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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.

Emerging evidence suggests that some cancers contain a population of stem-like TICs (tumor-initiating cells) and eliminating TICs may offer a new strategy to develop successful anti-cancer therapies. As molecular mechanisms underlying the maintenance of the TIC pool are poorly understood, the development of TIC-specific therapeutics remains a major challenge. We first identified and characterized TICs and non-TICs isolated from a mouse breast cancer model. TICs displayed increased tumorigenic potential, self-renewal, heterogeneous differentiation, and bipotency. Gene expression analysis and immunostaining of TICs and non-TICs revealed that FGFR2 was preferentially expressed in TICs. Loss of FGFR2 impaired self-renewal of TICs, thus resulting in marked decreases in the TIC population and tumorigenic potential. Restoration of FGFR2 rescued the defects in TIC pool maintenance, bipotency, and breast tumor growth driven by FGFR2 knockdown. In addition, pharmacological inhibition of FGFR2 kinase activity led to a decrease in the TIC population which resulted in suppression of breast tumor growth. Moreover, human breast TICs isolated from patient tumor samples were found enriched in a FGFR2+ population that was sufficient to initiate tumor growth. Our data suggest that FGFR2 is essential in sustaining the breast TIC pool through promotion of self-renewal and maintenance of bipotent TICs, and raise the possibility of FGFR2 inhibition as a strategy for anti-cancer therapy by eradicating breast TICs.

Hippo signaling represents a tumor suppressor pathway that regulates organ size and tumorigenesis through phosphorylation and inhibition of the transcription coactivator YAP. Here, we show that serum deprivation dramatically induces YAP Ser127 phosphorylation and cytoplasmic retention, independent of cell-cell contact. Through chemical isolation and activity profiling, we identified serum-derived sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) as small molecule activators of YAP. S1P induces YAP nuclear localization through S1P(2) receptor, Rho GTPase activation, and F-actin polymerization, independent of the core Hippo pathway kinases. Bioinformatics studies also showed that S1P stimulation induces YAP target gene expression in mouse liver and human embryonic stem cells. These results revealed potent small molecule regulators of YAP and suggest that S1P and LPA might modulate cell proliferation and tumorigenesis through YAP activation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Decoquinate has single-digit nanomolar activity against in vitro blood stage Plasmodium falciparum parasites, the causative agent of human malaria. In vitro evolution of decoquinate-resistant parasites and subsequent comparative genomic analysis to the drug-sensitive parental strain revealed resistance was conferred by two nonsynonymous single nucleotide polymorphisms in the gene encoding cytochrome b. The resultant amino acid mutations, A122T and Y126C, reside within helix C in the ubiquinol-binding pocket of cytochrome b, an essential subunit of the cytochrome bc(1) complex. As with other cytochrome bc(1) inhibitors, such as atovaquone, decoquinate has low nanomolar activity against in vitro liver stage P. yoelii and provides partial prophylaxis protection when administered to infected mice at 50 mg kg(-1). In addition, transgenic parasites expressing yeast dihydroorotate dehydrogenase are >200-fold less sensitive to decoquinate, which provides additional evidence that this drug inhibits the parasite's mitochondrial electron transport chain. Importantly, decoquinate exhibits limited cross-resistance to a panel of atovaquone-resistant parasites evolved to harbor various mutations in cytochrome b. The basis for this difference was revealed by molecular docking studies, in which both of these inhibitors were shown to have distinctly different modes of binding within the ubiquinol-binding site of cytochrome b.

Many proteins or other biological macromolecules are localized to more than one subcellular structure. The fraction of a protein in different cellular compartments is often measured by colocalization with organelle-specific fluorescent markers, requiring availability of fluorescent probes for each compartment and acquisition of images for each in conjunction with the macromolecule of interest. Alternatively, tailored algorithms allow finding particular regions in images and quantifying the amount of fluorescence they contain. Unfortunately, this approach requires extensive hand-tuning of algorithms and is often cell type-dependent. Here we describe a machine-learning approach for estimating the amount of fluorescent signal in different subcellular compartments without hand tuning, requiring only the acquisition of separate training images of markers for each compartment. In testing on images of cells stained with mixtures of probes for different organelles, we achieved a 93% correlation between estimated and expected amounts of probes in each compartment. We also demonstrated that the method can be used to quantify drug-dependent protein translocations. The method enables automated and unbiased determination of the distributions of protein across cellular compartments, and will significantly improve imaging-based high-throughput assays and facilitate proteome-scale localization efforts.

