Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met

Abstract Introduction Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical efficacy in lung, colon, and pancreatic cancers. In lung cancer, resistance to EGFR TKIs correlates with amplification of the hepatocyte growth factor (HGF) receptor tyrosine kinase Met. Breast cancers do not respond to EGFR TKIs, even though EGFR is overexpressed. This intrinsic resistance to EGFR TKIs in breast cancer does not correlate with Met amplification. In several tissue monoculture models of human breast cancer, Met, although expressed, is not phosphorylated, suggesting a requirement for a paracrine-produced ligand. In fact, HGF, the ligand for Met, is not expressed in epithelial cells but is secreted by fibroblasts in the tumor stroma. We have identified a number of breast cancer cell lines that are sensitive to EGFR TKIs. This sensitivity is in conflict with the observed clinical resistance to EGFR TKIs in breast cancers. Here we demonstrate that fibroblast secretion of HGF activates Met and leads to EGFR/Met crosstalk and resistance to EGFR TKIs in triple-negative breast cancer (TNBC). Methods The SUM102 and SUM149 TNBC cell lines were used in this study. Recombinant HGF as well as conditioned media from fibroblasts expressing HGF were used as sources for Met activation. Furthermore, we co-cultured HGF-secreting fibroblasts with Met-expressing cancer cells to mimic the paracrine HGF/Met pathway, which is active in the tumor microenvironment. Cell growth, survival, and transformation were measured by cell counting, clonogenic and MTS assays, and soft agar colony formation, respectively. Student\u27s t test was used for all statistical analysis. Results Here we demonstrate that treatment of breast cancer cells sensitive to EGFR TKIs with recombinant HGF confers a resistance to EGFR TKIs. Interestingly, knocking down EGFR abrogated HGF-mediated cell survival, suggesting a crosstalk between EGFR and Met. HGF is secreted as a single-chain pro-form, which has to be proteolytically cleaved in order to activate Met. To determine whether the proteases required to activate pro-HGF were present in the breast cancer cells, we utilized a fibroblast cell line expressing pro-HGF (RMF-HGF). Addition of pro-HGF-secreting conditioned fibroblast media to TNBC cells as well as co-culturing of TNBC cells with RMF-HGF fibroblasts resulted in robust phosphorylation of Met and stimulated proliferation in the presence of an EGFR TKI. Conclusions Taken together, these data suggest a role for Met in clinical resistance to EGFR TKIs in breast cancer through EGFR/Met crosstalk mediated by tumor-stromal interactions

The fibroblast Tiam1-osteopontin pathway modulates breast cancer invasion and metastasis

Background The tumor microenvironment has complex effects in cancer pathophysiology that are not fully understood. Most cancer therapies are directed against malignant cells specifically, leaving pro-malignant signals from the microenvironment unaddressed. Defining specific mechanisms by which the tumor microenvironment contributes to breast cancer metastasis may lead to new therapeutic approaches against advanced breast cancer. Methods We use a novel method for manipulating three-dimensional mixed cell co-cultures, along with studies in mouse xenograft models of human breast cancer and a histologic study of human breast cancer samples, to investigate how breast cancer-associated fibroblasts affect the malignant behaviors of breast cancer cells. Results Altering fibroblast Tiam1 expression induces changes in invasion, migration, epithelial-mesenchymal transition, and cancer stem cell characteristics in associated breast cancer cells. These changes are both dependent on fibroblast secretion of osteopontin and also long-lasting even after cancer cell dissociation from the fibroblasts, indicating a novel Tiam1-osteopontin pathway in breast cancer-associated fibroblasts. Notably, inhibition of fibroblast osteopontin with low doses of a novel small molecule prevents lung metastasis in a mouse model of human breast cancer metastasis. Moreover, fibroblast expression patterns of Tiam1 and osteopontin in human breast cancers show converse changes correlating with invasion, supporting the hypothesis that this pathway in tumor-associated fibroblasts regulates breast cancer invasiveness in human disease and is thus clinically relevant. Conclusions These findings suggest a new therapeutic paradigm for preventing breast cancer metastasis. Pro-malignant signals from the tumor microenvironment with long-lasting effects on associated cancer cells may perpetuate the metastatic potential of developing cancers. Inhibition of these microenvironment signals represents a new therapeutic strategy against cancer metastasis that enables targeting of stromal cells with less genetic plasticity than associated cancer cells and opens new avenues for investigation of novel therapeutic targets and agents.National Institute of General Medical Sciences (U.S.) (GM074825

