CIP4 promotes lung adenocarcinoma metastasis and is associated with poor prognosis.

Aberrant epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer (NSCLC) is linked to tumor progression, metastasis and poor survival rates. Here we report the role of Cdc42-interacting protein 4 (CIP4) in the regulation of NSCLC cell invasiveness and tumor metastasis. CIP4 was highly expressed in a panel of NSCLC cell lines and normal lung epithelial cell lines. Stable knockdown (KD) of CIP4 in lung adenocarcinoma H1299 cells, expressing wild-type EGFR, led to increased EGFR levels on the cell surface and defects in sustained activation of Erk kinase in H1299 cells treated with EGF. CIP4 localized to leading edge projections in NSCLC cells, and CIP4 KD cells displayed defects in EGF-induced cell motility and invasion through extracellular matrix. This correlated with reduced expression and activity of matrix metalloproteinase-2 (MMP-2) in CIP4 KD cells compared with control. In xenograft assays, CIP4 silencing had no effect on tumor growth but resulted in significant defects in spontaneous metastases to the lungs from these subcutaneous tumors. This correlated with reduced expression of the Erk target gene Zeb1 and the Zeb1 target gene MMP-2 in CIP4 KD tumors compared with control. CIP4 also enhanced rates of metastasis to the liver and lungs in an intrasplenic experimental metastasis model. In human NSCLC tumor sections, CIP4 expression was elevated greater than or equal to twofold in 43% of adenocarcinomas and 32% of squamous carcinomas compared with adjacent normal lung tissues. Analysis of microarray data for NSCLC patients also revealed that high CIP4 transcript levels correlated with reduced overall survival. Together, these results identify CIP4 as a positive regulator of NSCLC metastasis and a potential poor prognostic biomarker in lung adenocarcinoma.

Transducer of Cdc42-dependent actin assembly promotes breast cancer invasion and metastasis.

Metastatic breast adenocarcinomas display activation signatures for signaling pathways that trigger cell motility and tissue invasion. Here, we report that the adaptor protein transducer of Cdc42-dependent actin assembly-1 (Toca-1) is expressed in highly invasive breast cancers and regulates their metastatic phenotypes. We show that Toca-1 localizes to the filamentous actin-rich core of invadopodial protrusions actively degrading the extracellular matrix (ECM). Toca-1 colocalizes with Cortactin, and we show that this interaction is mediated by the SH3 domain of Toca-1. Stable knockdown (KD) of Toca-1 expression in MDA-MB-231 cells led to a significant defect in epidermal growth factor (EGF)-induced cell migration and invasion. Toca-1 KD cells also showed significant defects in EGF- and Src-induced ECM digestion and formation of invadopodial membrane protrusions. To test the role of Toca-1 in metastasis, we achieved stable Toca-1 KD in both human and rat metastatic breast adenocarcinoma cell lines. Orthotopic tumor xenografting of control and Toca-1 KD cells in natural-killer /B-/T-cell-deficient mice revealed a significant defect in spontaneous lung metastases with Toca-1 silencing in vivo. In contrast, no defects in primary tumor growth or lung seeding following tail vein injection of Toca-1 KD cells was observed, suggesting that Toca-1 functions at an early step in the dissemination of metastatic breast tumor cells. Taken together, our results identify Toca-1 as a proinvasive protein in breast adenocarcinoma and a potential therapeutic target to limit tumor metastasis.

Absence of Fer protein tyrosine kinase exacerbates endotoxin induced intestinal epithelial barrier dysfunction in vivo.

BACKGROUND AND AIMS: Fer kinase is activated by a number of growth factors and cytokines, and phosphorylates cortactin during cell shape change induced cortical actin reorganisation. In addition, Fer participates in cytoskeletal interactions mediated by cadherins, platelet endothelial cell adhesion molecule 1 (PECAM-1), and integrins, and has recently been implicated in limiting the innate immune response. Here we examined the role of Fer in modulating leucocyte recruitment and epithelial barrier function in the gut in response to lipopolysaccharide (LPS). METHODS: Mice targeted with a kinase inactivating mutation (FerDR) or strain matched wild-type (129Sv/J) mice were studied after intraperitoneal injection of LPS. Intravital microscopy was used to examine intestinal leucocyte kinetics, and leucocyte infiltration was assessed by fluorescence activated cell sorting. Systemic inflammation was assessed by measuring lung myeloperoxidase activity. Epithelial barrier function was assessed in vivo using blood to lumen 51Cr-EDTA clearance, with or without antibody based depletion of circulating neutrophils. RESULTS: LPS induced a significant increase in leucocyte adhesion and neutrophil infiltration into the intestinal tissue, and increased blood to lumen 51Cr-EDTA clearance. Pretreatment with neutrophil depleting antibody completely abrogated this response in wild-type mice. In FerDR mice, LPS induced leucocyte adhesion within the intestinal venules was exacerbated and associated with a trend towards increased neutrophil transmigration relative to wild-type mice. Surprisingly, LPS induced epithelial barrier permeability was increased 2.5-fold in FerDR mice relative to wild-type mice, and this barrier defect was only partly attenuated by depleting circulating neutrophils by >93 %. CONCLUSIONS: Fer plays a role in regulating LPS induced epithelial barrier dysfunction in vivo through both neutrophil dependent and neutrophil independent mechanisms.

Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation.

Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.