Daniel Besser

Stat3 activation is required for cellular transformation by v-src.

ABSTRACT Stat3 activation has been associated with cytokine-induced proliferation, anti-apoptosis, and transformation. Constitutively activated Stat3 has been found in many human tumors as well as v-abl- and v-src-transformed cell lines. Because of these correlations, we examined directly the relationship of activated Stat3 to cellular transformation and found that wild-type Stat3 enhances the transforming potential of v-src while three dominant negative Stat3 mutants inhibit v-srctransformation. Stat3 wild-type or mutant proteins did not affect v-ras transformation. We conclude that Stat3 has a necessary role in v-src transformation.

TRANCE, a TNF Family Member, Activates Akt/PKB through a Signaling Complex Involving TRAF6 and c-Src

TRANCE, a TNF family member, and its receptor, TRANCE-R, are critical regulators of dendritic cell and osteoclast function. Here, we demonstrate that TRANCE activates the antiapoptotic serine/threonine kinase Akt/PKB through a signaling complex involving c-Src and TRAF6. A deficiency in c-Src or addition of Src family kinase inhibitors blocks TRANCE-mediated PKB activation in osteoclasts. c-Src and TRAF6 interact with each other and with TRANCE-R upon receptor engagement. TRAF6, in turn, enhances the kinase activity of c-Src leading to tyrosine phosphorylation of downstream signaling molecules such as c-Cbl. These results define a mechanism by which TRANCE activates Src family kinases and PKB and provide evidence of cross-talk between TRAF proteins and Src family kinases.

E-cadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming

We report new functions of the cell‐adhesion molecule E‐cadherin in murine pluripotent cells. E‐cadherin is highly expressed in mouse embryonic stem cells, and interference with E‐cadherin causes differentiation. During cellular reprogramming of mouse fibroblasts by OCT4, SOX2, KLF4 and c‐MYC, fully reprogrammed cells were exclusively observed in the E‐cadherin‐positive cell population and could not be obtained in the absence of E‐cadherin. Moreover, reprogrammed cells could be established by viral E‐cadherin in the absence of exogenous OCT4. Thus, reprogramming requires spatial cues that cross‐talk with essential transcription factors. The cell‐adhesion molecule E‐cadherin has important functions in pluripotency and reprogramming.

Insulin regulates the activity of forkhead transcription factor Hnf-3β/Foxa-2 by Akt-mediated phosphorylation and nuclear/cytosolic localization

Hepatocyte nuclear factors 3 α, β, and γ (Foxa-1, -2, and -3) are transcriptional activators of important metabolic genes in the liver that are suppressed by the actions of insulin. Here, we show that the activation of phosphatidylinositol 3-kinase–Akt by insulin induces Foxa-2 phosphorylation, nuclear exclusion, and inhibition of Foxa-2-dependent transcriptional activity. Foxa-2 physically interacts with Akt, a key mediator of the phosphatidylinositol 3-kinase pathway and is phosphorylated at a single conserved site (T156) that is absent in Foxa-1 and Foxa-3 proteins. This Akt phosphorylation site in Foxa-2 is highly conserved from mammals to insects. Mutant Foxa-2T156A is resistant to Akt-mediated phosphorylation, nuclear exclusion, and transcriptional inactivation of Foxa-2-regulated gene expression. These results implicate an evolutionarily conserved mechanism in the regulation of Foxa-2-dependent transcriptional control by extracellular signals such as insulin.

Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells

Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9–HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency.

SH2 and SH3-containing adaptor proteins: redundant or independent mediators of intracellular signal transduction

Molecules which contain Src Homology 2 (SH2) and SH3 domains provide one of the principal ways by which signals are transduced in cells using protein–protein interactions between proline‐rich motifs and SH3 domains and induced interactions between phosphotyrosine residues and SH2 domains. The simplest of SH2/SH3‐containing proteins are the Crk, Grb2 and Nck adaptor proteins which contain SH2 and SH3 domains but no intrinsic catalytic activity. Whereas Grb2 connects activated receptor tyrosine kinases with Sos and activates p21ras, recent evidence suggests that this may not be the major mechanism by which Crk and Nck signal to downstream effectors. Identification of novel binding partners for Crk, Grb2 and Nck indicate that these adaptor proteins control distinct aspects of tyrosine kinase signalling.

