J Condeelis

..In this study, we test the hypothesis that heterogeneous cancer cells exhibit variable motility responses such as chemokinesis and chemotaxis that can be characterized molecularly...

..We have combined chemotaxis-based cell collection of the invasive subpopulation of cells within the primary tumor with array-based gene expression analysis to identify the genes necessary for the process of carcinoma cell invasion...

..However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy...

..We discuss how these new insights may lead to a better understanding of the molecular basis of the invasive behavior of tumor cells in vivo, which may result in new strategies for the diagnosis and treatment of metastasis...

..Thus, macrophages are at the center of the invasion microenvironment and are an important drug target for cancer therapy...

..Examples of new insights derived from the different scales of imaging and relevant probes are discussed in the context of cancer progression and metastasis...

..These results highlight the need for new methods that enable the direct observation of actin nucleation and so define precisely the relative contributions of the three processes to stimulated cell motility...

..We conclude that aligning cell behavior in vivo with patterns of gene expression can lead to new insights into the microenvironment of carcinoma cells in the primary tumor and the molecular mechanisms behind cell behavior...

..These results support a "tumor microenvironment invasion model" and provide new target opportunities for cancer therapy...

..Our data suggest that Cdc42 activation is crucial for the regulation of actin polymerization in carcinoma cells, and required for both EGF-stimulated protrusion and cell motility independently of effects on Rac...

..We propose a role for cofilin that is distinct from its role as an actin-depolymerizing factor...

..Therefore, our results do not support a model for the initial activation of cofilin by dephosphorylation in response to chemoattractant stimulation in metastatic carcinoma cells...

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..Taken together, we conjecture that invasive TAMs link angiogenesis and tumor invasion and that Wnt-signaling plays a role in mediating their activity...

..These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42...

..We found that ZBP1 expression actually reduced tumor cell motility and chemotaxis, presumably mediating their decreased metastatic potential by reducing their ability to respond to signals necessary for invasion...

..In particular, we show that the autocrine contribution to invasion is specifically amplified in vivo through a tumor microenvironment-induced upregulation of CSF-1R expression via the transforming growth factor-beta1...

..These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility...

..The Mena isoform switching pattern described here may provide a new biomarker for the presence of metastatic cancer cells and for prognosis...

..Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF...

..Importantly, these defining characteristics of cells undergoing EMT were discussed in the context of therapeutic and prognostic developments...

..LIMK1-mediated decreases or increases in the activity of the cofilin pathway are shown to cause proportional decreases or increases in motility, intravasation, and metastasis of tumor cells...

..Identification of effectors that are differently regulated by p110alpha versus p110beta will be important for understanding cell migration and its role in metastasis...

..In addition, these results indicate that tumor cells that are chemotactic and migratory in response to epidermal growth factor in the primary tumor have a survival advantage over stationary tumor cells...

..ZBP1 reexpression also inhibited metastasis from tumors. These experiments support the involvement in metastasis of the pathways identified in invasive cells, which are regulated by ZBP1...

..Disruption of this loop by blockade of either EGF receptor or CSF-1 receptor signaling is sufficient to inhibit both macrophage and tumor cell migration and invasion...

..Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development...

..Imaging of live tumors shows that MLC is organized in a similar ROCK-dependent fashion in vivo and that inhibition of ROCK but not matrix-metalloproteases reduces cancer cell motility in vivo...

..Our observations suggest that, when compared with kinase domain mutations in a genetically identical background, expression of helical domain mutants of p110alpha produce a more severe metastatic phenotype...

..Photoremoval of the cage generates an active protein, which cannot be switched off by endogenous protein kinases. Caged cofilin, and its irradiated counterpart, display the anticipated F-actin depolymerization and severing activities...

..This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient...

..These results indicate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement...

..eEF1A away from interactions with its known translational ligands, e.g. aminoacyl-tRNA and ribosomes...

..These animals provide a range of expression of GFP that is suitable for analysis of transgenic mammary tumors and metastases in vivo at the single cell level of resolution...

..The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins...

..The molar ratio of ABP-50 to actin is approximately 1:5 in vivo. Therefore, the abundance of ABP-50 suggests that it may be responsible for the majority of the bundling activity in these cells...

..Identifying regulatory sites on transport factors as well as novel physiological functions for well-known translation factors has provided significant advances in how spatially controlled translation impacts cell function...

..The present data provide new insight into the relevance of actin cytoskeleton regulatory proteins and, in particular, of hMena isoforms in coupling multiple signaling pathways involved in breast cancer...

..This analysis is predicted to allow a complete description of the molecular interactions required for the actin polymerization transients seen in a variety of cells during hormones stimulation. ..

..The specific aims of the proposal are: 1. Identify microenvironments involved in invasion in human breast tumors. 2. Determine gene expression profiles of invasive cells in human breast tumors. ..

..These reagents will be used in a variety of ways in cells to define the importance of the above mechanisms and their interactions in vivo. ..

..All projects require the capability to image cells or in vitro assay systems at high spatial resolution with minimum exposure to light or at high speeds. ..

..mRNAs for beta-actin and Arp3, when localized to the leading edge, determine cell polarity. 2. The EF1a-F-actin complex is the scaffold to which mRNAs, with a beta-actin mRNA-like zipcode, become anchored. ..

..4. Evaluate a variety of clustering algorithms, with DNA expression patterns obtained in specific aims 2 and 3, to identify genes related to cell behaviors believed necessary for metastasis. ..

..Use the reagents in 1 and 2 in vivo to determine the relationships between cofilin and the Arp2/3 complex in the regulation of actin nucleation in unstimulated and EGF-stimulated cells ..

..This uses nanotechnology to miniaturize an artificial blood vessel that can in dwell in human tumor xenografts and ultimately in human tumors that can report on the status of cell collection and diagnose invasive tumors. ..

..This program represents a unique perspective on invasion and metastasis that has the potential for generating broad new insights into the microenvironments of the primary tumor. ..

..abstract_text> ..

..The specific aims of the proposal are: 1. Identify microenvironments involved in invasion in human breast tumors. 2. Determine gene expression profiles of invasive cells in human breast tumors. ..

..As discussed in the budget justification, the system we request will solve the problems of speed, sensitivity, and quantitation for an expert user group. ..

..Nearest neighbor analysis using bifunctional reagents will be used to identify components on the vesicle membrane to which actin binds. ..

..This investigation will include experiments using drugs and antibodies to modulate in vitro activity and eventual isolation of the ligand stimulated nucleation activity for actin polymerization...

..The combined approaches of protein chemistry, bacterial expression and molecular genetics in Dictyostelium will provide a definitive analysis of the structure and function of aginactin...

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