Serge Roche

DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases.

The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.

Evidence that Resistance to Nilotinib May Be Due to BCR-ABL, Pgp, or Src Kinase Overexpression

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in chronic myeloid leukemia (CML) and in Bcr-Abl-positive acute lymphoblastic leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second-generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance.

A specific function for phosphatidylinositol 3-kinase α (p85α-p110α) in cell survival and for phosphatidylinositol 3-kinase β (p85α-p110β) in de novo DNA synthesis of human colon carcinoma cells

We have previously shown an important function of phosphatidylinositol 3-kinase (PI3K)α(p85α-p110α) and PI3Kβ (p85-α-p110β) for DNA synthesis induced by various mitogens in non transformed fibroblasts and we now report a specific role of these enzymes in human colon cancer cell growth. Using antibodies specific to p110α and to p110β catalytic subunits, increase in PI3Kα and PI3Kβ activities was detected in 15/19 human tumour biopsies relative to adjacent normal mucosa of human colon and bladder. Increase in such activities was also observed in adenocarcinoma cell lines CaCo2, CO115, HCT 116, LS 174T and WiDr relative to non-transformed fibroblasts. Maximal PI3Kα activity was observed for LS 174T and PI3Kβ activity for WiDr cells. This was partly correlated with an increase in p110α and p110β protein levels both in some primary tumours and established cell lines, suggesting that PI3K overexpression is involved in enzymatic deregulation. Functional consequence of such activation was assessed by a microinjection approach. An injection of neutralizing antibody specific to p110β in WiDr, HCT116 and CO 115 cells inhibited de novo DNA synthesis, whereas antibodies specific to p110γ had no effect. Neutralizing antibodies specific to p110α induced apoptosis, a response that was reverted by treating cells with the caspase inhibitor z-VAD-fmk. However anti-p110β and anti-p110γ antibodies did not affect cell survival. We concluded that PI3Kα and PI3Kβ play important roles in human colon cancer cell growth with a specific function for PI3Kβ in de novo DNA synthesis and an involvement of PI3Kα in cell survival.

A Function for Phosphatidylinositol 3-Kinase β (p85α-p110β) in Fibroblasts during Mitogenesis: Requirement for Insulin- and Lysophosphatidic Acid-Mediated Signal Transduction

ABSTRACT We have previously shown that phosphatidylinositol 3-kinase α (PI 3-Kα) (p85α-p110α) is required for DNA synthesis induced by various growth factors (S. Roche, M. Koegl, and S. A. Courtneidge, Proc. Natl. Acad. Sci. USA 91:9185–9189, 1994) in fibroblasts. In the present study, we have investigated the function of PI 3-Kβ (p85α-p110β) during mitogenesis. By using antibodies specific to p110β we showed that PI 3-Kβ is expressed in NIH 3T3 cells. PI 3-Kβ and PI 3-Kα have common features: PI 3-Kβ is tightly associated with a protein serine kinase that phosphorylates p85α, it interacts with the Src-middle T antigen complex and the activated platelet-derived growth factor (PDGF) receptor in fibroblasts in vivo, and it becomes tyrosine phosphorylated after PDGF stimulation. PI 3-Kβ was also activated in Swiss 3T3 and Cos7 cells stimulated with lysophosphatidic acid (LPA), a mitogen that interacts with a heterotrimeric G protein-coupled receptor. In contrast PI 3-Kα was activated to a lesser extent in these cells. Microinjection of neutralizing antibodies specific for p110β into quiescent fibroblasts inhibited DNA synthesis induced by both insulin and LPA but poorly affected PDGF receptor signaling. Therefore, PI 3-Kβ plays an important role in transmitting the mitogenic response induced by some, but not all, growth factors. Finally, we show that while oncogenic V12Ras interacts with type I PI 3-Ks, it could induce DNA synthesis in the absence of active PI 3-Kα and PI 3-Kβ, suggesting that Ras uses other effectors for DNA synthesis.