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.

Nuclear receptors (NRs) are transcription factors whose activity is modulated by ligand binding. These receptors are at the core of complex signaling pathways and act as integrators of many cellular signals. In the last decade our understanding of NRs has greatly evolved. In particular, regulation of NR subcellular dynamics has emerged as central to their activity. Research on the subcellular distribution of the thyroid hormone receptor (TR) has revealed new dimensions in the complexity of NR regulation, and points to the possibility that NR mislocalization plays a key role in oncogenesis. For many years, TR was thought to reside exclusively in the nucleus. It is now known that TR is a dynamic protein that shuttles between the nucleus and cytoplasm. TR is localized to the nucleus in a phosphorylated form, suggesting that compartment-specific phosphorylation mediates cross-talk between multiple cell signaling pathways. The oncoprotein v-ErbA, a viral-derived dominant negative variant of TR is actively exported to the cytoplasm by the CRM1 export receptor. Strikingly, the oncoprotein causes mislocalization of cellular TR and some of its coactivators by direct interaction. Here, we offer some perspectives on the role of subcellular trafficking in the oncogenic conversion of TR, and propose a new model for oncoprotein dominant negative activity.

The retroviral v-ErbA oncoprotein is a highly mutated variant of the thyroid hormone receptor alpha (TRalpha), which is unable to bind T(3) and interferes with the action of TRalpha in mammalian and avian cancer cells. v-ErbA dominant-negative activity is attributed to competition with TRalpha for T(3)-responsive DNA elements and/or auxiliary factors involved in the transcriptional regulation of T(3)-responsive genes. However, competition models do not address the altered subcellular localization of v-ErbA and its possible implications in oncogenesis. Here, we report that v-ErbA dimerizes with TRalpha and the retinoid X receptor and sequesters a significant fraction of the two nuclear receptors in the cytoplasm. Recruitment of TRalpha to the cytoplasm by v-ErbA can be partially reversed in the presence of ligand and when chromatin is disrupted by the histone deacetylase inhibitor trichostatin A. These results define a new mode of action of v-ErbA and illustrate the importance of cellular compartmentalization in transcriptional regulation and oncogenesis.

v-ErbA, an oncogenic derivative of the thyroid hormone receptor alpha (TRalpha) carried by the avian erythroblastosis virus, contains several alterations including fusion of a portion of avian erythroblastosis virus Gag to its N terminus, N- and C-terminal deletions, and 13 amino acid substitutions. Nuclear export of v-ErbA occurs through a CRM1-mediated pathway. In contrast, nuclear export of TRalpha and another isoform, TRbeta, is CRM1-independent. To determine which amino acid changes in v-ErbA confer CRM1-dependent nuclear export, we expressed a panel of green and yellow fluorescent protein-tagged mutant and chimeric proteins in mammalian cells. The sensitivity of subcellular trafficking of these mutants to leptomycin B (LMB), a specific inhibitor of CRM1, was assessed by fluorescence microscopy. Our data showed that a nuclear export sequence resides within a 70-amino acid domain in the C-terminal portion of the p10 region of Gag, and in vitro binding assays demonstrated that Gag interacts directly with CRM1. However, a panel of ligand-binding domain mutants of v-ErbA lacking the Gag sequence exhibited greater nuclear localization in the presence of LMB, suggesting that the various amino acid substitutions/deletions may cause a conformation shift, unmasking an additional CRM1-dependent nuclear export sequence. In contrast, the altered DNA-binding domain of the oncoprotein did not contribute to CRM1-dependent nuclear export. Heterokaryon experiments revealed that v-ErbA did not undergo nucleocytoplasmic shuttling when the CRM1 export pathway was blocked by LMB treatment, suggesting that the ability to follow the export pathway used by TRalpha has been lost by the oncoprotein during its evolution. Our findings thus point to the intriguing possibility that acquisition of altered nuclear export capabilities contributes to the oncogenic properties of v-ErbA.