Identification of Selective Inhibitors of Cancer Stem Cells by High-Throughput Screening

Screens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here an approach to screening for agents with epithelial CSC-specific toxicity. We implemented this method in a chemical screen and discovered compounds showing selective toxicity for breast CSCs. One compound, salinomycin, reduces the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates the ability to identify agents with specific toxicity for epithelial CSCs.National Cancer Institute (U.S.). Initiative for Chemical GeneticsBreast Cancer Research FoundationRoot, DavidBroad Institute of MIT and Harvard (RNAi Platform

Human breast progenitor cell numbers are regulated by WNT and TBX3

BACKGROUND: Although human breast development is mediated by hormonal and non-hormonal means, the mechanisms that regulate breast progenitor cell activity remain to be clarified. This limited understanding of breast progenitor cells has been due in part to the lack of appropriate model systems to detect and characterize their properties. METHODS: To examine the effects of WNT signaling and TBX3 expression on progenitor activity in the breast, primary human mammary epithelial cells (MEC) were isolated from reduction mammoplasty tissues and transduced with lentivirus to overexpress WNT1 or TBX3 or reduce expression of their cognate receptors using shRNA. Changes in progenitor activity were quantified using characterized assays. We identified WNT family members expressed by cell populations within the epithelium and assessed alterations in expression of WNT family ligands by MECs in response to TBX3 overexpression and treatment with estrogen and progesterone. RESULTS: Growth of MECs on collagen gels resulted in the formation of distinct luminal acinar and basal ductal colonies. Overexpression of TBX3 in MECs resulted in increased ductal colonies, while shTBX3 expression diminished both colony types. Increased WNT1 expression led to enhanced acinar colony formation, shLRP6 decreased both types of colonies. Estrogen stimulated the formation of acinar colonies in control MEC, but not shLRP6 MEC. Formation of ductal colonies was enhanced in response to progesterone. However, while shLRP6 decreased MEC responsiveness to progesterone, shTBX3 expression did not alter this response. CONCLUSIONS: We identified two phenotypically distinguishable lineage-committed progenitor cells that contribute to different structural elements and are regulated via hormonal and non-hormonal mechanisms. WNT signaling regulates both types of progenitor activity. Progesterone favors the expansion of ductal progenitor cells, while estrogen stimulates the expansion of acinar progenitor cells. Paracrine WNT signaling is stimulated by estrogen and progesterone, while autocrine WNT signaling is induced by the embryonic T-box transcription factor TBX3

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.United States. Dept. of Defense. Breast Cancer Research Program (Pre-doctoral Traineeship Award)Raymond and Beverly Sackler FoundationBreast Cancer Research FoundationThe Slomo and Cindy Silvian Foundation, Inc.National Cancer Institute (U.S.) (CA125554)National Cancer Institute (U.S.) (CA092644)Raymond and Beverley Sackler Foundatio

BCL11B Drives Human Mammary Stem Cell Self-Renewal In Vitro by Inhibiting Basal Differentiation

The epithelial compartment of the mammary gland contains basal and luminal cell lineages, as well as stem and progenitor cells that reside upstream in the differentiation hierarchy. Stem and progenitor cell differentiation is regulated to maintain adult tissue and mediate expansion during pregnancy and lactation. The genetic factors that regulate the transition of cells between differentiation states remain incompletely understood. Here, we present a genome-scale method to discover genes driving cell-state specification. Applying this method, we identify a transcription factor, BCL11B, which drives stem cell self-renewal in vitro, by inhibiting differentiation into the basal lineage. To validate BCL11B's functional role, we use two-dimensional colony-forming and three-dimensional tissue differentiation assays to assess the lineage differentiation potential and functional abilities of primary human mammary cells. These findings show that BCL11B regulates mammary cell differentiation and demonstrate the utility of our proposed genome-scale strategy for identifying lineage regulators in mammalian tissues. Miller et al. describe a strategy to identify candidate master regulators of cell lineage specification. This approach identified BCL11B as a key regulator of human mammary stem cell self-renewal in in vitro progenitor and differentiation assays. Using a combination of 2D and 3D primary cell culture techniques, they show that BCL11B drives stem cell self-renewal by inhibiting basal lineage commitment.National Science Foundation (U.S.) (Grant 1122374

