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.

Genome-Engineering Tools to Establish Accurate Reporter Cell Lines That Enable Identification of Therapeutic Strategies to Treat Friedreich's Ataxia.

Friedreich's ataxia is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin. This deficiency results from expansion of a trinucleotide repeat in the first intron of the frataxin gene. Because this repeat expansion resides in an intron and hence does not alter the amino acid sequence of the frataxin protein, gene reactivation could be of therapeutic benefit. High-throughput screening for frataxin activators has so far met with limited success because current cellular models may not accurately assess endogenous frataxin gene regulation. Here we report the design and validation of genome-engineering tools that enable the generation of human cell lines that express the frataxin gene fused to a luciferase reporter gene from its endogenous locus. Performing a pilot high-throughput genomic screen in a newly established reporter cell line, we uncovered novel negative regulators of frataxin expression. Rational design of small-molecule inhibitors of the identified frataxin repressors and/or high-throughput screening of large siRNA or compound libraries with our system may yield treatments for Friedreich's ataxia.

Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells.

INTRODUCTION: Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. METHODS: We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. RESULTS: We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFß) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. CONCLUSIONS: The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer.

Elevated protein kinase D3 (PKD3) expression supports proliferation of triple-negative breast cancer cells and contributes to mTORC1-S6K1 pathway activation.

Here, we show that the expression of the Golgi-localized serine-threonine kinase protein kinase D3 (PKD3) is elevated in triple-negative breast cancer (TNBC). Using an antibody array, we identified PKD3 to trigger the activation of S6 kinase 1 (S6K1), a main downstream target of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Accordingly, PKD3 knockdown in TNBC cells led to reduced S6K1 phosphorylation, which was associated with impaired activation of mTORC1 at endolysosomal membranes, the accumulation of the mannose 6-phosphate receptor in and the recruitment of the autophagy marker light chain 3 to enlarged acidic vesicles. We further show that PKD3 depletion strongly inhibited cell spreading and proliferation of TNBC cells, identifying this kinase as a potential novel molecular therapeutic target in TNBC. Together, our data suggest that PKD3 in TNBC cells provides a molecular connection between the Golgi and endolysosomal compartments to enhance proliferative mTORC1-S6K1 signaling.

Mammary tumor formation and metastasis evoked by a HER2 splice variant.

The HER2 gene is amplified and overexpressed in approximately 20% of invasive breast cancers where it is associated with metastasis and poor prognosis. Here, we describe a constitutively active splice variant of HER2 (Delta-HER2) in human mammary epithelial cells that evokes aggressive breast cancer phenotypes. Delta-HER2 overexpression in mammary epithelial cells was sufficient to reduce apoptosis, increase proliferation, and induce expression of mesenchymal markers, features that were associated with greater invasive potential in three-dimensional cultures in vitro and more aggressive tumorigenicity and metastasis in vivo. In contrast, overexpression of wild-type HER2 was insufficient at evoking such effects. Unbiased protein-tyrosine phosphorylation profiling in Delta-HER2-expressing cells revealed increased phosphorylation of several signaling proteins not previously known to be controlled by the HER2 pathway. Furthermore, microarray expression analysis revealed activation of genes known to be highly expressed in ER-negative, high-grade, and metastatic primary breast tumors. Together, our results provide mechanistic insights into the activity of a highly pathogenic splice variant of HER2.

Parity induces differentiation and reduces Wnt/Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium.

INTRODUCTION: Early pregnancy has a strong protective effect against breast cancer in humans and rodents, but the underlying mechanism is unknown. Because breast cancers are thought to arise from specific cell subpopulations of mammary epithelia, we studied the effect of parity on the transcriptome and the differentiation/proliferation potential of specific luminal and basal mammary cells in mice. METHODS: Mammary epithelial cell subpopulations (luminal Sca1-, luminal Sca1+, basal stem/progenitor, and basal myoepithelial cells) were isolated by flow cytometry from parous and age-matched virgin mice and examined by using a combination of unbiased genomics, bioinformatics, in vitro colony formation, and in vivo limiting dilution transplantation assays. Specific findings were further investigated with immunohistochemistry in entire glands of parous and age-matched virgin mice. RESULTS: Transcriptome analysis revealed an upregulation of differentiation genes and a marked decrease in the Wnt/Notch signaling ratio in basal stem/progenitor cells of parous mice. Separate bioinformatics analyses showed reduced activity for the canonical Wnt transcription factor LEF1/TCF7 and increased activity for the Wnt repressor TCF3. This finding was specific for basal stem/progenitor cells and was associated with downregulation of potentially carcinogenic pathways and a reduction in the proliferation potential of this cell subpopulation in vitro and in vivo. As a possible mechanism for decreased Wnt signaling in basal stem/progenitor cells, we found a more than threefold reduction in the expression of the secreted Wnt ligand Wnt4 in total mammary cells from parous mice, which corresponded to a similar decrease in the proportion of Wnt4-secreting and estrogen/progesterone receptor-positive cells. Because recombinant Wnt4 rescued the proliferation defect of basal stem/progenitor cells in vitro, reduced Wnt4 secretion appears to be causally related to parity-induced alterations of basal stem/progenitor cell properties in mice. CONCLUSIONS: By revealing that parity induces differentiation and downregulates the Wnt/Notch signaling ratio and the in vitro and in vivo proliferation potential of basal stem/progenitor cells in mice, our study sheds light on the long-term consequences of an early pregnancy. Furthermore, it opens the door to future studies assessing whether inhibitors of the Wnt pathway may be used to mimic the parity-induced protective effect against breast cancer.

