Marganit Farago

The Type III Secretion Effector NleE Inhibits NF-κB Activation

The complex host-pathogen interplay involves the recognition of the pathogen by the hosts innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-kappaB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-kappaB activation. We show that inhibition of NF-kappaB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

Oct‐3/4 regulates stem cell identity and cell fate decisions by modulating Wnt/β‐catenin signalling

Although the transcriptional regulatory events triggered by Oct‐3/4 are well documented, understanding the proteomic networks that mediate the diverse functions of this POU domain homeobox protein remains a major challenge. Here, we present genetic and biochemical studies that suggest an unexpected novel strategy for Oct‐3/4‐dependent regulation of embryogenesis and cell lineage determination. Our data suggest that Oct‐3/4 specifically interacts with nuclear β‐catenin and facilitates its proteasomal degradation, resulting in the maintenance of an undifferentiated, early embryonic phenotype both in Xenopus embryos and embryonic stem (ES) cells. Our data also show that Oct‐3/4‐mediated control of β‐catenin stability has an important function in regulating ES cell motility. Down‐regulation of Oct‐3/4 increases β‐catenin protein levels, enhancing Wnt signalling and initiating invasive cellular activity characteristic of epithelial‐mesenchymal transition. Our data suggest a novel mode of regulation by which a delicate balance between β‐catenin, Tcf3 and Oct‐3/4 regulates maintenance of stem cell identity. Altering the balance between these proteins can direct cell fate decisions and differentiation.

Clonal allelic predetermination of immunoglobulin-κ rearrangement.

Although most genes are expressed biallelically, a number of key genomic sites—including immune and olfactory receptor regions—are controlled monoallelically in a stochastic manner, with some cells expressing the maternal allele and others the paternal allele in the target tissue. Very little is known about how this phenomenon is regulated and programmed during development. Here, using mouse immunoglobulin-κ (Igκ) as a model system, we demonstrate that although individual haematopoietic stem cells are characterized by allelic plasticity, early lymphoid lineage cells become committed to the choice of a single allele, and this decision is then stably maintained in a clonal manner that predetermines monoallelic rearrangement in B cells. This is accompanied at the molecular level by underlying allelic changes in asynchronous replication timing patterns at the κ locus. These experiments may serve to define a new concept of stem cell plasticity.

Vav1 fine tunes p53 control of apoptosis versus proliferation in breast cancer.

Vav1 functions as a signal transducer protein in the hematopoietic system, where it is exclusively expressed. Vav1 was recently implicated in several human cancers, including lung, pancreatic and neuroblasoma. In this study, we analyzed the expression and function of Vav1 in human breast tumors and breast cancer cell lines. Immunohistochemical analysis of primary human breast carcinomas indicated that Vav1 is expressed in 62% of 65 tumors tested and is correlated positively with estrogen receptor expression. Based on published gene profiling of 50 breast cancer cell lines, several Vav1-expressing cell lines were identified. RT-PCR confirmed Vav1 mRNA expression in several of these cell lines, yet no detectable levels of Vav1 protein were observed due to cbl-c proteasomal degradation. We used two of these lines, MCF-7 (Vav1 mRNA negative) and AU565 (Vav1 mRNA positive), to explore the effect of Vav1 expression on breast cell phenotype and function. Vav1 expression had opposite effects on function in these two lines: it reduced proliferation and enhanced cell death in MCF-7 cells but enhanced proliferation in AU565 cells. Consistent with these findings, transcriptome analysis revealed an increase in expression of proliferation-related genes in Vav1-expressing AU565 cells compared to controls, and an increase in apoptosis-related genes in Vav1-expressing MCF-7 cells compared with controls. TUNEL and γ-H2AX foci assays confirmed that expression of Vav1 increased apoptosis in MCF-7 cells but not AU565 cells and shRNA experiments revealed that p53 is required for this pro-apoptotic effect of Vav1 in these cells. These results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer and the p53 dependence of its pro-apoptotic effect in breast cells.

