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.ion channel | CaCCinh-A01 | TMEM16A | HNSCC | ESCC

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 Ca 2+ 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.

New cancer therapies are likely to arise from an in-depth understanding of the signaling networks influencing tumor initiation, progression and metastasis. We show a fundamental role for Src-homology 2 domain-containing phosphatase 2 (SHP2) in these processes in human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancers. Knockdown of SHP2 eradicated breast tumor-initiating cells in xenograft models, and SHP2 depletion also prevented invasion in three-dimensional cultures and in a transductal invasion assay in vivo. Notably, SHP2 knockdown in established breast tumors blocked their growth and reduced metastasis. Mechanistically, SHP2 activated stemness-associated transcription factors, including v-myc myelocytomatosis viral oncogene homolog (c-Myc) and zinc finger E-box binding homeobox 1 (ZEB1), which resulted in the repression of let-7 microRNA and the expression of a set of 'SHP2 signature' genes. We found these genes to be simultaneously activated in a large subset of human primary breast tumors that are associated with invasive behavior and poor prognosis. These results provide new insights into the signaling cascades influencing tumor-initiating cells as well as a rationale for targeting SHP2 in 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.

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 a-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. Cancer Res; 71(13); 4344-51. Ó2011 AACR.

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.

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.

Dendritic spines form the postsynaptic contact sites for most excitatory synapses in the brain. Spines occur in a wide range of different shapes that can vary depending on an animal's experience or behavioral status. Recently we showed that spines on living neurons can change shape within seconds in a process that depends on actin polymerization. We have now found that this morphological plasticity is blocked by inhalational anesthetics at concentrations at which they are clinically effective. These volatile compounds also block actin-based motility in fibroblasts, indicating that their action is independent of neuron-specific components and thus identifying the actin cytoskeleton as a general cellular target of anesthetic action. These observations imply that inhibition of actin dynamics at brain synapses occurs during general anesthesia and that inhalational anesthetics are capable of inf luencing the morphological plasticity of excitatory synapses in the brain.