Src and Ras are involved in separate pathways in epithelial cell scattering

We have demonstrated previously that Src controls the epidermal growth factor (EGF)‐induced dispersion of NBT‐II carcinoma epithelial cells. Here we show that while only Src and Yes were expressed and activated by EGF, microinjected kinase‐inactive mutants of Src (SrcK−) and Fyn (FynK−) were able to exert a dominant‐negative effect on the scattering response. Both SH2 and SH3 domains of FynK− were required for inhibition of cell scattering. Expression of dominant‐negative N17Ras also abrogated EGF‐induced dispersion, showing that Ras is another regulator of cell dispersion. Expression of SrcK− did not alter EGF‐evoked Shc tyrosine phosphorylation, Shc–Grb2 complex formation and MAPK activation, three elements of the Ras pathway. Furthermore, the expression of Jun–Fos and Slug rescued the block induced by N17Ras but not by SrcK−, showing that Src kinases and Ras operate in separate pathways. In addition, actinomycin D inhibition of RNA synthesis repressed the ability of the activated mutant L61Ras but not that of F527Src to induce epithelial cell scattering. Since tyrosine phosphorylation of cytoskeleton‐associated proteins pp125FAK and cortactin were abolished in EGF‐stimulated SrcK− cells, we concluded that, in contrast to Ras, Src kinases may control epithelial cell dispersion in the absence of gene expression and by directly regulating the organization of the cortical cytoskeleton.

Requirement of phospholipase C gamma, the tyrosine phosphatase Syp and the adaptor proteins Shc and Nck for PDGF-induced DNA synthesis: evidence for the existence of Ras-dependent and Ras-independent pathways.

We have investigated the roles of the phosphotyrosine phosphatase Syp (also called SH‐PTP2), phospholipase C (PLC) gamma1, rasGTPase Activating Protein (rasGAP) and the adapter molecules Nck and Shc in the mitogenic response induced by PDGF in fibroblasts. Two separate approaches were used to inhibit the biological activity of these signalling proteins in vivo. Either glutathione S‐transferase (GST) fusion proteins containing the SH2 domains of these proteins, or antibodies specific for these polypeptides, were microinjected into cells. GST‐SH2 fusion proteins are expected to act as dominant inhibitors by competing for physiological SH2‐mediated interactions, while microinjected antibodies can directly block protein functions. Inhibition of PLCgamma, Syp, Shc and Nck signals blocked PDGF‐stimulated cells in G1 showing a requirement for these proteins for S‐phase entry. Inhibition of rasGAP, in contrast, had no effect on S‐phase entry. We next examined which of these signals were required for PDGF‐induced cFos expression, a Ras‐dependent event important for signalling. By using the same approaches with cells expressing beta‐galactosidase under the control of a c‐fos promoter, we showed that PLCgamma, Syp and Shc were necessary for ligand‐induced cFos expression whereas Nck and phosphatidylinositol 3‐kinase alpha were not. From these results we concluded that PDGF generates Ras‐dependent and Ras‐independent pathways important for DNA synthesis.

c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis

The mechanism by which the ubiquitously expressed Src family kinases regulate mitogenesis is not well understood. Here we report that cytoplasmic tyrosine kinase c‐Abl is an important effector of c‐Src for PDGF‐ and serum‐induced DNA synthesis. Inactivation of cytoplasmic c‐Abl by the kinase‐ inactive Abl‐PP‐K− (AblP242E/P249E/K290M) or by microinjection of Abl neutralizing antibodies inhibited mitogenesis. The kinase‐inactive SrcK295M induced a G1 block that was overcome by the constitutively active Abl‐PP (AblP242E/P249E). Conversely, the inhibitory effect of Abl‐PP‐K− was not compensated by Src. c‐Src‐induced c‐Abl activation involves phosphorylation of Y245 and Y412, two residues required for c‐Abl mitogenic function. Finally, we found that p53 inactivation and c‐myc expression, two cell cycle events regulated by Src during mitogenesis, also implied c‐Abl: c‐Abl function was dispensable in cells deficient in active p53 and inhibition of c‐Abl reduced mitogen‐induced c‐myc expression. These data identify a novel function of cytoplasmic c‐Abl in the signalling pathways regulating growth factor‐induced c‐myc expression and we propose the existence of a tyro sine kinase signalling cascade (PDGFR/c‐Src/c‐Abl) important for mitogenesis.