GTP exchange factor Vav regulates guided cell migration by coupling guidance receptor signalling to local Rac activation

Summary Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Here, using a multidisciplinary approach, we identify the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). We show that elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. Our results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements.

Tyrosine Residues at the Carboxyl Terminus of Vav1 Play an Important Role in Regulation of Its Biological Activity

The guanine nucleotide exchange factor (GEF) Vav1 is an essential signal transducer protein in the hematopoietic system, where it is expressed physiologically. It is also involved in several human malignancies. Tyrosine phosphorylation at the Vav1 amino terminus plays a central role in regulating its activity; however, the role of carboxyl terminal tyrosine residues is unknown. We found that mutation of either Tyr-826 (Y826F) or Tyr-841 (Y841F) to phenylalanine led to loss of Vav1 GEF activity. When these Vav1 mutants were ectopically expressed in pancreatic cancer cells lacking Vav1, they failed to induce growth in agar, indicating loss of transforming potential. Furthermore, although Y841F had no effect on Vav1-stimulated nuclear factor of activated T cells (NFAT) activity, Y826F doubled NFAT activity when compared with Vav1, suggesting that Tyr-826 mediates an autoinhibitory effect on NFAT activity. SH2 profiling revealed that Shc, Csk, Abl, and Sap associate with Tyr-826, whereas SH2-B, Src, Brk, GTPase-activating protein, and phospholipase C-γ associate with Tyr-841. Although the mutations in the Tyr-826 and Tyr-841 did not affect the binding of the carboxyl SH3 of Vav1 to other proteins, binding to several of the proteins identified by the SH2 profiling was lost. Of interest is Csk, which associates with wild-type Vav1 and Y841F, yet it fails to associate with Y826F, suggesting that loss of binding between Y826F and Csk might relieve an autoinhibitory effect, leading to increased NFAT. Our data indicate that GEF activity is critical for the function of Vav1 as a transforming protein but not for NFAT stimulation. The association of Vav1 with other proteins, detected by SH2 profiling, might affect other Vav1-dependent activities, such as NFAT stimulation.

Direct proteasome binding and subsequent degradation of unspliced XBP-1 prevent its intracellular aggregation.

MINT‐7302186: XBP1‐u (uniprotkb:P17861‐1) binds (MI:0407) to Proteasome subunit alpha 6 (uniprotkb:P60900) by pull down (MI:0096)

Programming asynchronous replication in stem cells

Many regions of the genome replicate asynchronously and are expressed monoallelically. It is thought that asynchronous replication may be involved in choosing one allele over the other, but little is known about how these patterns are established during development. We show that, unlike somatic cells, which replicate in a clonal manner, embryonic and adult stem cells are programmed to undergo switching, such that daughter cells with an early-replicating paternal allele are derived from mother cells that have a late-replicating paternal allele. Furthermore, using ground-state embryonic stem (ES) cells, we demonstrate that in the initial transition to asynchronous replication, it is always the paternal allele that is chosen to replicate early, suggesting that primary allelic choice is directed by preset gametic DNA markers. Taken together, these studies help define a basic general strategy for establishing allelic discrimination and generating allelic diversity throughout the organism.

PO-196 Vav1 and Mutant K-Ras synergize in pancreatic ductal adenocarcinoma development: lessons from mouse models

