Ina Klebba

Genetic predisposition directs breast cancer phenotype by dictating progenitor cell fate

Women with inherited mutations in the BRCA1 gene have increased risk of developing breast cancer but also exhibit a predisposition for the development of aggressive basal-like breast tumors. We report here that breast epithelial cells derived from patients harboring deleterious mutations in BRCA1 (BRCA1(mut /+) give rise to tumors with increased basal differentiation relative to cells from BRCA1+/+ patients. Molecular analysis of disease-free breast tissues from BRCA1(mut /+) patients revealed defects in progenitor cell lineage commitment even before cancer incidence. Moreover, we discovered that the transcriptional repressor Slug is an important functional suppressor of human breast progenitor cell lineage commitment and differentiation and that it is aberrantly expressed in BRCA1(mut /+) tissues. Slug expression is necessary for increased basal-like phenotypes prior to and after neoplastic transformation. These findings demonstrate that the genetic background of patient populations, in addition to affecting incidence rates, significantly impacts progenitor cell fate commitment and, therefore, tumor phenotype.

Defining the cellular precursors to human breast cancer

Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM+) and basal/myoepithelial (CD10+). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM+ epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10+ cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10+ breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues.

Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

IntroductionNormal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues.MethodsWe used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages.ResultsHuman breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions.ConclusionsAs a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues.

Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis.

Transplantation studies have demonstrated the existence of mammary progenitor cells with the ability to self-renew and regenerate a functional mammary gland. Although these progenitors are the likely targets for oncogenic transformation, correlating progenitor populations with certain oncogenic stimuli has been difficult. Cyclin D1 is required for lobuloalveolar development during pregnancy and lactation as well as MMTV-ErbB2- but not MMTV-Wnt1-mediated tumorigenesis. Using a kinase-deficient cyclin D1 mouse, we identified two functional mammary progenitor cell populations, one of which is the target of MMTV-ErbB2. Moreover, cyclin D1 activity is required for the self-renewal and differentiation of mammary progenitors because its abrogation leads to a failure to maintain the mammary epithelial regenerative potential and also results in defects in luminal lineage differentiation.

Distinct Roles of the Three Akt Isoforms in Lactogenic Differentiation and Involution

The three Akt isoforms differ in their ability to transduce oncogenic signals initiated by the Neu and PyMT oncogenes in mammary epithelia. As a result, ablation of Akt1 inhibits and ablation of Akt2 accelerates mammary tumor development by both oncogenes, while ablation of Akt3 is phenotypically almost neutral. Since the risk of breast cancer development in humans correlates with multiple late pregnancies, we embarked on a study to determine whether individual Akt isoforms also differ in their ability to transduce hormonal and growth factor signals during pregnancy, lactation and post‐lactation involution. The results showed that the ablation of Akt1 delays the differentiation of the mammary epithelia during pregnancy and lactation, and that the ablation of Akt2 has the opposite effect. Finally, ablation of Akt3 results in minor defects, but its phenotype is closer to that of the wild type mice. Whereas the phenotype of the Akt1 ablation is cell autonomous, that of Akt2 is not. The ablation of Akt1 promotes apoptosis and accelerates involution, whereas the ablation of Akt2 inhibits apoptosis and delays involution. Mammary gland differentiation during pregnancy depends on the phosphorylation of Stat5a, which is induced by prolactin, a hormone that generates signals transduced via Akt. Here we show that the ablation of Akt1, but not the ablation of Akt2 or Akt3 interferes with the phosphorylation of Stat5a during late pregnancy and lactation. We conclude that the three Akt isoforms have different roles in mammary gland differentiation during pregnancy and this may reflect differences in hormonal signaling. J. Cell. Physiol. 217: 468–477, 2008.

