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Triple-negative breast cancer (TNBC) accounts for 15–20% of breast cancer cases in the United States, lacks targeted therapeutic options, and is associated with a 40–80% risk of recurrence. Thus, identifying actionable targets in treatment-naïve and chemoresistant TNBC is a critical unmet medical need. To address this need, we performed high-throughput drug viability screens on human tumor cells isolated from 16 patient-derived xenograft models of treatment-naïve primary TNBC. The models span a range of TNBC subtypes and exhibit a diverse set of putative driver mutations, thus providing a unique patient-derived, molecularly annotated pharmacologic resource that is reflective of TNBC. We identified therapeutically actionable targets including kinesin spindle protein (KSP). The KSP inhibitor targets the mitotic spindle through mechanisms independent of microtubule stability and showed efficacy in models that were resistant to microtubule inhibitors used as part of the current standard of care for TNBC. We also observed subtype selectivity of Prima-1Met, which showed higher levels of efficacy in the mesenchymal subtype. Coupling pharmacologic data with genomic and transcriptomic information, we showed that Prima-1Met activity was independent of its canonical target, mutant p53, and was better associated with glutathione metabolism, providing an alternate molecularly defined biomarker for this drug.

Background Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC. Methods A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3β inhibitors were identified as EMT inhibitors. The effects of GSK3β inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3β correlates with the overall survival of breast cancer patients. Results We identified a GSK3β inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3β inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3β reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3β inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24− in cells after exposure to GSK3β inhibitors. We found that GSK3β inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3β inhibitors and found that GSK3β inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3β correlated with poorer overall patient survival. Conclusions Taken together, our data demonstrate that GSK3β is a potential target for TNBCs and suggest that GSK3β inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3β inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.

Retinoid X receptor (RXR) belongs to a family of ligand-activated transcription factors that regulate many aspects of metazoan life. A class of nuclear receptors requires RXR as heterodimerization partner for their function. This places RXR in the crossroad of multiple distinct biological pathways. This and the fact that the debate on the endogenous ligand requirement for RXR is not yet settled make RXR still an enigmatic transcription factor. Here, we review some of the biology of RXR. We place RXR into the evolution of nuclear receptors, review structural details and ligands of the receptor. Then processes regulated by RXR are discussed focusing on the developmental roles deduced from studies on knockout animals and metabolic roles in diseases such as diabetes and atherosclerosis deduced from pharmacological studies. Finally, aspects of RXR's involvement in myeloid differentiation and apoptosis are summarized along with issues on RXR's suitability as a therapeutic target.

Vitamin A and its derivatives (collectively referred to as retinoids) are required for many fundamental life processes, including vision, reproduction, metabolism, cellular differentiation, hematopoesis, bone development, and pattern formation during embryogenesis. There is also considerable evidence to suggest that natural and synthetic retinoids have therapeutical effects due to their antiproliferative and apoptosis-inducing effects in human diseases such as cancer. Therefore it is not surprising that a significant amount of research was dedicated to probe the molecular and cellular mechanisms of retinoid action during the past decade. One of the cellular mechanisms retinoids have been implicated in is the initiation and modulation of apoptosis in normal development and disease. This review provides a brief overview of the molecular basis of retinoid signaling, and focuses on the retinoid-regulation of apoptotic cell death and gene expression during normal development and in pathological conditions in vivo and in various tumor cell lines in vitro.

Tissue transglutaminase is a multifunctional enzyme that accumulates to high levels in cells undergoing apoptosis. Retinoids act as an acute and direct regulator of tissue transglutaminase gene transcription. The studies reported here were carried out to elucidate the molecular mechanisms involved in the regulation of tissue transglutaminase expression. We have isolated and characterized the mouse tissue transglutaminase gene promoter and 3.8 kb of 5'-flanking DNA. A large fragment of the promoter that includes both the core promoter and 3.8 kb of 5'-flanking DNA shows retinoid-dependent transcriptional activity when stably transfected into HeLa cells. In these stably transfected HeLa cells both the endogenous tissue transglutaminase gene and transfected mouse tissue transglutaminase promoter are activated by all-trans retinoic acid and by retinoic acid receptor (RAR)-specific and retinoid X receptor (RXR)-specific retinoids. In embryos made transgenic with a transglutaminase promoter-beta-galactosidase reporter gene, the transgene shows specific patterns of expression during limb development. The transglutaminase transgene is expressed in cartilage, the cells of the apical ectodermal ridge, and in regions of apoptotic cell death of the interdigital mesenchyme. It appears that cis-acting elements responsible for the complex retinoid regulation, tissue- and apoptosis-specific expression are embedded within the proximal 3.8 kb of DNA flanking the 5'-end of the mouse tissue transglutaminase gene.

The receptor-mediated endocytosis of alpha 2-macroglobulin can be inhibited by a diverse group of chemical compounds all of which share the property of being inhibitors of one form of cellular transglutaminase. The present results strongly suggest that protein cross-linking may be essential for receptor-mediated endocytosis of some protein and polypeptide hormones.

The function of intracellular transglutaminases remains to be clarified. In fibroblasts the links between the activity of this enzyme and receptor-mediated endocytosis are complex and open to interpretation. However, the issue cannot be firmly laid to rest until the structural specificity of the alkylamine inhibitors of endocytosis is explained. In macrophages, there is substantial evidence that the enzyme plays some role in receptor-mediated phagocytosis, but what this role is and how it might relate to endocytosis in other types of cells is at present an unresolved issue.

