Summary EGF induces signal transduction between EGFR and FAK, and FAK is required for EGF-induced cell migration. It is unknown, however, what factor mediates the interaction between EGFR and FAK and leads to EGF-induced FAK phosphorylation. Here we identify SRC-3Δ4, a splicing isoform of the SRC-3 oncogene, as a signaling adaptor that links EGFR and FAK and promotes EGF-induced phosphorylations of FAK and c-Src. We identify three PAK1-mediated phosphorylations in SRC-3Δ4 that promote the localization of SRC-3Δ4 to the plasma membrane and mediate the interactions with EGFR and FAK. Importantly, over-expression of SRC-3Δ4 promotes MDA-MB231-induced breast tumor metastasis. Our findings identify phosphorylated SRC-3Δ4 as a missing adaptor between EGFR and its downstream signaling molecule FAK, to coordinately regulate EGF-induced cell migration. Our study also reveals the new concept that a nuclear receptor coactivator can act in the periphery of a cell to directly mediate activation of an enzyme.

Background: Previously, proteomic methods were applied to characterise differentially expressed proteins in microdissected pancreatic ductal adenocarcinoma cells. Aims: To report that CapG and a related protein, gelsolin, which have established roles in cell motility, are overexpressed in metastatic pancreatic cancer; and to describe their pattern of expression in pancreatic cancer tissue and their effect on cell motility in pancreatic cancer cell lines. Methods: CapG was identified by mass spectrometry and immunoblotting. CapG and gelsolin expression was assessed by immunohistochemical analysis on a pancreatic cancer tissue microarray and correlated with clinical and pathological parameters. CapG and gelsolin levels were reduced using RNA interface in Suit-2, Panc-1 and MiaPaCa-2 cells. Cell motility was assessed using modified Boyden chamber or wound-healing assays.Results: Multiple isoforms of CapG were detected in pancreatic cancer tissue and cell lines. Immunohistochemical analysis of benign (n = 44 patients) and malignant (n = 69) pancreatic ductal cells showed significantly higher CapG staining intensity in nuclear (p,0.001) and cytoplasmic (p,0.001) compartments of malignant cells. Similarly, gelsolin immunostaining of benign (n = 24 patients) and malignant (n = 68 patients) pancreatic ductal cells showed higher expression in both compartments (both p,0.001). High nuclear CapG was associated with increased tumour size (p = 0.001). High nuclear gelsolin was associated with reduced survival (p = 0.01). Reduction of CapG or gelsolin expression in cell lines by RNAi was accompanied by significantly impaired motility. Conclusions: Up regulation of these actin-capping proteins in pancreatic cancer and their ability to modulate cell motility in vitro suggest their potentially important role in pancreatic cancer cell motility and consequently dissemination.

Summary Environmental exposures to chemically heterogeneous endocrine disrupting chemicals (EDCs) mimic or interfere with hormone actions, and negatively impact human health. Despite public interest and the prevalence of EDCs in the environment, methods to mechanistically classify these diverse chemicals in a high throughput (HT) manner have not been actively explored. Here, we describe the use of multi-parametric, HT microscopy-based platforms to examine how a prototypical EDC, Bisphenol A (BPA), and eighteen poorly studied analogs (BPXs), affect estrogen receptor (ER). We show that short exposure to BPA and most BPXs induce ERα and/or ERβ and change levels of target gene transcription. Many BPXs exhibit higher affinity for ERβ and act as ERβ antagonists, while they act largely as agonists or mixed agonists/antagonists on ERα. Finally, despite binding to ERs, some BPXs exhibit lower levels of activity. Our comprehensive view of BPXs activities allows their classification and evaluation of potential harmful effects. The strategy described here used on a large scale basis likely offers a faster, more cost-effective way to identify safer BPA alternatives.

BACKGROUND: High levels of S100A6 have been associated with poor outcome in pancreatic cancer patients. The functional role of S100A6 is, however, poorly understood. METHODS: Immunoprecipitation followed by two-dimensional gel electrophoresis and mass spectrometry were undertaken to identify S100A6 interacting proteins in pancreatic cancer cells. Immunohistochemistry and coimmunofluorescence were performed to examine expression or colocalisation of proteins. siRNA was used to deplete specific proteins and effects on motility were measured using Boyden Chamber and wound healing assays. RESULTS: Our proteomic screen to identify S100A6 interacting proteins revealed annexin 11, annexin 2, tropomyosin b and a candidate novel interactor lamin B1. Of these, annexin 2 was considered particularly interesting, as, like S100A6, it is expressed early in the development of pancreatic cancer and overexpression occurs with high frequency in invasive cancer. Reciprocal immunoprecipitation confirmed the interaction between annexin 2 and S100A6 and the proteins colocalised, particularly in the plasma membrane of cultured pancreatic cancer cells and primary pancreatic tumour tissue. Analysis of primary pancreatic cancer specimens (n ¼ 55) revealed a strong association between high levels of cytoplasmic S100A6 and the presence of annexin 2 in the plasma membrane of cancer cells (P ¼ 0.009). Depletion of S100A6 was accompanied by diminished levels of membrane annexin 2 and caused a pronounced reduction in the motility of pancreatic cancer cells. CONCLUSION: These findings point towards a functional role for S100A6 that may help explain the link between S100A6 expression in pancreatic cancer and aggressive disease.

