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Supplemental material, sj-pdf-2-lan-10.1177_00236772221138942 for Identification of vacuolar autophagic aggregates in the skeletal muscles of inbred C57BL/6NCrl mice by Davide De Biase, Teresa Bruna Pagano, Donatella Malanga, Valeria Russo, Giuseppe Piegari, Ilaria d’Aquino, Valentina Iovane, Marzia Scarfò, Serenella Papparella, Slawomir Wojcik, Orlando Paciello in Laboratory Animals

Supplemental material, sj-pdf-1-lan-10.1177_00236772221138942 for Identification of vacuolar autophagic aggregates in the skeletal muscles of inbred C57BL/6NCrl mice by Davide De Biase, Teresa Bruna Pagano, Donatella Malanga, Valeria Russo, Giuseppe Piegari, Ilaria d’Aquino, Valentina Iovane, Marzia Scarfò, Serenella Papparella, Slawomir Wojcik, Orlando Paciello in Laboratory Animals

Supplemental material, sj-pdf-1-lan-10.1177_00236772221138942 for Identification of vacuolar autophagic aggregates in the skeletal muscles of inbred C57BL/6NCrl mice by Davide De Biase, Teresa Bruna Pagano, Donatella Malanga, Valeria Russo, Giuseppe Piegari, Ilaria d’Aquino, Valentina Iovane, Marzia Scarfò, Serenella Papparella, Slawomir Wojcik, Orlando Paciello in Laboratory Animals

Intraoperative auto-transfusion with the use of cell saver systems is routinely used to reduce the rate of packed red blood transfusion in major surgery. Nevertheless some concerns have been raised on possible risks of coagulation disorders. The aim of the study was to analyze the blood processed by the cell saver, ready to be re-infused to the patient, in order to individuate unexpected cellular components, that can favor coagulopathy. We tested the blood processed by the cell saver in thirteen patients undergoing coronary bypass surgery with Cellsearch(®), ScreenCell(®), Cytology and Immunofluorescence. Those four methods allowed us to look for the presence of unexpected cells, quantify and characterize them. Furthermore, the blood processed by the cell saver was mixed with the patient’s peripheral blood and analyzed with the ROTEM(®) thromboelastography. The Cellsearch(®) revealed and counted a mean number of 1241 unexpected cells/7.5 ml in the blood processed by the cell saver. The ScreenCell(®) and Cytology confirmed the presence of non-hematological cells. Immunofluorescence showed positivity for Calretinin and WT-1, confirming the mesothelial origin. Moreover we detected a peculiar arrangement of the platelets around the mesothelial cells in a “cloud” form, suggesting platelet activation. The ROTEM(®) analysis showed a significantly longer clot formation time, smaller clot amplitude and maximum clot firmness, compared to controls. In conclusion we demonstrated the presence of mesothelial cells in the cell saving blood, ready to be auto-transfused. This finding can contribute to develop a platelet depletion coagulopathy, with coagulation factors consumption.

In the publication of this article [1], there is an error in Fig. 7. The minus and plus signals are inverted which impairs understanding of the results described. Figure 7 should instead be read as indicated in this correction.

Background: Purpose of this study was to evaluate the contribution of the Extracellular-regulated protein kinase (ERK)-1/2 pathway to oncogenic signaling elicited by the tyrosine kinase receptor HER2 in Non-Small Cell Lung Cancer (NSCLC) and to assess the prognostic value of these oncoproteins in NSCLC patients. Methods: Immunohistochemistry was performed to determine expression and activation of HER2 and ERK1/2 (detected by phosphorylation of Y1248 and T202/Y204, respectively) using Tissue Micro Arrays (TMA) containing matched normal and neoplastic tissues from 132 NSCLC patients. Survival analysis was carried out using the Kaplan-Meier method. Univariate and multivariate analysis were used to evaluate the prognostic value of pERK1/2, pHER2 and a combination thereof with clinical-pathological parameters such as age, lymph node status (N), size (T), stage (TNM) and grade. Results: We found that HER2 was overexpressed in 33/120 (27%) and activated in 41/114 (36%) cases; ERK1/2 was activated in 44/102 (43%) cases. A direct association was found between pERK1/2 and pHER2 (23/41; p=0.038). In addition, patients positive for pERK1/2 and for both pHER2 and pERK1/2 showed significantly worse overall survival (OS) and disease-free survival (DFS) compared with negative patients. Univariate and multivariate analysis of patients' survival revealed that positivity for pHER2-pERK1/2 and for pERK1/2 alone were independent prognostic factors of poor survival in NSCLC patients. In particular, this association was significantly important for DFS in stage I+II patients. Conclusion: This study provides evidence that activated ERK1/2 and/or the combined activation of HER2 and ERK1/2 are good indicators of poor prognosis in NSCLC patients, not only in unselected patients but also in early stage disease.