The role of fibroblast Tiam1 in tumor cell invasion and metastasis

The co-evolution of tumors and their microenvironment involves bidirectional communication between tumor cells and tumor-associated stroma. Various cell types are present in tumor-associated stroma, of which fibroblasts are the most abundant. The Rac exchange factor Tiam1 is implicated in multiple signaling pathways in epithelial tumor cells and lack of Tiam1 in tumor cells retards tumor growth in Tiam1 knockout mouse models. Conversely, tumors arising in Tiam1 knockout mice have increased invasiveness. We have investigated the role of Tiam1 in tumor-associated fibroblasts as a modulator of tumor cell invasion and metastasis, using retroviral delivery of short hairpin RNA to suppress Tiam1 levels in three different experimental models. In spheroid co-culture of mammary epithelial cells and fibroblasts, Tiam1 silencing in fibroblasts led to increased epithelial cell outgrowth into matrix. In tissue-engineered human skin, Tiam1 silencing in dermal fibroblasts led to increased invasiveness of epidermal keratinocytes with pre-malignant features. In a model of human breast cancer in mice, co-implantation of mammary fibroblasts inhibited tumor invasion and metastasis, which was reversed by Tiam1 silencing in co-injected fibroblasts. These results suggest that stromal Tiam1 may have a role in modulating the effects of the tumor microenvironment on malignant cell invasion and metastasis. This suggests a set of pathways for further investigation, with implications for future therapeutic targets.

Estrogen Promotes ER-Negative Tumor Growth and Angiogenesis through Mobilization of Bone Marrow–Derived Monocytes

Estrogen has a central role in the genesis and progression of breast cancers whether they are positive or negative for the estrogen receptor (ER). While therapies that disrupt estrogen biosynthesis or ER activity can treat these diseases in postmenopausal women, in younger women where ovarian function remains intact, these anti-estrogen therapies are not as effective. Moreover, emerging clinical evidence suggests that estrogen may promote other cancers. Thus, circulating estrogens may participate in cancer pathogenesis in ways that are not yet understood. In this study, we show that estrogen can promote the outgrowth of murine xenograft tumors established from patient-derived ER-negative breast cancer cells by influencing the mobilization and recruitment of a proangiogenic population of bone marrow-derived myeloid cells. ERα expression was necessary and sufficient in the bone marrow-derived cells themselves to promote tumor formation in response to estrogen. Our findings reveal a novel way in which estrogen promotes tumor formation, with implications for the development and application of anti-estrogen therapies to treat cancer in premenopausal women.

Functional heterogeneity of breast fibroblasts is defined by a prostaglandin secretory phenotype that promotes expansion of cancer-stem like cells.

Fibroblasts are important in orchestrating various functions necessary for maintaining normal tissue homeostasis as well as promoting malignant tumor growth. Significant evidence indicates that fibroblasts are functionally heterogeneous with respect to their ability to promote tumor growth, but markers that can be used to distinguish growth promoting from growth suppressing fibroblasts remain ill-defined. Here we show that human breast fibroblasts are functionally heterogeneous with respect to tumor-promoting activity regardless of whether they were isolated from normal or cancerous breast tissues. Rather than significant differences in fibroblast marker expression, we show that fibroblasts secreting abundant levels of prostaglandin (PGE2), when isolated from either reduction mammoplasty or carcinoma tissues, were both capable of enhancing tumor growth in vivo and could increase the number of cancer stem-like cells. PGE2 further enhanced the tumor promoting properties of fibroblasts by increasing secretion of IL-6, which was necessary, but not sufficient, for expansion of breast cancer stem-like cells. These findings identify a population of fibroblasts which both produce and respond to PGE2, and that are functionally distinct from other fibroblasts. Identifying markers of these cells could allow for the targeted ablation of tumor-promoting and inflammatory fibroblasts in human breast cancers.

