Non small cell lung cancer and PI3K pathway

The phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT)/mammalian target of rapamycin (mTOR) pathway is upregulated in a number of human cancers, including non-small cell lung cancer (NSCLC). Its potential role in NSCLC progression provides an attractive target for anticancer therapy. The expression of phosphorylated mTOR (p-mTOR), phosphorylated AKT (p-AKT), p85α and p110γ subunits of PI3K, phosphorylated p70S6K (p-p70S6K), phosphatase and tensin homolog (PTEN) and phosphorylated 4E-BP1 (p-4E-BP1) was examined by immunohistochemistry in 102 NSCLC specimens. The results were correlated withclinicopathological features. We also examined 61 of our cases for the presence of PIK3CA, AKT1, PTEN and K-RAS mutations. A common PIK3CA mutation was detected at exon 9 in 2 samples (p.E545K), whereas another sample displayed a rare mutation (p.D1018N). Furthermore, 10 out of 54 cases (18.5%) had a K-RAS mutation at codon 12, 5 had a PTEN mutation (exons 7 and 8) and 1 case had an AKT1 mutation (p.E17K). PTEN mutations were associated with nodal metastases. The expression of p-mTOR positively correlated with that of p-AKT and p-p70S6K and was higher in adenocarcinomas along with nuclear p110γPI3K expression, whereas p-4E-BP1 expression was higher in squamous cell carcinomas. We also established a positive association between p85αPI3K or p110γPI3K and cytoplasmic p-AKT and its downstream effectors. An inverse correlation was noted between p-4E-BP1 immunoexpression and tumour status and nuclear p-AKT expression as regards tumour stage. Univariate survival analysis demonstrated that p-4E-BP1 expression, either alone or in combination with cytoplasmic p-AKT expression had an adverse prognostic significance in adenocarcinomas. The combination of p-4E-BP1 and cytoplasmic p-AKT expression remained significant in the multivariate analysis as a function of their interaction with histological type. Our data demonstrate the significance of p-4E-BP1 immunoexpression as a molecular marker of prognostic value in adenocarcinomas, particularly when combined with p-AKT.Το μονοπάτι phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT)/mammalian target of rapamycin (mTOR) ενεργοποιείται σε μεγάλο αριθμό ανθρώπινων καρκινωμάτων, συμπεριλαμανομένου του μη μικροκυτταρικού καρκίνου του πνεύμονα. Ο πιθανολογούμενος ρόλος του στην εξέλιξη των μη μικροκυτταρικών καρκινωμάτων του πνεύμονα το καθιστά στόχο αντικαρκινικής θεραπείας. Μελετήθηκε ανοσοϊστοχημικά η έκφραση του φωσφορυλιωμένου mTOR (p-mTOR), του φωσφορυλιωμένου AKT (p-AKT), των p85 και p110γ υπομονάδων του PI3K, του φωσφορυλιωμένου p70S6K (p-p70S6K), του ομολόγου φωσφατάσης και τενσίνης (PTEN) και του φωσφορυλιωμένου 4E-BP1 (p-4E-BP1) σε 102 περιπτώσεις μη μικροκυτταρικού καρκίνου του πνεύμονα. Τα αποτελέσματα συσχετίσθηκαν με τα κλινικοπαθολογοανατομικά χαρακτηριστικά. Μελετήσαμε επίσης 61 από τις περιπτώσεις μας για την παρουσία PIK3CA, AKT1, PTEN και K-RAS μεταλλαγών. Σε δύο δείγματα εντοπίστηκε μια PIK3CA μεταλλαγή στο εξόνιο 9 (p.E545K), ενώ σε ένα άλλο δείγμα αναγνωρίστηκε μια σπάνια μεταλλαγή (p.D1018N). Επίσης, 10 από τις 54 περιπτώσεις (18.5%) εμφάνισαν K-RAS μεταλλαγή στο κωδικόνιο 12, 5 είχαν PTEN μεταλλαγή (εξόνια 7 και 8) και σε μία περίπτωση αναγνωρίστηκε AKT1 μεταλλαγή (p.E17K). Οι PTEN μεταλλαγές συνοδεύονταν από λεμφαδενικές μεταστάσεις. Η έκφραση του p-mTOR συσχετίσθηκε θετικά με αυτήν των p-AKT και p-p70S6K και ήταν υψηλότερη στα αδενοκαρκινώματα, όπως και η πυρηνική p110γPΙ3Κ έκφραση, ενώ η p-4E-BP1 έκφραση ήταν υψηλότερη στα πλακώδη καρκινώματα. Αποδείξαμε επίσης θετική συσχέτιση μεταξύ p85αPI3K/p110γPI3K και κυτταροπλασματικού p-AKT και των υποκείμενων τελεστών του. Αντίστροφος συσχετισμός παρατηρήθηκε μεταξύ της ανοσοέκφρασης του p-4E-BP1 και του σταδίου Τ του όγκου καθώς και της πυρηνικής έκφρασης του p-AKT με το στάδιο Τ του όγκου. Η μονοπαραγοντική ανάλυση επιβίωσης ανέδειξε πως η έκφραση του p-4E-BP1, είτε μεμονωμένη είτε σε συνδυασμό με την κυτταροπλασματική έκφραση του p-AKT, είχε αντίστροφη προγνωστική σημασία στα αδενοκαρκινώματα. Ο συνδυασμός της έκφρασης του p-4E-BP1 και της κυτταροπλασματικής έκφρασης του p-AKT παρέμεινε σημαντικός στην πολυπαραγοντική ανάλυση ως αποτέλεσμα της αλληλεπίδρασης τους με τον ιστολογικό τύπο. Τα στοιχεία μας ανέδειξαν τη σημασία της ανοσοέκφρασης του p-4E-BP1 ως μοριακό δείκτη με προγνωστική αξία στα αδενοκαρκινώματα, ειδικά σε συνδυασμό με το p-AKT