Cytoplasmic signalling by the c-Abl tyrosine kinase in normal and cancer cells

c‐Abl is a non‐receptor tyrosine kinase which is localized both in the nucleus and cytoplasm, and is involved in the regulation of cell growth, survival and morphogenesis. Although c‐Abl nuclear function has been extensively studied, recent data also indicate an important role in cytoplasmic signalling through mitogenic and adhesive receptors. Here, we review the mechanisms by which growth factors promote cytoplasmic c‐Abl activation and signalling and its function in the induction of DNA synthesis, changes in cell morphology and receptor endocytosis. The importance of de‐regulated c‐Abl cytoplasmic signalling in solid tumours is also discussed.

The hepatitis B virus HBx protein induces adherens junction disruption in a src-dependent manner

Chronic hepatitis B virus infection is strongly associated with the development of hepatocellular carcinoma (HCC). Epithelial tumors are frequently characterized by loss of cadherin expression or function. Cadherin-dependent adhesion prevents the acquisition of a migratory and invasive phenotype, and loss of its function is itself enough for the progression from adenoma to carcinoma. The HBx protein of hepatitis B virus is thought to contribute to the development of the carcinoma, however, its role in the oncogenic and metastatic processes is far from being fully understood. We report herein the ability of HBx to disrupt intercellular adhesion in three different cell lines stably transfected with an inducible HBx expression vector. The linkage between the actin cytoskeleton and cadherin complex, which is essential for its function, is disrupted in the presence of HBx, as indicated by detergent solubility and immunoprecipitation experiments. In addition, β-catenin was tyrosine phosphorylated in HBx-expressing cells. Inhibition of the src family of tyrosine kinases resulted in the prevention of the disruption of adherens junctions. These results suggest that HBx is able to disrupt intercellular adhesion in a src-dependent manner, and provide a novel mechanism by which HBx may contribute to the development of HCC.

Adherens junctions and tight junctions are regulated via different pathways by progastrin in epithelial cells

Adhesion between neighbouring epithelial cells is a crucial and tightly controlled process. In the gastrointestinal tract, the integrity of cell-cell contacts is essential for the regulation of electrolyte absorption and for the prevention of tumour metastasis. We recently showed that migration of the gastric epithelial cell line IMGE-5 is stimulated by the nonamidated form of the hormone gastrin17. Here, we examine the effect on cell-cell adhesion of the prohormone progastrin, the concentration of which is increased in the plasma of patients with colorectal carcinoma. Progastrin induced the dissociation of both tight junction (TJ) and adherens junction (AJ) complexes in IMGE-5 cells. In progastrin-secreting DLD-1 human colorectal carcinoma cells, expression of an antisense gastrin construct restored membrane localisation of zonula occludens-1 (ZO-1), occludin, β-catenin and E-cadherin. This restoration was reversed by treatment with exogenous progastrin. Endogenous or exogenous progastrin also increased the paracellular flux of mannitol, and induced cell migration of several gastrointestinal cell lines. In addition, progastrin enhanced Src tyrosine kinase activity and induced a spatial delocalisation of protein kinase Cα. Using dominant-negative mutants and pharmacological inhibitors, we showed that the stimulation of Src kinase activity was essential for the regulation of TJs. By contrast, the dissociation of AJs involved phosphatidylinositol 3-kinase, partly through the formation of a complex with protein kinase Cα. We conclude that separate pathways mediate the disruption of AJs and TJs by progastrin. Either pathway may contribute to the co-carcinogenic role of this prohormone in colorectal carcinoma.