1+/PDGFRα+ cells, hence renamed as PZP cells, are enriched in the normal breast tissues of AA compared to EA or LA women. *In vitro*, PZP cells trans-differentiated into adipocytes or osteocytes. In co-culture conditions, PZP:epithelial cell communication resulted in luminal epithelial cells acquiring basal/stem cell characteristics and increased expression of IL-6 suggesting the impact of this communication on breast epithelial hierarchy and the microenvironment. Consistent with this possibility, the level of phospho-STAT3, which is a downstream target of IL-6, was higher in the normal and cancerous breast tissues of AA compared to EA women. PZP cells transformed with HRas^G12V^ ± SV40-T/t antigens generated metaplastic carcinoma in NSG mice suggesting that these cells could be the cell-of-origin of metaplastic breast cancers. Collectively, these results identify a stromal cell component that could influence the biology of breast cancer in AA women.