Introduction The overall 5 year survival rate of Pancreatic Ductal Adenocarcinoma (PDAC) is less than 5% and has remained stubbornly unchanged long time ago, despite the efforts in preclinical and clinical science. PDAC, the main form of pancreatic cancer, develops via acinar-ductal metaplasia (ADM) and neoplastic precursor lesions, such as pancreatic intraepithelial neoplasia (PanIN). Mutant K-Ras is present in >90% of PDAC and is the most frequent and the earliest genetic alteration, being found in low-grade lesions. Identification of other molecular lesions that affect PDAC is of cardinal importance. One such potential protein is Vav1, a hematopietic specific signal transducer. Overexpression of wild-type Vav1 is implicated in human cancers, such as neuroblastoma, lung and PDAC. The expression of Vav1 in PDAC is indicative of a worse survival rate. Our goal was to determine whether Vav1 plays a causative or facilitatory role in-vivo in PDAC development. Material and methods We generated several transgenic mouse models that express Vav1, K-RasG12D, or Vav1 and K-RasG12D in the pancreas. K-Ras was induced by tamoxifen and Vav1 by Dox. Pancreata were analysed at different times post transgenes induction. Results and discussions Vav1 Expression together with K-RasG12D in the pancreas has a dramatic synergistic effect enhancing ADM formation already at 3 months post transgene induction, resulting in at least 3 times the number of lesions in the pancreata of Vav1;K-RasG12D mice compared to K-RasG12 mice. No lesions were observed in the pancreas of Vav1 mice. The number of Ki-67 positive cells in Vav1;K-RasG12D mice was significantly higher than in Vav1 or K-RasG12D transgenic mice. The increase of pancreatic lesions was also accompanied with an increase in staining of Sox9 and Keratin and various pathways such as pERK, pEGFR and Rac1-GTP., highlighting the synergistic effect of Vav1 and K-RasG12D in the development of PDAC. Notably, removal of Dox, thus ablating the expression of Vav1 in the pancreas of Vav1;K-RasG12D led to a significant reduction in malignant lesions, thus further highlighting the necessity of expression of both oncogenes to cancer development in the pancreas. Conclusion Vav1 contributes to the development of malignant lesions in the pancreas when expressed with mutant K-Ras. Identification of Vav1 as a protein that synergizes with mutant K-Ras in PDAC development might pave the way to choosing good candidates for therapeutic drug design.

PO-135 Analysis of the transforming activity of human cancer identified vav1 mutants prove its classification as an oncogene

Introduction The activity of Vav1 as a mutant oncogene in human tumours has remained questionable for decades. Although mutants of Vav1 were recently identified in human cancers of various origins, the functional activity of these mutants is not fully studied. Vav1 is physiologically active as a GDP/GTP nucleotide exchange factor (GEF) in the hematopoietic system. In this study, we addressed the contribution of several cancer-identified Vav1 mutants to tumorigenic processes. Material and methods We introduced several amino-acid substitutions at residues identified in human lung cancer as follows: glutamic acid (position 59; calponin homology region) to lysine (E59K); aspartic acid (position 517; C1 domain) to glutamic acid (D517E); and leucine (position 801; carboxySH3) to proline (L801P). The biochemical and transforming activities of these mutants were tested following transfection into NIH3T3 cells. Results and discussions Among the mutants produced, E59K generated a truncated protein, which preserved its expected size once cells are incubated with MG132, a specific proteasome inhibitor. E59K, D517E and oncVav1 are active as GEF towards Rho/RacGTPases, albeit E59K exhibited the uppermost activity. This result was illustrated by Pymol, a computer software for molecular visualisation, that predicts its increased activity as a GEF. This activity is also manifested in changes in cytoskeleton organisation indicative of transformation. Analysis of protein stability using cycloheximide decay assay revealed that D517E mutant protein is more stable than the other mutants, thus explaining its increased expression and activity. Furthermore, NIH3T3 cells expressing E59K, D517E and oncVav1 mutants exhibit increased cell proliferation, elevated number of transformed foci and increased number of generated tumours in NOD/Scid mice. Of note is the fact that tumours generated by E59K exhibit the most aggressive phenotype among the mutant proteins used in this study, reminiscent of epithelial morphology. Conclusion Our results convincingly attest to the transforming potential of the Vav1 mutants, E59K and D517E, thus providing compelling evidence that Vav1 mutants can act as ‘real’ oncogenes in human cancer.