Chromogranin A activates diverse pathways mediating inducible nitric oxide expression and apoptosis in primary microglia

Chromogranin A (CgA) is associated with microglial activation cascades implicated in neurodegeneration in Alzheimer's, Pick's and Parkinson's diseases. In primary rat microglia, CgA-mediated inducible nitric oxide (iNOS) expression, nitric oxide (NO) production, mitochondrial depolarisation and apoptosis were inhibited by PP2 (Src kinase inhibitor). CgA-mediated iNOS expression and NO production were also inhibited by U0126 (MEK inhibitor), but mitochondrial depolarisation and apoptosis were not. PP2 inhibited ERK phosphorylation; therefore, Src mediates CgA-induced ERK phosphorylation leading to iNOS expression and NO production. Glutamate release induced by CgA was independent of both pathways. These findings provide insights into the way microglia are activated by CgA and the microglial signalling mechanisms associated with neurological disorders such as Alzheimer's disease.     

Suppression of protein kinase C is associated with inhibition of PYK2 tyrosine phosphorylation and enhancement of PYK2 interaction with Src in thrombin-activated platelets

Blood platelets have recently been shown to express PYK2, a nonreceptor tyrosine kinase belonging to the FAK gene family. In this study, we examined the involvement of protein kinase C (PKC) in PYK2-related responses in human platelets. While PYK2 tyrosine phosphorylation induced by thrombin was inhibited by preincubation of platelets with PKC inhibitors, staurosporine and Ro31-8220, PYK2 association with Src was markedly enhanced under the same conditions. Platelet intracellular Ca²⁺ mobilization induced by thrombin was hardly inhibited by these PKC inhibitors. p130^Cas is a docking protein that associates with FAK or PYK2 through the SH3 domain. Although we identified p130^Cas in platelets for the first time, this docking protein failed to interact with PYK2. These results suggest that PKC activation (but not Ca²⁺ mobilization) is involved in PYK2 tyrosine phosphorylation and that PYK2 associates with Src without PYK2 tyrosine phosphorylation or p130^Cas involvement in platelets.     

C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK)—endogenous negative regulators of Src-family protein kinases

C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) are endogenous inhibitors of the Src-family protein tyrosine kinases (SFKs). Since constitutive activation of SFKs contributes to cancer formation and progression, to prevent excessive activation of SFKs, their activity in normal cells is kept at the basal level by CSK and CHK. CSK and CHK inactivate SFKs by specifically phosphorylating a consensus tyrosine (called Y_T) near their C-termini. Upon phosphorylation, the phospho-Y_T engages in intramolecular interactions that lock the SFK molecule in an inactive conformation. SFKs are anchored to the plasma membrane, while CSK and CHK are localized predominantly in the cytosol. To inhibit SFKs, CSK and CHK need to translocate to the plasma membrane. Recruitment of CSK and CHK to the plasma membrane is mediated by the binding of their SH2, SH3 and/or kinase domains to specific transmembrane proteins, G-proteins and adaptor proteins located near the plasma membrane. For CSK, membrane recruitment often accompanies activation. CSK and CHK employ two types of direct interactions with SFKs to achieve efficient Y_T phosphorylation: (i) short-range interactions involving binding of the active sites of CSK and CHK to specific residues near Y_T, (ii) long-range non-catalytic interactions involving binding of SFKs to motifs located distally from the active sites of CSK and CHK. The interactions between CSK and SFKs are transient in nature. Unlike CSK, CHK binds tightly to SFKs to form stable protein complexes. The binding is non-catalytic as it is independent of Y_T. More importantly, the tight binding alone is sufficient to completely inhibit SFKs. This non-catalytic inhibitory binding represents a novel mechanism employed by CHK to inhibit SFKs. Given that SFKs are implicated in cancer development, compounds mimicking the non-catalytic inhibitory mechanism of CHK are potential anti-cancer therapeutics.     

Elevated Src family kinase activity stabilizes E-cadherin-based junctions and collective movement of head and neck squamous cell carcinomas

EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread.     