Estrogen receptor-α (ER) is an important target both for therapeutic compounds and endocrine disrupting chemicals (EDCs); however, the mechanisms involved in chemical modulation of regulating ER transcriptional activity are inadequately understood. Here, we report the development of a high content analysis-based assay to describe ER activity that uniquely exploits a microscopically visible multicopy integration of an ER-regulated promoter. Through automated single-cell analyses we simultaneously quantified promoter occupancy, recruitment of transcriptional cofactors and large-scale chromatin changes in response to a panel of ER ligands and EDCs. Image-derived multi-parametric data was used to classify a panel of ligand responses at high resolution. We propose this system as a novel technology providing new mechanistic insights into EDC activities in a manner useful for both basic mechanistic studies and drug testing.

Cell migration requires the regulated disassembly of focal adhesions, but the underlying mechanisms remain poorly defined. We have previously shown that focal adhesion disassembly requires the dynamin 2-and clathrin-dependent endocytosis of ligand-activated ␤1 integrins. Here, we identify type I phosphatidylinositol phosphate kinase beta (PIPKI␤), an enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI4,5P 2 ), as a key regulator of this process. We found that knockdown of PIPKI␤ by RNA interference blocks the internalization of active ␤1 integrins and impairs focal adhesion turnover and cell migration. These defects are caused by the failure to target the endocytic machinery, including clathrin adaptors and dynamin 2, to focal adhesion sites. As a consequence, depletion of PIPKI␤ blocks clathrin assembly at adhesion plaques and prevents complex formation between dynamin 2 and focal adhesion kinase (FAK), a critical step in focal adhesion turnover. Together, our findings identify PIPKI␤ as a novel regulator of focal adhesion disassembly and suggest that PIPKI␤ spatially regulates integrin endocytosis at adhesion sites to control cell migration.

Bisphenols and phthalates, chemicals frequently used in plastic products, promote obesity in cell and animal models. However, these well-known metabolism-disrupting chemicals (MDCs) represent only a minute fraction of all compounds found in plastics. To gain a comprehensive understanding of plastics as a source of exposure to MDCs, we characterized the chemicals present in 34 everyday products using nontarget high-resolution mass spectrometry and analyzed their joint adipogenic activities by high-content imaging. We detected 55,300 chemical features and tentatively identified 629 unique compounds, including 11 known MDCs. Importantly, the chemicals extracted from one-third of the products caused murine 3T3-L1 preadipocytes to proliferate, and differentiate into adipocytes, which were larger and contained more triglycerides than those treated with the reference compound rosiglitazone. Because the majority of plastic extracts did not activate the peroxisome proliferator-activated receptor γ and the glucocorticoid receptor, the adipogenic effects are mediated via other mechanisms and, thus, likely to be caused by unknown MDCs. Our study demonstrates that daily-use plastics contain potent mixtures of MDCs and can, therefore, be a relevant yet underestimated environmental factor contributing to obesity.

Epithelial organization is maintained by cell proliferation, migration, and differentiation. In the case of the gastric epithelium, at least some of these events are regulated by the hormone gastrin. In addition, gastric epithelial cells are organized into characteristic tubular structures (the gastric glands), but the cellular mechanisms regulating the organization of tubular structures (sometimes called branching morphogenesis) are uncertain. In the present study, we examined the role of the gastrin-cholecystokinin(B) receptor in promoting branching morphogenesis of gastric epithelial cells. When gastric cancer AGS-G(R) cells were cultured on plastic, gastrin and PMA stimulated cell adhesion, formation of lamellipodia, and extension of long processes in part by activation of protein kinase C (PKC) and phosphatidylinositol (PI)-3 kinase. Branching morphogenesis was not observed in these circumstances. However, when cells were cultured on artificial basement membrane, the same stimuli increased the formation of organized multicellular arrays, exhibiting branching morphogenesis. These effects were reversed by inhibitors of PKC but not of PI-3 kinase. We conclude that, in the presence of basement membrane, activation of PKC by gastrin stimulates branching morphogenesis.