EGF receptor (EGFR) promotes tumor growth as well as radio-and chemoresistance in various human malignancies including squamous cell carcinomas (SCC). In addition to deactivation of prosurvival signaling, cetuximab-mediated EGFR targeting might concomitantly induce self-attenuating signaling bypasses. Identification of such bypass mechanisms is key to improve the efficacy of targeted approaches. Here, we show great similarity of EGFR signaling and radiation survival in cetuximab-treated SCC cells grown in a more physiologic three-dimensional extracellular matrix and as tumor xenografts in contrast to conventional monolayer cell cultures. Using phosphoproteome arrays, we observed strong induction of JNK2 phosphorylation potentially resulting from cetuximab-inhibited EGFR through c-jun-NH 2 -kinase (JNK)-interacting protein-4 (JIP-4), which was identified using an immunoprecipitation-mass spectrometric approach. Inhibition of this signaling bypass by JIP-4 or JNK2 knockdown or pharmacologic JNK2 inhibition enhanced cetuximab efficacy and tumor cell radiosensitivity. Our findings add new facets to EGFR signaling and indicate signaling bypass possibilities of cancer cells to improve their survival on cetuximab treatment. By deactivation of cetuximab-self-attenuating JNK2-dependent signaling, the cytotoxicity, and radiosensitizing potential of cetuximab can be augmented.

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BACKGROUND: Intrauterine infection and/or inflammation (Triple I) is an important cause of preterm birth (PTB) and adverse newborn outcomes. N-acetylcysteine (NAC) is a Food and Drug Administration (FDA)-approved drug safely administered to pregnant women with acetaminophen toxicity. METHODS: We conducted a single-center, quadruple-blind, placebo-controlled trial of pregnant women with impending PTB due to confirmed Triple I. Participants (n = 67) were randomized to an intravenous infusion of NAC or placebo mimicking the FDAapproved regimen. Outcomes included clinical measures and mechanistic biomarkers. RESULTS: Newborns exposed to NAC (n = 33) had significantly improved status at birth and required less intensive resuscitation compared to placebo (n = 34). Fewer NAC-exposed newborns developed two or more prematurity-related severe morbidities [NAC: 21% vs. placebo: 47%, relative risk, 0.45; 95% confidence interval (CI) 0.21-0.95] with the strongest protection afforded against bronchopulmonary dysplasia (BPD, NAC: 3% vs. placebo: 32%, relative risk, 0.10; 95% CI: 0.01-0.73). These effects were independent of gestational age, birth weight, sex, or race. Umbilical cord plasma NAC concentration correlated directly with cysteine, but not with plasma or whole blood glutathione. NAC reduced the placental expression of histone deacetylase-2, suggesting that epigenetic mechanisms may be involved. CONCLUSIONS: These data provide support for larger studies of intrapartum NAC to reduce prematurity-related morbidity.

What is already known about this subject • Isoprostanes are the product of free radical oxidation of arachidonic acid bound to phospholipids.• Their hydrolysis from phospholipids is presumably catalysed by phospholipases A2.• Atorvastatin reduces protein concentrations of secretory PLA2s and concentrations of LDL, with which PAF-AH (group VII phospholipase) is associated. What this study adds• Atorvastatin affects PAF-AH activity and this effect is strongly associated with its lipid-lowering effect, but it has no effect on groups IIA and V PLA2s' activity.• Thus, PAF-AH is no independent risk factor of cardiovascular diseases.• Moreover, a role of PAF-AH in the liberation of 15-F2t-isoP from phospholipids is excluded. AimsIsoprostanes are the product of free radical oxidation of arachidonic acid, whose hydrolysis from phospholipids is presumably catalysed by phospholipases A2 (PLA2s) such as group IIA or V PLA2s, or group VII PLA2 [platelet-activating factor acetylhydrolase (PAF-AH), lipoprotein-associated phospholipase]. Atorvastatin reduces concentrations of low-density lipoprotein (LDL), with which PAF-AH is associated, and PLA2s' protein concentrations. We investigated the effect of atorvastatin on PLA2s and PAF-AH activity and the urinary excretion of 15-F2trans-isoprostane (15-F2t-IsoP, 8-iso-PGF2a, iPF2a-III). MethodsTwenty-four hypercholesterolaemic individuals naive to lipid-lowering therapy were randomized to atorvastatin 40 mg or placebo for 6 weeks. The 15-F2t-isoP urinary excretion (gas chromatography/mass spectrometry), PAF-AH and group IIA and V PLA2 activities (photometry) were assessed at baseline and end-point. ResultsAt end-point, 15-F2t-isoP urinary excretion concentrations as well as PLA2s' activity were unchanged under atorvastatin (mean change 0.21 Ϯ 1.79 ng h -1 , 95% confidence interval -0.92, 1.35 and 0.33 Ϯ 0.94 nmol min -1 ml -1 , -0.27, 0.93) and under placebo (mean change 0.69 Ϯ 1.69 ng h -1 , -0.52, 1.90 and 1.29 Ϯ 2.16 nmol min -1 ml -1 , -0.25, 2.84). Atorvastatin treatment decreased total (P < 0.001) and LDL-cholesterol (P < 0.001) but had no effect on high-density lipoprotein. PAF-AH activity was lowered in the atorvastatin group (mean change -5.27Ϯ 1.96 nmol min -1 ml -1 , -6.51, -4.03, P < 0.001) but not in the placebo group (mean change 1.02 Ϯ 1.64 nmol min -1 ml -1 , 0.15, 2.20), and the change in PAF-AH activity was correlated with that in total (P = 0.03) and LDL-cholesterol (P = 0.03). ConclusionOur results show a lowering effect of atorvastatin on PAF-AH activity associated with its lipid-lowering effect and exclude a key role of PAF-AH in the liberation of 15-F2t-isoP from phospholipids.