A detailed immunohistochemical analysis of the PI3K/AKT/mTOR pathway in lung cancer: Correlation with PIK3CA, AKT1, K-RAS or PTEN mutational status and clinicopathological features

The phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT)/mammalian target of rapamycin (mTOR) pathway is upregulated in a number of human cancers, including non-small cell lung cancer (NSCLC). Its potential role in NSCLC progression provides an attractive target for anticancer therapy. The expression of phosphorylated mTOR (p-mTOR), phosphorylated AKT (p-AKT), p85 alpha and p110 gamma subunits of PI3K, phosphorylated p70S6K (p-p70S6K), phosphatase and tensin homolog (PTEN) and phosphorylated 4E-BP1 (p-4E-BP1) was examined by immunohistochemistry in 102 NSCLC specimens. The results were correlated with clinicopathological features. We also examined 61 of our cases for the presence of PIK3CA, AKT1, PTEN and K-RAS mutations. A common PIK3CA mutation was detected at exon 9 in 2 samples (p.E545K), whereas another sample displayed a rare mutation (p.D1018N). Furthermore, 10 out of 54 cases (18.5%) had a K-RAS mutation at codon 12, 5 had a PTEN mutation (exons 7 and 8) and 1 case had an AKT1 mutation (p.E17K). PTEN mutations were associated with nodal metastases. The expression of p-mTOR positively correlated with that of p-AKT and p-p70S6K and was higher in adenocarcinomas along with nuclear p110 gamma PI3K expression, whereas p-4E-BP1 expression was higher in squamous cell carcinomas. We also established a positive association between p85 alpha PI3K or p110 gamma PI3K and cytoplasmic p-AKT and its downstream effectors. An inverse correlation was noted between p-4E-BP1 immunoexpression and tumour status and nuclear p-AKT expression as regards tumour stage. Univariate survival analysis demonstrated that p-4E-BP1 expression, either alone or in combination with cytoplasmic p-AKT expression had an adverse prognostic significance in adenocarcinomas. The combination of p-4E-BP1 and cytoplasmic p-AKT expression remained significant in the multivariate analysis as a function of their interaction with histological type. Our data demonstrate the significance of p-4E-BP1 immunoexpression as a molecular marker of prognostic value in adenocarcinomas, particularly when combined with p-AKT

Impact of diet-induced obesity in male mouse reproductive system: The role of advanced glycation end product-receptor for advanced glycation end product axis