A Single Amino Acid Substitution in the v-Eyk Intracellular Domain Results in Activation of Stat3 and Enhances Cellular Transformation

ABSTRACT The receptor tyrosine kinase Eyk, a member of the Axl/Tyro3 subfamily, activates the STAT pathway and transforms cells when constitutively activated. Here, we compared the potentials of the intracellular domains of Eyk molecules derived from c-Eyk and v-Eyk to transform rat 3Y1 fibroblasts. The v-Eyk molecule induced higher numbers of transformants in soft agar and stronger activation of Stat3; levels of Stat1 activation by the two Eyk molecules were similar. A mutation in the sequence Y933VPL, present in c-Eyk, to the v-Eyk sequence Y933VPQ led to increased activation of Stat3 and increased transformation efficiency. However, altering another sequence, Y862VNT, present in both Eyk molecules to F862VNT markedly decreased transformation without impairing Stat3 activation. These results indicate that activation of Stat3 enhances transformation efficiency and cooperates with another pathway to induce transformation.

Protein kinase B beta/Akt2 plays a specific role in muscle differentiation.

Insulin-like growth factors positively regulate muscle differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway. Here, we compare the role of the two closely related α (Akt1) and β (Akt2) isoforms of PKB in muscle differentiation. During differentiation of C2.7 or L6D2 myoblasts, PKBβ was up-regulated whereas expression of PKBα was unaltered. Although the two isoforms were found active in both myoblasts and myotubes, cell fractionation experiments indicated that they displayed distinct subcellular localizations in differentiated cells with only PKBβ localized in the nuclei. In a transactivation assay, PKBβ (either wild-type or constitutively active) was more efficient than PKBα in activating muscle-specific gene expression. Moreover, microinjection of specific antibodies to PKBβ inhibited differentiation of muscle cells, whereas control or anti-PKBα antibodies did not. On the other hand, microinjection of the anti-PKBα antibodies caused a block in cell cycle progression in both non muscle and muscle cells, whereas anti-PKBβ antibodies had no effect. Taken together, these results show that PKBβ plays a crucial role in the commitment of myoblasts to differentiation that cannot be substituted by PKBα.

A Colorectal Cancer Expression Profile That Includes Transforming Growth Factor β Inhibitor BAMBI Predicts Metastatic Potential

BACKGROUND & AIMS Much is known about the genes and mutations that cause colorectal cancer (CRC), yet only a few have been associated with CRC metastasis. We performed expression-profiling experiments to identify genetic markers of risk and to elucidate the molecular mechanisms of CRC metastasis. METHODS We compared gene expression patterns between metastatic and nonmetastatic stage-matched human colorectal carcinomas by microarray analysis. Correlations between BAMBI and metastasis-free survival were examined by quantitative real-time polymerase chain reaction (PCR) using an independent set of human colon carcinomas. Human colon cancer cell lines were analyzed for BAMBI regulation, cell motility, and experimental metastasis. RESULTS We established a signature of 115 genes that differentiated metastatic from nonmetastatic primary tumors. Among these, the transforming growth factor (TGF) beta inhibitor BAMBI was highly expressed in approximately half of metastatic primary tumors and metastases but not in nonmetastatic tumors. BAMBI is a target of canonical Wnt signaling that involves the beta-catenin coactivator BCL9-2. We observed an inverse correlation between level of BAMBI expression and metastasis-free survival time of patients. BAMBI inhibits TGF-beta signaling and increases migration in colon cancer cells. In mice, overexpression of BAMBI caused colon cancer cells to form tumors that metastasized more frequently to liver and lymph nodes than control cancer cells. CONCLUSIONS BAMBI regulates CRC metastasis by connecting the Wnt/beta-catenin and TGF-beta-signaling pathways. The metastatic expression signature we describe, along with BAMBI levels, can be used in prognosis. Developmental signaling pathways appear to act in hierarchies and cooperate in tumor cell migration, invasion, and metastasis.

The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development

The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.