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling.

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.

Co-expression of HER2 and HER3 receptor tyrosine kinases enhances invasion of breast cells via stimulation of interleukin-8 autocrine secretion.

INTRODUCTION: The tyrosine kinase receptors HER2 and HER3 play an important role in breast cancer. The HER2/HER3 heterodimer is a critical oncogenic unit associated with reduced relapse-free and decreased overall survival. While signaling cascades downstream of HER2 and HER3 have been studied extensively at the level of post-translational modification, little is known about the effects of HER2/HER3 overexpression and activation on gene expression in breast cancer. We have now defined the genetic landscape induced by activation of the HER2/HER3 unit in mammary cells, and have identified interleukin (IL)8 and CXCR1 as potential therapeutic targets for the treatment of HER2/HER3-overexpressing breast cancers. METHODS: Three-dimensional (3D) cultures, invasion and migration assays were used to determine the effects of HER2 and HER3 co-expression and activation. Gene expression analysis was performed to identify the gene network induced by HER2/HER3 in 3D cultures. Bioinformatic analysis and neutralizing antibodies were used to identify key mediators of HER2/HER3-evoked invasion. RESULTS: Co-expression of the tyrosine kinase receptors HER2 and HER3 induced migration and invasion of MCF10A cells. Microarray analysis of these cells revealed a specific "HER2/HER3 signature" comprising 80 upregulated transcripts, with IL8 being the highest (11-fold upregulation). Notably, examination of public datasets revealed high levels of IL8 transcripts in HER2-enriched as well as basal-like primary breast tumors, two subtypes characterized by a particularly poor prognosis. Moreover, IL8 expression correlated with high tumor grade and ER-negative status. Importantly, treatment with IL8-neutralizing antibodies prevented invasion of MCF10A-HER2/HER3 and BT474 cells in 3D cultures, highlighting the importance of IL8 autocrine signaling upon HER2/HER3 activation. CONCLUSIONS: Our findings demonstrate that HER2 and HER3 co-expression induces IL8 autocrine signaling, leading to the invasion of mammary cells. Agents targeting IL8 or its receptor CXCR1 may be useful for the treatment of HER2/HER3/IL8-positive breast cancers with invasive traits.

Hippo inactivation feeds tumor-initiating cells.

The Hippo pathway has emerged as a well-conserved kinase cascade controlling cell proliferation and survival and has recently gained much attention for its key activity as a tumor suppressor. In a study published in Cell, Cordenonsi and colleagues link TAZ, a downstream effector of the Hippo pathway, to attributes of putative breast cancer stem cells, epithelial-to-mesenchymal transition and cell polarity.

Loss of the ceramide transfer protein augments EGF receptor signaling in breast cancer.

Triple-negative breast cancers (TNBC) are especially refractory to treatment due to their negative hormone receptor and ErbB2/HER2 status. Therefore, the identification of cancer-associated deregulated signaling pathways is necessary to develop improved targeted therapies. Here, we show that expression of the ceramide transfer protein CERT is reduced in TNBCs. CERT transfers ceramide from the endoplasmic reticulum to the Golgi complex for conversion into sphingomyelin (SM). We provide evidence that by regulating cellular SM levels, CERT determines the signaling output of the EGF receptor (EGFR/ErbB1), which is upregulated in approximately 70% of TNBCs. CERT downregulation in breast cancer cells enhanced ErbB1 lateral mobility, ligand-induced autophosphorylation, internalization, and chemotaxis. Together, our findings provide a link between lipid metabolism at the Golgi with signaling at the plasma membrane, thereby implicating CERT loss in the progression of TNBCs.

Stem cells and the stem cell niche in the breast: an integrated hormonal and developmental perspective.