Obesity represents a route to broad physiological dysfunction affecting major organs including male urogenital system. Hyperglycemia, hyperlipidemia, and oxidative stress associated with obesity augment the formation of reactive metabolic by-products, namely advanced glycation end products (AGEs), leading to increased tissue deposition and damage. The exogenous intake and the endogenous accumulation of AGEs contribute to metabolic and reproductive abnormalities in both women and men. The present study assessed the effects of a diet high in saturated fatty acids (SAFA) on the lipid and metabolic profile (AGE levels, oxidative stress) as well as pathogenic (AGE, receptor for AGEs [RAGE] expression, apoptosis) and morphometric parameters of male reproductive system in vivo. Effects of switching to a diet rich in monounsaturated fatty acids (MUFA) or equal in the proportion MUFA to SAFA were further investigated. SAFA-fed animals were characterized by increased serum lipid concentrations (p < .05) compared to controls, but AGEs and peroxide levels were not significantly different across the different experimental groups. Elevated AGE deposition was detected for the first time in germ cells with a higher staining intensity in animals on the SAFA diet, compared to MUFA or MUFA-SAFA-fed animals or the control samples (p = .018). In Leydig cells, AGE localization was higher in the entire cohort of high-fat-fed animals compared to controls (p < .05). High-fat-fed mice displayed enhanced apoptosis compared to controls (p < .005). Furthermore, prostatic tissue demonstrated reduction in epithelial folding, an effect which was significantly reversed after MUFA diet administration. Our findings provide the basis for further investigation of AGE-RAGE axis in testicular and prostatic disturbances associated with diet-induced obesity. Simple dietetic intervention has beneficial effects on metabolic dysfunction of reproductive system before overt manifestations, indicating glycation as a promising therapeutic target

Phosphorylated 4E-binding protein 1 (p-4E-BP1): a novel prognostic marker in human astrocytomas

International audienceKorkolopoulou P, Levidou G, El-Habr E A, Piperi C, Adamopoulos C, Samaras V, Boviatsis E, Thymara I, Trigka E-A, Sakellariou S, Kavantzas N, Patsouris E & Saetta A A ?(2012) Histopathology similar to 61, 293305 Phosphorylated 4E-binding protein 1 (p-4E-BP1): a novel prognostic marker in human astrocytomas Aims: To investigate the significance of the mammalian target of rapamycin (mTOR) pathway in astrocytic tumours, published information in this context being limited, especially regarding phosphorylated 4E-binding protein (p-4E-BP) 1. Methods and results: Paraffin-embedded tissue from 111 patients with astroglial tumours (grades IIIV) was investigated for the association of phosphorylated mTOR (p-mTOR) signalling components with phosphorylated extracellular signal-related kinase 1/2 (p-ERK1/2) and phosphorylated AKT (p-AKT) expression, clinicopathological features, angiogenesis, isocitrate dehydrogenase 1 (IDH1)-R132H, and survival. Expression was also quantified by western blot analysis in 12 cases and in three primary glioma cell cultures following rapamycin treatment. p-mTOR expression correlated with p-4E-BP1 expression and marginally with p-p70S6K expression. p-4E-BP1 expression increased with tumour grade. Rapamycin induced a decline in phosphorylation levels of all three proteins. Nuclear p-AKT and cytoplasmic p-ERK1/2 immunoexpression correlated with p-4E-BP1 expression, whereas cytoplasmic p-AKT expression correlated with p-p70S6K expression. All three proteins were associated with increased angiogenesis but not with IDH1-R132H expression status. p-mTOR adversely affected overall and disease-free survival in univariate analysis. In multivariate survival analysis, the presence of p-4E-BP1 predicted shortened overall survival in the entire cohort and glioblastomas. Conclusions: mTOR signalling components are differentially involved in the acquisition of a more aggressive and angiogenic phenotype in astrocytic tumours. Moreover, p-4E-BP1 emerges as a novel prognostic marker, which might aid in the selection of patients who are more likely to benefit from therapy with mTOR inhibitors

Beneficial Effect of U-74389 G and Sildenafil in An Experimental Model of Flap Ischemia/Reperfusion Injury in Swine. Histological and Biochemical Evaluation of the Model

Purpose of the study: Tissue reconstruction after burns, tumor excisions, infections or injuries is a frequent surgical challenge to avoid Ischemia-reperfusion injury. Lazaroids and sildenafil, through their mechanisms of action, have been studied for their protective effects on various organs subjected to IRI. In this study, we aimed to evaluate the therapeutic potential of U-74389G and sildenafil in a swine model of ischemia and reperfusion injury of latissimus dorsi flap. Materials and methods: Forty-two Landrace male pigs, weighing 28–35 kg, were equally (n = 6) randomized into the following groups: (a) Group I: control, (b) Group II: administration of U-74389G after ischemia, (c) Group III: administration of sildenafil after ischemia, (d) Group IV: administration of U-74389G and sildenafil after ischemia, (e) Group V: administration of U-74389G prior to ischemia, (f) Group VI: administration of sildenafil prior to ischemia, and (g) Group VII: administration of U-74389G and sildenafil prior to ischemia. Blood and tissue sampling was conducted before ischemia, 15 and 30 min after occlusion, 30, 60, 90, and 120 min after reperfusion. Results: Statistically significant reduction (p < 0.05) was detected in lymphocytes and polymorphonuclear leukocytes concentrations as well as in the appearance of edema after histopathologic evaluation of the ischemic tissue, especially in the groups of combined treatment. Measurements of malondialdeyde and tumour necrosis factor alpha in tissues revealed a significant decrease (p < 0.001) of these markers in the treatment groups when compared to the control, particularly in the latest estimated timepoints. Conclusions: The synergistic action of U-74389G and sildenafil seems protective and promising in cases of flap IRI during tissue reconstruction surgery