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

Introduction: Lineage 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. Methods: Mammary 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. Results: Integrating 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. Conclusions: These 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 material The online version of this article (doi:10.1186/s13058-014-0453-3) contains supplementary material, which is available to authorized users

Pregnancy-Associated Breast Cancers are Driven by Differences in Adipose Stromal Cells Present During Lactation

Introduction The prognosis of breast cancer is strongly influenced by the developmental stage of the breast when the tumor is diagnosed. Pregnancy-associated breast cancers (PABCs), cancers diagnosed during pregnancy, lactation, or in the first postpartum year, are typically found at an advanced stage, are more aggressive and have a poorer prognosis. Although the systemic and microenvironmental changes that occur during post-partum involution have been best recognized for their role in the pathogenesis of PABCs, epidemiological data indicate that PABCs diagnosed during lactation have an overall poorer prognosis than those diagnosed during involution. Thus, the physiologic and/or biological events during lactation may have a significant and unrecognized role in the pathobiology of PABCs. Methods Syngeneic in vivo mouse models of PABC were used to examine the effects of system and stromal factors during pregnancy, lactation and involution on mammary tumorigenesis. Mammary adipose stromal cell (ASC) populations were isolated from mammary glands and examined by using a combination of in vitro and in vivo functional assays, gene expression analysis, and molecular and cellular assays. Specific findings were further investigated by immunohistochemistry in mammary glands of mice as well as in functional studies using ASCs from lactating mammary glands. Additional findings were further investigated using human clinical samples, human stromal cells and using in vivo xenograft assays. Results ASCs present during lactation (ASC-Ls), but not during other mammary developmental stages, promote the growth of carcinoma cells and angiogenesis. ASCs-Ls are distinguished by their elevated expression of cellular retinoic acid binding protein-1 (crabp1), which regulates their ability to retain lipid. Human breast carcinoma-associated fibroblasts (CAFs) exhibit traits of ASC-Ls and express crabp1. Inhibition of crabp1 in CAFs or in ASC-Ls abolished their tumor-promoting activity and also restored their ability to accumulate lipid. Conclusions These findings imply that (1) PABC is a complex disease, which likely has different etiologies when diagnosed during different stages of pregnancy; (2) both systemic and local factors are important for the pathobiology of PABCs; and (3) the stromal changes during lactation play a distinct and important role in the etiology and pathogenesis of PABCs that differ from those during post-lactational involution

Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases

Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger-finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy-finger stitching-that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger-finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo, demonstrating that stitching yields ZFAs with robust recognition properties

A Novel Lung Metastasis Signature Links Wnt Signaling with Cancer Cell Self-Renewal and Epithelial-Mesenchymal Transition in Basal-like Breast Cancer

The establishment of metastasis depends on the ability of cancer cells to acquire a migratory phenotype combined with their capacity to recreate a secondary tumor in a distant tissue. In epithelial cancers, such as those of the breast, the epithelial-mesenchymal transition (EMT) is associated with basal-like breast cancers, generates cells with stem-like properties, and enables cancer cell dissemination and metastasis. However, the molecular mechanism(s) that connects stem cell–like characteristics with EMT has yet to be defined. Using an orthotopic model of human breast cancer metastasis to lung, we identified a poor prognosis gene signature, in which several components of the wnt signaling pathway were overexpressed in early lung metastases. The wnt genes identified in this signature were strongly associated with human basal-like breast cancers. We found that inhibiting wnt signaling through LRP6 reduced the capacity of cancer cells to self-renew and seed tumors in vivo. Furthermore, inhibition of wnt signaling resulted in the reexpression of breast epithelial differentiation markers and repression of EMT transcription factors SLUG and TWIST. Collectively, these results provide a molecular link between self-renewal, EMT, and metastasis in basal-like breast cancers