Vav guanine nucleotide exchange factors (GEFs) have been implicated in cell adhesion by integrin and immune response receptors through the regulation of Rho GTPases. Here, we examine the role of Vav and Rho GTPases in phagocytosis by using primary murine macrophages. The genetic deletion of Rac1 and Rac2 prevents phagocytosis mediated by integrin and Fcgamma receptors (FcgammaR), whereas the genetic deletion of Vav1 and Vav3 only prevents integrin-mediated phagocytosis through the complement receptor alpha(M)beta(2). In addition, a Rac1/2 or Vav1/3 deficiency blocks Arp2/3 recruitment and actin polymerization at the complement-induced phagosome, indicating that these proteins regulate early steps in phagocytosis. Moreover, constitutively active Rac is able to rescue actin polymerization and complement-mediated phagocytosis in Vav-deficient macrophages. These studies indicate that Rac is critical for complement- and FcgammaR-mediated phagocytosis. In contrast, Vav is specifically required for complement-mediated phagocytosis, suggesting that Rac is regulated by GEFs other than Vav downstream of the FcgammaR.

Sprouty was originally identified in a genetic screen in Drosophila as an antagonist of fibroblast (FGF) and epidermal growth factor (EGF) signaling. Subsequently, four vertebrate homologs were discovered; among these, the human homolog Sprouty 2 (hSpry2) contains the highest degree of sequence homology to the Drosophila protein. It has been shown that hSpry2 interacts directly with c-Cbl, an E3-ubiquitin ligase, which promotes the downregulation of receptor tyrosine kinases (RTKs). In this study, we have investigated the functional consequences of the association between hSpry2 and c-Cbl. We have found that hSpry2 is ubiquitinated by c-Cbl in an EGF-dependent manner. EGF stimulation induces the tyrosine phosphorylation of hSpry2, which in turn enhances the interaction of hSpry2 with c-Cbl. The c-Cbl-mediated ubiquitination of hSpry2 targets the protein for degradation by the 26S proteasome. An enhanced proteolytic degradation of hSpry2 is also observed in response to FGF stimulation. The FGF-induced degradation of hSpry2 limits the duration of the inhibitory effect of hSpry2 on extracellular signal-regulated kinase (ERK) activation and enables the cells to recover their sensitivity to FGF stimulation. Our results indicate that the interaction of hSpry2 with c-Cbl might serve as a mechanism for the downregulation of hSpry2 during receptor tyrosine kinase signaling.

Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients.

This work describes a 3D, paper-based assay that can isolate subpopulations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O 2 ). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. Stacking the layers of paper assembles them into 3D tissue-like constructs of defined thickness and composition. The plastic holder ensures the layers of paper are in conformal contact; this geometry allows the cells to migrate between adjacent layers through the embedded hydrogel. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel (into which the cells migrate). Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients both in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates subpopulations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells. Page 3 of 35 Significance StatementThe invasion of cancerous cells from a tumor into surrounding tissues is one contribution to metastasis-a major contributor to death for patients with cancer. There is a strong link between the directed invasion of cancer cells and the gradients of molecules formed in the microenvironment of the tumor. Using a paper-based invasion assay, this work demonstrates that oxygen-a nutrient known to induce significant behavioral changes to cells within a tumor in a concentration-dependent manner-can also act as a chemoattractant, resulting in the migration of cancer cells towards higher concentrations of oxygen. This finding, and the invasion assay described, could lead to a better understanding of oxygen-based chemotaxis in cancer, and ultimately new strategies for managing metastasis.

Signal transduction through epidermal growth factor receptors (EGFRs) is essential for the growth and development of multicellular organisms. A genetic screen for regulators of EGFR signaling has led to the identification of Sprouty, a cell autonomous inhibitor of EGF signaling that is transcriptionally induced by the pathway. However, the molecular mechanisms by which Sprouty exerts its antagonistic effect remain largely unknown. Here we have used transient expression in human cells to investigate the functional properties of human Sprouty (hSpry) proteins. Ectopically expressed full-length hSpry1 and hSpry2 induce the potentiation of EGFR-mediated mitogenactivated protein (MAP) kinase activation. In contrast, truncation mutants of hSpry1 and hSpry2 containing the highly conserved carboxyl-terminal cysteine-rich domain inhibit EGF-induced MAP kinase activation. The potentiating effect of the full-length hSpry2 proteins on EGF signaling is mediated by the amino-terminal domain and results from the sequestration of c-Cbl, which in turn leads to the inhibition of EGFR ubiquitination and degradation. These results indicate that hSpry2 can function both as a negative and positive regulator of EGFR-mediated MAP kinase signaling in a domaindependent fashion. A dual function of this kind could provide a mechanism for achieving proper balance between the activation and repression of EGFR signaling. R eceptor tyrosine kinase-mediated signaling pathways are used in both invertebrates and vertebrates to control critical aspects of organ morphogenesis, patterning, cellular proliferation, and differentiation (1). These signaling pathways are tightly regulated by positive and negative inputs that ensure the proper cellular fate (2). The development of the Drosophila compound eye is regulated primarily through inductive signaling emanating from the Drosophila homolog of the epidermal growth factor receptor (EGFR) (DER; ref. 3). Recently, a genetic screen for modifiers of DER signaling in the Drosophila eye has identified Sprouty (dSpry) as a negative modulator of this pathway (4). Loss-of-function dSpry mutations were able to overcome the phenotype resulting from the overexpression of another negative regulator of EGFR signaling, Argos, whereas the overexpression of dSpry induced a phenotype similar to that exhibited by EGFR loss-of-function mutants (4). In addition, dSpry function is required for the proper development of other tissues in Drosophila that require DER signaling, including embryonic chordotonal organ precursors, the wing imaginal disk, midline glia, and ovarian follicle cells (5, 6). Overexpression of dSpry in these tissues mimics a DER loss-of-function phenotype, whereas loss of dSpry leads to a hyperactivation of the DER pathway.The activation of DER by EGF leads to the stimulation of the Ras͞mitogen-activated protein (MAP) kinase pathway (7). In Drosophila, dSpry seems to impinge on the DER pathway upstream of MAP kinase as overexpression of dSpry leads to a decrease in activated MAP kinase (6). Epistasis exp...