Antibody mediated CDCP1 degradation as mode of action for cancer targeted therapy

CUB-domain-containing-protein-1 (CDCP1) is an integral membrane protein whose expression is up-regulated in various cancer types. Although high CDCP1 expression has been correlated with poor prognosis in lung, breast, pancreas, and renal cancer, its functional role in tumor formation or progression is incompletely understood. So far it has remained unclear, whether CDCP1 is a useful target for antibody therapy of cancer and what could be a desired mode of action for a therapeutically useful antibody. To shed light on these questions, we have investigated the cellular effects of a therapeutic antibody candidate (RG7287). In focus formation assays, prolonged RG7287 treatment prevented the loss of contact inhibition caused by co-transformation of NIH3T3 cells with CDCP1 and Src. In a xenograft study, MCF7 cells stably overexpressing CDCP1 reached the predefined tumor volume faster than the parental MCF7 cells lacking endogenous CDCP1. This tumor growth advantage was abolished by RG7287 treatment. In vitro, RG7287 induced rapid tyrosine phosphorylation of CDCP1 by Src, which was accompanied by translocation of CDCP1 to a Triton X-100 insoluble fraction of the plasma membrane. Triggering these effects required bivalency of the antibody suggesting that it involves CDCP1 dimerization or clustering. However, this initial activation of CDCP1 was only transient and prolonged RG7287 treatment induced internalization and down-regulation of CDCP1 in different cancer cell lines. Antibody stimulated CDCP1 degradation required Src activity and was proteasome dependent. Also in three different xenograft models with endogenous CDCP1 expression RG7287 treatment resulted in significant tumor growth inhibition concomitant with substantially reduced CDCP1 levels as judged by immunohistochemistry and Western blotting. Thus, despite transiently activating CDCP1 signaling, the RG7287 antibody has a therapeutically useful mode of action.     

Overcoming EMT-associated resistance to anti-cancer drugs via Src/FAK pathway inhibition

Epithelial to mesenchymal transition (EMT) is a key process in embryonic development and has been associated with cancer metastasis and drug resistance. For example, in EGFR mutated non-small cell lung cancers (NSCLC), EMT has been associated with acquired resistance to the EGFR inhibitor erlotinib. Moreover, “EGFR-addicted” cancer cell lines induced to undergo EMT become erlotinib-resistant in vitro. To identify potential therapeutic vulnerabilities specifically within these mesenchymal, erlotinib-resistant cells, we performed a small molecule screen of ~200 established anti-cancer agents using the EGFR mutant NSCLC HCC827 cell line and a corresponding mesenchymal derivative line. The mesenchymal cells were more resistant to most tested agents; however, a small number of agents showed selective growth inhibitory activity against the mesenchymal cells, with the most potent being the Abl/Src inhibitor, dasatinib. Analysis of the tyrosine phospho-proteome revealed several Src/FAK pathway kinases that were differentially phosphorylated in the mesenchymal cells, and RNAi depletion of the core Src/FAK pathway components in these mesenchymal cells caused apoptosis. These findings reveal a novel role for Src/FAK pathway kinases in drug resistance and identify dasatinib as a potential therapeutic for treatment of erlotinib resistance associated with EMT.     

Urotensin II induces transactivation of the epidermal growth factor receptor via transient oxidation of SHP-2 in the rat renal tubular cell line NRK-52E

Urotensin-II (UII) is a potent vasoactive peptide that has been implicated in cardiac fibrosis and renal diseases. However, the role played by UII in renal tissues is largely unknown. In this study, we investigated the effects of human UII (hUII) on rat renal proximal tubular cells of the NRK-52E line and the role of Src homology 2-containing phosphotyrosine phosphatase (SHP-2) in the hUII-induced transactivation of the epidermal growth factor receptor (EGFR). Exposure to hUII at low concentrations significantly induced proliferation in NRK-52E cells; this effect was inhibited by treatment with an ERK1/2 inhibitor (PD98059). UII treatment increased the phosphorylation of EGFR and induced the generation of reactive oxygen species (ROS). Treatment of the ROS scavenger N-acetyl-cysteine (NAC) inhibited EGFR transactivation and ERK phosphorylation induced by hUII. SHP-2 was found to interact with EGFR and be transiently oxidized following the hUII treatment. In SHP-2 knockdown cells, UII-induced phosphorylation of EGFR was less influenced by NAC, and significantly suppressed by heparin binding (HB)-EGF neutralizing antibody. Our data suggest that the ROS-mediated oxidation of SHP-2 is essential for the hUII-induced mitogenic pathway in NRK-52E cells.