Abstract 916: BRCA1 mutations impair breast epithelial differentiation through upregulation of the transcriptional repressor Slug

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tumor histopathology is a strong predictor of patient mortality, although the molecular and cellular factors that are responsible for this phenotypic diversity remain poorly understood. Women with inherited mutations in the BRCA1 gene have increased risk for the development of breast cancer, but also exhibit a specific predisposition for the development of aggressive basal-like breast cancers. Here we study the interplay between inherited mutations in BRCA1 and tumor differentiation by examining the regulation of progenitor cell fate in disease-free breast tissues from BRCA1 mutation carriers. We demonstrate for the first time that cell populations derived from patients harboring mutations in BRCA1 (BRCA1mut/+) give rise to tumors with increased basal differentiation, relative to cells obtained from BRCA1+/+ patients. Molecular analysis of disease-free breast tissues from BRCA1mut/+ patients revealed significant defects in epithelial progenitor cells that are present prior to cancer incidence. Moreover, we discovered that the transcriptional repressor Slug is an important functional regulator of human breast progenitor cell lineage commitment and differentiation and that it is aberrantly expressed in BRCA1mut/+ tissues and cells prior to neoplastic transformation. In addition, Slug expression is necessary for the increased basal-like phenotypes prior to and following neoplastic transformation. These findings demonstrate that the genetic background of patient populations, in addition to affecting incidence rates, significantly impacts progenitor cell fate commitment and therefore, tumor phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 916. doi:10.1158/1538-7445.AM2011-916

Abstract LB-265: Mutations in BRCA1 impair breast epithelial differentiation through the transcriptional repressor Slug

Human breast cancers can be broadly classified based on their molecular and gene expression profiles into luminal and basal-like tumors. These tumor subtypes express markers corresponding to the two major differentiation states of epithelial cells in the breast: luminal cells that line the breast ducts and the outer myoepithelial/basal cells that provide contractile functions. Women with inherited mutations in the BRCA1 gene have increased breast cancer risk and also exhibit a specific predisposition to the development of aggressive basal-like breast cancers. Accumulating evidence suggests that BRCA1 has a role in breast epithelial differentiation and we wanted to further understand how this could contribute to the formation of basal-like tumors. Microarray, flow cytometry and immunohistochemical analysis of breast epithelial cells from disease-free women harboring deleterious mutations in BRCA1 (BRCA1 mut/+) compared to those from BRCA1 +/+ reduction mammoplasties showed an increase in markers of basal differentiation and a decrease in markers of luminal differentiation. We also created breast cancers from single cell suspensions of BRCA1 mut/+ and BRCA1 +/+ epithelial cells that had been transformed with identical oncogenes and injected into humanized mammary fat pads. Tumors derived from BRCA1 mut/+ cells had increased basal differentiation relative to cells obtained from BRCA1 +/+ patients, indicating that the perturbed differentiation evident prior to neoplastic transformation was mirrored in the tumors. Pathway analysis of the microarray data comparing BRCA1 mut/+ and BRCA1 +/+ cells from disease-free tissue indicated that signaling components relating to the transcriptional repressor Slug were overexpressed in BRCA1 mut/+ tissues. We confirmed that Slug protein levels were elevated in BRCA1 mut/+ tissues and in human tumors from BRCA1 mutation carriers. RNAi-mediated downregulation of slug in primary breast epithelial cells and cell lines derived from BRCA1 mut/+ tissue as well as breast cancer cell lines known to harbor BRCA1 mutations, led to a decrease in markers of basal differentiation, indicating that elevated Slug protein levels seen in BRCA1-associated tissues and tumors are contributing to the basal phenotype. Furthermore, RNAi-mediated knockdown of BRCA1 led to an increase in Slug protein expression and breast cancer cell lines harboring BRCA1 mutations showed an increase in Slug protein stability, indicating that loss of BRCA1 protein by mutation contributes to elevated Slug protein levels. These results reveal an important mechanism by which BRCA1 can regulate breast epithelial differentiation and may explain how, in addition to affecting incidence rates, the genetic background of patients could impact tumor phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-265.