Heike Brinkhaus

Glutamate receptors regulate actin-based plasticity in dendritic spines.

Dendritic spines at excitatory synapses undergo rapid, actin-dependent shape changes which may contribute to plasticity in brain circuits. Here we show that actin dynamics in spines are potently inhibited by activation of either AMPA or NMDA subtype glutamate receptors. Activation of either receptor type inhibited actin-based protrusive activity from the spine head. This blockade of motility caused spines to round up so that spine morphology became both more stable and more regular. Inhibition of spine motility by AMPA receptors was dependent on postsynaptic membrane depolarization and influx of Ca2+ through voltage-activated channels. In combination with previous studies, our results suggest a two-step process in which spines initially formed in response to NMDA receptor activation are subsequently stabilized by AMPA receptors.

JAK2/STAT5 Inhibition Circumvents Resistance to PI3K/mTOR Blockade: A Rationale for Cotargeting These Pathways in Metastatic Breast Cancer

Hyperactive PI3K/mTOR signaling is prevalent in human malignancies and its inhibition has potent antitumor consequences. Unfortunately, single-agent targeted cancer therapy is usually short-lived. We have discovered a JAK2/STAT5-evoked positive feedback loop that dampens the efficacy of PI3K/mTOR inhibition. Mechanistically, PI3K/mTOR inhibition increased IRS1-dependent activation of JAK2/STAT5 and secretion of IL-8 in several cell lines and primary breast tumors. Genetic or pharmacological inhibition of JAK2 abrogated this feedback loop and combined PI3K/mTOR and JAK2 inhibition synergistically reduced cancer cell number and tumor growth, decreased tumor seeding and metastasis, and also increased overall survival of the animals. Our results provide a rationale for combined targeting of the PI3K/mTOR and JAK2/STAT5 pathways in triple-negative breast cancer, a particularly aggressive and currently incurable disease.

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.

Actin dynamics in dendritic spines: A form of regulated plasticity at excitatory synapses

Dendritic spines form the postsynaptic element at most excitatory synapses in the brain. The spine cytoskeleton consists of actin filaments which, in time‐lapse recordings of living neurons expressing actin labeled with green fluorescent protein, can be seen to undergo rapid, dynamic changes. Because actin dynamics are associated with changes in cell shape, these cytoskeletal rearrangements may form a molecular basis for the morphological plasticity at brain synapses. The rapidity of these dynamic events in dendritic spines raises new questions. First, do the changes in actin cytoskeleton that are visible by light microscopy really correspond to changes in spine morphology, or do they represent changes in the relationship between actin and its many binding partners at postsynaptic sites? Second, how are these changes regulated by synaptic transmission? Third, to what extent do these changes occur in organized brain tissue? Answers to these questions are now beginning to emerge. Hippocampus 2000;10:555–560.

Luminal Expression of PIK3CA Mutant H1047R in the Mammary Gland Induces Heterogeneous Tumors

The phosphoinositide 3-kinase (PI3K) signaling cascade, a key mediator of cellular survival, growth, and metabolism, is frequently altered in human cancer. Activating mutations in PIK3CA, which encodes the α-catalytic subunit of PI3K, occur in approximately 30% of breast cancers. These mutations result in constitutive activity of the enzyme and are oncogenic, but it is not known whether they are sufficient to induce mammary carcinomas in mice. In the present study, we show that the expression of mutant PIK3CA H1047R in the luminal mammary epithelium evokes heterogeneous tumors that express luminal and basal markers and are positive for the estrogen receptor. Our results suggest that the PIK3CA H1047R oncogene targets a multipotent progenitor cell and, furthermore, show that this model recapitulates features of human breast tumors with PIK3CA H1047R.

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

IntroductionEarly 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.MethodsMammary 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.ResultsTranscriptome 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.ConclusionsBy 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.

Interactions between drebrin and Ras regulate dendritic spine plasticity

Dendritic spines are major sites of morphological plasticity in the CNS, but the molecular mechanisms that regulate their dynamics remain poorly understood. Here we show that the association of drebrin with actin filaments plays a major role in regulating dendritic spine stability and plasticity. Overexpressing drebrin or the internal actin‐binding site of drebrin in rat hippocampal neurons destabilized mature dendritic spines so that they lost synaptic contacts and came to resemble immature dendritic filopodia. Drebrin‐induced spine destabilization was dependent on Ras activation: expression of constitutively active Ras destabilized spine morphology whereas drebrin‐induced spine destabilization was rescued by co‐expressing dominant negative Ras. Conversely, RNAi‐mediated drebrin knockdown prevented Ras‐induced destabilization and promoted spine maturation in developing neurons. Together these data demonstrate a novel mechanism in which the balance between stability and plasticity in dendritic spines depends on binding of drebrin to actin filaments in a manner that is regulated by Ras.

Influx of extracellular calcium regulates actin-dependent morphological plasticity in dendritic spines.

Dendritic spines contain a specialized cytoskeleton composed of dynamic actin filaments capable of producing rapid changes in their motility and morphology. Transient changes in Ca2+ levels in the spine cytoplasm have been associated with the modulation of these effects in a variety of ways. To characterize the contribution of Ca2+ fluxes originating through different pathways to these phenomena, we used time-lapse imaging of cultured hippocampal neurons expressing GFP-actin to follow the influence of postsynaptic neurotransmitter receptors, voltage-activated Ca2+ channels and release from internal Ca2+ stores on spine actin dynamics. Stimulation of AMPA receptors produced a rapid blockade of actin-dependent spine motility that was immediately reversible when AMPA was removed. Stimulation of NMDA receptors also blocked spine motility but in this case suppression of actin dynamics was delayed by up to 30 min depending on NMDA concentration and motility was never seen to recover when NMDA was removed. These effects could be mimicked by depolarizing neurons under appropriate circumstances demonstrating the involvement of voltage-activated Ca2+ channels in AMPA receptor-mediated effects and the receptor associated Ca2+ channel in the effects of NMDA. Caffeine, an agent that releases Ca2+ from internal stores, had no immediate effect on spine actin, a result compatible with the lack of caffeine-releasable Ca2+ in cultured hippocampal neurons under resting conditions. Blocking internal store function by thapsigargin led to a delayed suppression of spine actin dynamics that was dependent on extracellular Ca2+. Together these results indicate the common involvement of changes in Ca2+ levels in modulating actin-dependent effects on dendritic spine motility and morphology through several modes of electrophysiological activation.

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.

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

IntroductionStromal-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.MethodsWe 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.ResultsWe 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 (TGFβ) 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.ConclusionsThe 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.