High-frequency p16 INK4A promoter methylation is associated with histone methyltransferase SETDB1 expression in sporadic cutaneous melanoma

Epigenetic mechanisms participate in melanoma development and progression. The effect of histone modifications and their catalysing enzymes over euchromatic promoter DNA methylation in melanoma remains unclear. This study investigated the potential association of p16(INK4A) promoter methylation with histone methyltransferase SETDB1 expression in Greek patients with sporadic melanoma and their correlation with clinicopathological characteristics. Promoter methylation was detected by methylation-specific PCR in 100 peripheral blood samples and 58 melanoma tissues from the same patients. Cell proliferation (Ki-67 index), p16(INK4A) and SETDB1 expression were evaluated by immunohistochemistry. High-frequency promoter methylation (25.86%) was observed in tissue samples and correlated with increased cell proliferation (P=0.0514). p16(INK4A) promoter methylation was higher in vertical growth-phase (60%) melanomas than in radial (40%, P=0.063) and those displaying epidermal involvement (P=0.046). Importantly, p16(INK4A) methylation correlated with increased melanoma thickness according to Breslow index (P=0.0495) and marginally with increased Clark level (I/II vs III/IV/V, P=0.070). Low (1-30%) p16(INK4A) expression was detected at the majority (19 of 54) of melanoma cases (35.19%), being marginally correlated with tumor lymphocytic infiltration (P=0.078). SETDB1 nuclear immunoreactivity was observed in 47 of 57 (82.46%) cases, whereas 27 of 57 (47.37%) showed cytoplasmic immunoexpression. Cytoplasmic SETDB1 expression correlated with higher frequency of p16(INK4A) methylation and p16(INK4A) expression (P=0.033, P=0.011, respectively). Increased nuclear SETDB1 levels were associated with higher mitotic count (0-5/mm(2) vs &gt;5/mm(2), P=0.0869), advanced Clark level (III-V, P=0.0380), epidermal involvement (P=0.0331) and the non-chronic sun exposure-associated melanoma type (P=0.0664). Our data demonstrate for the first time the association of histone methyltransferase SETDB1 with frequent methylation of the euchromatic p16(INK4A) promoter and several prognostic parameters in melanomas

Complex interactions between the components of the PI3K/AKT/mTOR pathway, and with components of MAPK, JAK/STAT and Notch-1 pathways, indicate their involvement in meningioma development

International audienceWe investigated the significance of PI3K/AKT/mTOR pathway and its interactions with MAPK, JAK/STAT and Notch pathways in meningioma progression. Paraffin-embedded tissue from 108 meningioma patients was analysed for the presence of mutations in PIK3CA and AKT1. These were correlated with the expression status of components of the PI3K/AKT/mTOR pathway, including p85 alpha and p110 gamma subunits of PI3K, phosphorylated (p)-AKT, p-mTOR, p-p70S6K and p-4E-BP1, as well as of p-ERK1/2, p-STAT3 and Notch-1, clinicopathological data and patient survival. A mutation in PIK3CA or AKT1 was found in around 9 % of the cases. Higher grade meningiomas displayed higher nuclear expression of p-p70S6K; higher nuclear and cytoplasmic expression of p-4E-BP1 and of Notch-1; lower cytoplasmic expression of p85 alpha PI3K, p-p70S6K and p-ERK1/2; and lower PTEN Histo-scores (H-scores). PTEN H-score was inversely correlated with recurrence probability. In univariate survival analysis, nuclear expression of p-4E-BP1 and absence of p-ERK1/2 expression portended adverse prognosis, whereas in multivariate survival analysis, p-ERK1/2 expression emerged as an independent favourable prognostic factor. Treatment of the human meningioma cell line HBL-52 with the PI3K inhibitor LY294002 resulted in reduction of p-AKT, p-p70S6K and p-ERK1/2 protein levels. The complex interactions established between components of the PI3K/AKT/mTOR pathway, or with components of the MAPK, JAK/STAT and Notch-1 pathways, appear to be essential for facilitating and fuelling meningioma progression