The reefless tract directly behind the ribbon reefs on the outer shelf off Cooktown supports a luxuriant growth of Halimeda that, during the Holocene, has developed into bioherms. These mounded biodies of unconsolidated sediment have formed banks that vary in height between 2 and 20 m. Combined shallow, high-resolution seismic reflection profiles and side-scan sonar have diferentiated three areas of biohermal complexes behind the ribbon reefs of Cooktown. Observations by SCUBA and submersible plus the sedimentology of the bioherms indicate that they are in situ accumulations. Evidence from dating of cores suggests that the Halimeda bioherms began to grow about 10 000 years B.P. and their growth has continued to the present time, even though their tops are presently restricted to a depth of -20 m. It is suggested that the origin and morphology of the bioherms are related to a specific hydrodynamic phenomenon, involving jets of nutrient-rich, upwelled oceanic water intruding onto the outer shelf via the narrow passes between the ribbon reefs, and forming eddies behind the ribbons.

α2-MACROGLOBULIN (α2M), insulin and epidermal growth factor (EGF) have a common mechanism for internalisation1. We have proposed a model for this mechanism1–3 in which the ligands bind to diffusely distributed receptors followed by the collection of the mobile ligand–receptor complexes over coated pits. These coated pits pinch off from the plasma membrane to form endocytic vesicles. The diffuse initial distribution of the α2M receptors has been shown by binding α2M to fixed cells and observing the location of α2M by an electron microscopic immunocytochemical method2. The mobility of the ligand–receptor complexes has been demonstrated using fluorescence photobleaching recovery4, and the clustering of such complexes has been demonstrated by fluorescence microscopy1,3,5. Electron microscopic immunocytochemical localisation has been used to show that the clustering occurs over coated pits which pinch off to form endocytic vesicles2. The mechanism proposed for the endocytosis of α2M, insulin and EGF is similar to that described for the internalisation of low density lipoprotein (LDL), except that unoccupied LDL receptors may cluster in coated pits before binding LDL6. It seems likely that many molecules, including other polypeptide hormones and the lysosomal hydrolases7 which bind to specific receptors on the cell surface, are internalised by a similar mechanism. To determine the role of clustering and internalisation in hormone action it would be useful to have an agent that inhibits these processes. It has been found that the internalisation of lysosomal enzymes is noncompetitively inhibited by some amines and ammonia (E. Neufeld, personal communication). It has also been reported that ammonia prevents the internalisation of diptheria toxin by HeLa cells8. In view of these effects, we examined the possibility that amines and ammonia would block the clustering of α2M and EGF, and we report here that these agents are effective in blocking clustering and internalisation.

GEBELEIN and Hoffman1 drew attention to the association of dolomite with stromatolitic sequences, and suggested that the precipitation of dolomite is related to the presence of organic material. Their experiments confirmed previous results2,3 that large quantities of magnesium may be absorbed from seawater by organic material. Magnesium-rich algal filaments may form sites for the precipitation of high magnesium calcite with 17–20 mol % MgCO3 and although dolomite has not been precipitated in such conditions, it is possible that high-Mg calcite could be diagenetically altered to dolomite because of the high Mg–Ca ratio associated with the organic material1.

Studies of modem coral reefs have shown that Holocene reef growth is relatively thin, and that in many instances it has developed on an older limestone surface1–4. This surface is commonly associated with leaching and pedogenic processes, indicative of subaerial exposure, and has been termed the solution unconformity5. Investigations into reef growth history have interpreted the pre-Holocene substrate as an older reef surface2–4,6–8 that may or may hot have undergone karst erosion during periods of lower sea level6,9. Uranium-series dating of corals directly below the uppermost solution unconformity at Eniwetok10 and Mururoa11 has shown that previous reef growth, before the Holocene, occurred ∼120 kyr ago. We present here 230Th/234U dates that confirm a last interglacial age for reefal limestones directly beneath the uppermost solution unconformity in the southern Great Barrier Reef.

The growth of coral reefs has been a controversial subject ever since Darwin's1 subsidence theory. Scientists have argued over the major factors affecting growth2–4, but most have agreed that reefs grow dominantly along their windward margins5–7, a characteristic displayed in the Permian8 and Devonian9 reefs of New Mexico and Western Australia. Recently, however, Adey10 has indicated that the growth characteristics of windward margins are energy related, such that different growth characteristics are to be expected under different energy regimes. The Great Barrier Reef is a high-energy epicontinental shelf reef system on which it has been suggested11–13 that, in the prevailing conditions, windward growth has been subordinate to vertical and leeward growth. We present here results from drilling and monitoring studies demonstrating that reef growth in a transgressive epicontinental shelf environment is first vertical and then backwards, especially on the leeward margin, when the reef reaches the stillstand sea level position.

MYOSIN, a major contractile element of muscle, has been found in a variety of non-muscle cells such as platelets1,2, fibroblasts3,4, brain tissue5, amoebae6 and slime mould7. It is localised in the plasma membrane8 as well as in the cytosol4 of cultured fibroblasts. Although no definite function has been established for non-muscle myosins, they may participate in cellular movement, mitosis, changes in cell shape and related functions. We have examined the protein subunit composition of plasma membranes from normal and transformed fibroblasts with a particular emphasis on determining the content of plasma membrane-associated myosin. We present here evidence that the content of plasma membrane-associated myosin is diminished in transformed fibroblasts. This decrease may in part account for the abnormal shape and loss of contact inhibition of movement of transformed cells9.