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PED (phosphoprotein enriched in diabetes) is a death-effector domain (DED) family member with a broad anti-apoptotic action. PED inhibits the assembly of the death-inducing signalling complex (DISC) of death receptors following stimulation. Recently, we reported that the expression of PED is increased in breast cancer cells and determines the refractoriness of these cells to anticancer therapy. In the present study, we focused on the role of PED in non-small cell lung cancer (NSCLC), a tumour frequently characterized by evasion of apoptosis and drug resistance. Immunohistochemical analysis of a tissue microarray, containing 160 lung cancer samples, indicated that PED was strongly expressed in different lung tumour types. Western blotting performed with specimens from NSCLC-affected patients showed that PED was strongly up-regulated (>6 fold) in the areas of tumour compared to adjacent normal tissue. Furthermore, PED expression levels in NSCLC cell lines correlated with their resistance to tumour necrosis factor related apoptosis-inducing ligand (TRAIL)-induced cell death. The involvement of PED in the refractoriness to TRAIL-induced cell death was investigated by silencing PED expression in TRAIL-resistant NSCLC cells with small interfering (si) RNAs: transfection with PED siRNA, but not with cFLIP siRNA, sensitized cells to TRAIL-induced cell death. In conclusion, PED is specifically overexpressed in lung tumour tissue and contributes to TRAIL resistance.

MicroRNAs (miRNAs) control cell cycle progression by targeting the transcripts encoding for cyclins, CDKs and CDK inhibitors, such as p27(KIP1) (p27). p27 expression is controlled by multiple transcriptional and posttranscriptional mechanisms, including translational inhibition by miR-221/222 and posttranslational regulation by the SCF(SKP2) complex. The oncosuppressor activity of miR-340 has been recently characterized in breast, colorectal and osteosarcoma tumor cells. However, the mechanisms underlying miR-340-induced cell growth arrest have not been elucidated. Here, we describe miR-340 as a novel tumor suppressor in non-small cell lung cancer (NSCLC). Starting from the observation that the growth-inhibitory and proapoptotic effects of miR-340 correlate with the accumulation of p27 in lung adenocarcinoma and glioblastoma cells, we have analyzed the functional relationship between miR-340 and p27 expression. miR-340 targets three key negative regulators of p27. The miR-340-mediated inhibition of both Pumilio family RNA-binding proteins (PUM1 and PUM2), required for the miR-221/222 interaction with the p27 3'-UTR, antagonizes the miRNA-dependent downregulation of p27. At the same time, miR-340 induces the stabilization of p27 by targeting SKP2, the key posttranslational regulator of p27. Therefore, miR-340 controls p27 at both translational and posttranslational levels. Accordingly, the inhibition of either PUM1 or SKP2 partially recapitulates the miR-340 effect on cell proliferation and apoptosis. In addition to the effect on tumor cell proliferation, miR-340 also inhibits intercellular adhesion and motility in lung cancer cells. These changes correlate with the miR-340-mediated inhibition of previously validated (MET and ROCK1) and potentially novel (RHOA and CDH1) miR-340 target transcripts. Finally, we show that in a small cohort of NSCLC patients (n=23), representative of all four stages of lung cancer, miR-340 expression inversely correlates with clinical staging, thus suggesting that miR-340 downregulation contributes to the disease progression.

BackgroundExtracellular superoxide dismutase (SOD3), which dismutates superoxide anion to hydrogen peroxide, has been shown to reduce the free radical stress derived apoptosis in tissue injuries. Since both superoxide anion and hydrogen peroxide have a marked impact on signal transduction pathways and could potentially explain a number of apoptosis and survival -related phenomena in different pathological conditions, we clarified the impact of SOD3 on Akt and Erk1/2 cell survival pathways in rat hind limb injury model.Methodology and principal findingsBased on our data, the hind limb ischemic rats treated with virally delivered sod3 have milder injury and less apoptosis than control animals that could be due to parallel activation of pro-proliferative and anti-apoptotic Erk1/2 and Akt pathways. The common downstream factor of both signaling pathways, the apoptosis related forkhead box protein O3a (FoxO3a), was phosphorylated and translocated to the cytoplasm in sod3 treated tissues and cell line. Additionally, we obtained increased mRNA production of elk-1, ets-1, and microRNA 21 (miR-21), whereas synthesis of bim mRNA was decreased in sod3 overexpressing tissues. We further showed that overexpression of sod3 modulated redox related gene expression by downregulating nox2 and inos when compared to injured control animals.Conclusions and significanceThe study shows the complexity of SOD3-derived effects on tissue injury recovery that are not limited to the reduction of superoxide anion caused cellular stress but highlights the impact of SOD3 related signal transduction on tissue functions and suggests an important role for SOD3 in attenuating cell stress effects in different pathological conditions.

The balance between specific signals from different growth factors dictates the biological response of mammalian cells including cell proliferation, differentiation and survival. PC12 cells represent a model of choice to compare the signalling of differentiative growth factors, as NGF, and of mitogenic growth factors, as EGF. In these cells the prolonged activity of the ERK kinase dictates the decision of cells to differentiate. Here we focused on the cytosolic tyrosine phosphatase Shp2 as an established regulator of the Ras–ERK cascade, to elucidate its involvement in determining the stimulation-dependent PC12 cell fate. To this end, we generated PC12 derived cell lines that express the interfering mutant of Shp2 under a tetracycline-inducible promoter. Our findings show that Shp2 participates to the opposite effects induced in PC12 cells by EGF and NGF and that the interactions with the multidocking Gab2 protein mediate such effects.