The mammary gland is a unique organ in that it undergoes most of its development after birth under the control of systemic hormones. Whereas in most other organs stem cells divide in response to local stimuli, to replace lost cells, in the mammary gland large numbers of cells need to be generated at specific times during puberty, estrous cycles and pregnancy to generate new tissue structures. This puts special demands on the mammary stem cells and requires coordination of local events with systemic needs. Our aim is to understand how the female reproductive hormones control mammary gland development and influence tumorigenesis. We have shown that steroid hormones act in a paracrine fashion in the mammary gland delegating different functions to locally produced factors. These in turn, affect cell-cell interactions that result in changes of cell behavior required for morphogenesis and differentiation. Here, we discuss how these hormonally regulated paracrine interactions may impinge on stem cells and the stem cell niche and how this integration of signals adds extra levels of complexity to current mammary stem cell models. We propose a model whereby the stem cell niches change depending on the developmental stages and the hormonal milieu. According to this model, repeated hormone stimulation of stem cells and their niches in the course of menstrual cycles may be an important early event in breast carcinogenesis and may explain the conundrum why breast cancer risk increases with the number of menstrual cycles experienced prior to a first pregnancy.

An oestrogen-dependent model of breast cancer created by transformation of normal human mammary epithelial cells.

INTRODUCTION: About 70% of breast cancers express oestrogen receptor alpha (ESR1/ERalpha) and are oestrogen-dependent for growth. In contrast with the highly proliferative nature of ERalpha-positive tumour cells, ERalpha-positive cells in normal breast tissue rarely proliferate. Because ERalpha expression is rapidly lost when normal human mammary epithelial cells (HMECs) are grown in vitro, breast cancer models derived from HMECs are ERalpha-negative. Currently only tumour cell lines are available to model ERalpha-positive disease. To create an ERalpha-positive breast cancer model, we have forced normal HMECs derived from reduction mammoplasty tissue to express ERalpha in combination with other relevant breast cancer genes. METHODS: Candidate genes were selected based on breast cancer microarray data and cloned into lentiviral vectors. Primary HMECs prepared from reduction mammoplasty tissue were infected with lentiviral particles. Infected HMECs were characterised by Western blotting, immunofluorescence microscopy, microarray analysis, growth curves, karyotyping and SNP chip analysis. The tumorigenicity of the modified HMECs was tested after orthotopic injection into the inguinal mammary glands of NOD/SCID mice. Cells were marked with a fluorescent protein to allow visualisation in the fat pad. The growth of the graft was analysed by fluorescence microscopy of the mammary glands and pathological analysis of stained tissue sections. Oestrogen dependence of tumour growth was assessed by treatment with the oestrogen antagonist fulvestrant. RESULTS: Microarray analysis of ERalpha-positive tumours reveals that they commonly overexpress the Polycomb-group gene BMI1. Lentiviral transduction with ERalpha, BMI1, TERT and MYC allows primary HMECs to be expanded in vitro in an oestrogen-dependent manner. Orthotopic xenografting of these cells into the mammary glands of NOD/SCID mice results in the formation of ERalpha-positive tumours that metastasise to multiple organs. The cells remain wild type for TP53, diploid and genetically stable. In vivo tumour growth and in vitro proliferation of cells explanted from tumours are dependent on oestrogen. CONCLUSION: We have created a genetically defined model of ERalpha-positive human breast cancer based on normal HMECs that has the potential to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasis.

Identification of molecular apocrine breast tumours by microarray analysis.

Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).

Antisense to the early growth response-1 gene (Egr-1) inhibits prostate tumor development in TRAMP mice.

Egr-1 is a transcription factor induced by stress or injury, mitogens, and differentiation factors. Egr-1 regulates the expression of genes involved in growth control or survival. Expression of Egr-1 results in either promotion or regression of cell proliferation, depending on cell type and environment. Egr-1 acts as a tumor suppressor in many cell types and loss of Egr-1 has been proposed to contribute to cancer progression. There is strong new evidence however suggesting that Egr-1 overexpression is involved in prostate cancer progression. For example, Egr-1 expression levels are elevated in human prostate carcinomas in proportion to grade and stage. Furthermore, prostate cancer progression was significantly delayed in two models of prostate cancer mice lacking Egr-1. Our objective in the present study is to test whether inhibition of Egr-1 function would block cell proliferation and inhibit the transformed phenotype of prostate cancer cells in vitro and in vivo. We describe the development of high affinity and high specificity antisense oligonucleotides that efficiently inhibit Egr-1 expression. We show that inhibition of Egr-1 expression in mouse or human prostate cancer cells decreased proliferation and reduced the capacity of these cells to form colonies and to grow in soft agar. Conversely, stable expression of Egr-1 in normal human prostate epithelial 267B1 cells promoted transformation. In TRAMP mice, treatment with Egr-1 antisense oligonucleotides delayed the occurrence of prostate tumors. Importantly, Egr-1 antisense showed little or no toxicity when injected into animals. Finally, we identified a few genes such as cyclin D2, p19ink4d, and Fas that are directly regulated by Egr-1 in prostate cancer cells and that control cell cycle and survival.