OBJECTIVE: Compelling animal data suggests restoration of maternalfetal oxidative balance by N-acetylcysteine (NAC) may reduce risk of adverse neonatal outcome in pregnancies complicated by intrauterine infection/inflammation. We aimed to evaluate if maternal administration of NAC lowers risk of morbidity or death in infants of women with intra-amniotic infection (IAI). STUDY DESIGN: In a randomized, placebo-controlled, double-blind trial we assigned women from 23-33 weeks' gestation with an indication for delivery in the context of IAI to receive NAC or placebo. IAI was diagnosed in all cases by trans-abdominal clinically indicated amniocentesis. NAC was administered intravenously (IV) 150 mg/kg loading dose (1 h), followed by 50 mg/kg IV continuous infusion rate for 4 h, and followed by 100 mg/kg IV continuous infusion rate until delivery. The primary outcome was a composite of mortality and severe short-term neonatal morbidities at discharge from NICU. Maternal and umbilical cord plasma levels of NAC, endogenous free thiols (cysteine [CYS], cysteine-glycine [CYS-GLY], glutathione [GSH]) were analyzed by HPLC. Cytokines were quantified by multiplex immunoassay. RESULTS: A total of 68 women pregnant with singletons were randomized. Demographics and clinical characteristics of the groups were similar (Table). Bolus administration was achieved in 98% of women with 42% of women completing the 4 h dose. The rate of the primary outcome was significantly different between the NAC (12%) and placebo (32%) groups: RR: 0.31 (95% CI 0.11-0.85), p¼.023, with the highest protection achieved for bronchopulmonary dysplasia (BPD), RR: 0.09 (95% CI 0.01-0.07), p¼.019], independent of gestational age or sex. In both maternal and fetal compartments, the achieved concentration of NAC significantly correlated to CYS (r¼0.92, p<.001) and CYS-GLY (0.63, p¼.005). NAC did not impact on the fetal cytokine profile or red blood cells GSH. CONCLUSION: Fetal exposure to NAC prior to an impending preterm birth in the context of IAI significantly reduced the rate of the primary composite neonatal outcome, with the highest protection against BPD. Protective mechanism of NAC may involve increase in anti-oxidative pool of endogenous thiols. Intrapartum NAC infusion is practically feasible, safe and does not increase frequency of neonatal sepsis. OBJECTIVE:The role of host immunity and maternal psychosocial stress in the pathogenesis of spontaneous preterm birth (sPTB) remains unclear. Antimicrobial proteins in the cervicovaginal CV) space, such as beta-defensins (bD), modulate immune responses to bacteria. While stress is known to induce immunological changes, no study has examined the interplay between stress and the CV immune response in association with sPTB. STUDY DESIGN: From the Motherhood & Microbiome cohort (n¼2000), we performed a nested case-control study of 519 pregnant women (110 sPTB and 409 term). Stress and CV-bD were measured at 16-20 weeks' gestation. Stress was dichotomized at 30 on Cohen's Perceived Stress Scale (PSS-14). We me...