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The search for target genes involved in unbalanced acquired chromosomal abnormalities has been largely unsuccessful, because the breakpoints of these rearrangements are too variable. Here, we use the example of dicentric chromosomes in B cell precursor acute lymphoblastic leukemia to show that, despite this heterogeneity, single genes are targeted through a variety of mechanisms. FISH showed that, although they were heterogeneous, breakpoints on 9p resulted in the partial or complete deletion of PAX5. Molecular copy number counting further delineated the breakpoints and facilitated cloning with long-distance inverse PCR. This approach identified 5 fusion gene partners with PAX5: LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21), and C20orf112 (20q11.1). In each predicted fusion protein, the DNA-binding paired domain of PAX5 was present. Using quantitative PCR, we demonstrated that both the deletion and gene fusion events resulted in the same underexpression of PAX5, which extended to the differential expression of the PAX5 target genes, EBF1, ALDH1A1, ATP9A, and FLT3. Further molecular analysis showed deletion and mutation of the homologous PAX5 allele, providing further support for the key role of PAX5. Here, we show that specific gene loci may be the target of heterogeneous translocation breakpoints in human cancer, acting through a variety of mechanisms. This approach indicates an application for the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent and few genes have been identified. It can be extrapolated that this strategy will reveal that the same mechanisms operate in cancer pathogenesis in general.ALL ͉ breakpoint cloning ͉ molecular copy number counting C hromosomal rearrangements are recurrent findings in human cancer and result in aberrant restructuring of the genome (1). Reciprocal (balanced) translocations may lead to abnormal gene function by direct disruption of coding sequences, such as the formation of chimaeric fusion genes. To date, 358 fusion genes have been identified in human malignancy, the majority of which are the result of balanced chromosomal rearrangements (2). However, the majority of translocations described in human cancer are unbalanced (3), suggesting that other cancer genes remain to be identified. The analysis of unbalanced translocations has largely failed to identify target genes because of the heterogeneity of the chromosomal breakpoints and the multiplicity of partner chromosomes. Thus, it has been assumed that these rearrangements affect gene function through the loss or gain of chromosomal material. Identification of the key molecular events resulting from unbalanced rearrangements would be a significant step toward understanding their role in cancer pathogenesis.Examples among which both breakpoint heterogeneity and multiplicity of partners are found are dicentric chromosomes: one of the cytogenetic features found in patients with B cell precursor acute lymphoblastic leukaemias (B...

Glucocorticoids (GCs) specifically induce apoptosis in malignant lymphoblasts and are thus pivotal in the treatment of acute lymphoblastic leukemia (ALL). However, GC-resistance is a therapeutic problem with an unclear molecular mechanism. We generated approximately 70 GC-resistant sublines from a GC-sensitive B- and a T-ALL cell line and investigated their mechanisms of resistance. In response to GCs, all GC-resistant subclones analyzed by real-time polymerase chain reaction (PCR) showed a deficient up-regulation of the GC-receptor (GR) and its downstream target, GC-induced leucine zipper. This deficiency in GR up-regulation was confirmed by Western blotting and on retroviral overexpression of GR in resistant subclones GC-sensitivity was restored. All GC-resistant subclones were screened for GR mutations using denaturing high-pressure liquid chromatography (DHPLC), DNA-fingerprinting, and fluorescence in situ hybridization (FISH). Among the identified mutations were some previously not associated with GC resistance: A484D, P515H, L756N, Y663H, L680P, and R714W. This approach revealed three genotypes, complete loss of functional GR in the mismatch repair deficient T-ALL model, apparently normal GR genes in B-ALLs, and heterozygosity in both. In the first genotype, deficiency in GR up-regulation was fully explained by mutational events, in the second by a putative regulatory defect, and in the third by a combination thereof. In all instances, GC-resistance occurred at the level of the GR in both models.