Introduction: Increasing evidence suggest that an interplay between T cells and fibroblasts plays a pivotal role in promoting matrix accumulation in systemic sclerosis (SSc). Aim: We investigated if the co-culturing of fibroblasts derived from SSc patients with autologous PBMCs could induce peculiar modifications in any cell types. Materials and methods: Fibroblasts were obtained from the skin of patients undergoing biopsy for diagnostic purposes. PBMCs were obtained from the same patients. All the patients were fully informed of the meaning of the study and accepted to donate blood. Cells were taken in culture at 37 ˚C5% CO2 for ten days, then cells were stained with monoclonal antibodies for HLA-DR, CD3, CD4, CD56, CD95, TCRab, TCRcd, After the staining, cells were analyzed by flow-cytometry using a FACScalibur. Results: We found that T cells bearing an ab receptor were expanded in the co-cultures. Moreover, these cells were positive for the expression of HLA-DR, suggesting an activation of T cells induced by co-culturing with autologous fibroblasts. No expansion of cd T cells nor CD56 positive T cells was found. SSc fibroblasts, which have already been reported to have a basal high expression of CD95 (FAS), were found to up-regulate FAS in these experiments. Conclusions: We recently showed that peripheral blood T cells from SSc patients expand when co-cultured with autologous fibroblasts and acquire the same T cell clonotypes that are increased in the affected skin. The results presented here further support that such expansion may be due to a specific antigenic activity of fibroblasts on T cells. In addition, the up-regulation of FAS expression on SSc fibroblasts may be related to phenotypic changes that indicate an increased ability of these cells to escape normal pathways leading to cell death. The meaning of these findings in the pathogenesis of SSc remains to be further investigated.

Simple Summary The main finding reported in this manuscript is that the gain-of-function mutation AKT1E17K is a bona fide oncogene for mammary epithelium, being able to efficiently initiate breast cancer in mice. On the basis of high-molecular-weight cytokeratins expressed by AKT1E17K-derived tumors supported by additional integrative gene expression analysis these tumors resulted similar to human basal-like cancer, phenotypically and molecularly. These results indicate that the AKTE17K strain may represent an appropriate model of human basal-like breast cancer for the identification of novel therapies specific for this type of tumor. The gain-of-function mutation in the pleckstrin homology domain of AKT1 (AKT1E17K) occurs in lung and breast cancer. Through the use of human cellular models and of a AKT1E17K transgenic Cre-inducible murine strain (R26-AKT1E17K mice), we have demonstrated that AKT1E17K is a bona fide oncogene for lung epithelial cells. However, the role of AKT1E17K in breast cancer remains to be determined. Here, we report the generation and the characterization of a MMTV-CRE; R26-AKT1E17K mouse strain that expresses the mutant AKT1E17K allele in the mammary epithelium. We observed that AKT1E17K stimulates the development of mammary tumors classified as ductal adenocarcinoma of medium-high grade and presented a variety of proliferative alterations classified as adenosis with low-to-high grade dysplasia in the mammary epithelium. A subsequent immunohistochemical characterization suggested they were PR-/HER2(-)/ER+, basal-like and CK8(-)/CK10(-)/CK5(+)/CK14(+). We also observed that, in parallel with an increased proliferation rate, tumors expressing mutant AKT1E17K presented an activation of the GSK3/cyclin D1 pathway in the mammary epithelium and cluster significantly with the human basal-like tumors. In conclusion, we demonstrate AKT1E17K is a bona fide oncogene that can initiate tumors at high efficiency in murine mammary epithelium in vivo.

// Duarte Mendes Oliveira 1, * , Carmelo Laudanna 1, * , Simona Migliozzi 1 , Pietro Zoppoli 1 , Gianluca Santamaria 1 , Katia Grillone 1 , Laura Elia 2 , Chiara Mignogna 3 , Flavia Biamonte 1 , Rosario Sacco 2 , Francesco Corcione 4 , Giuseppe Viglietto 1 , Donatella Malanga 1 and Antonia Rizzuto 2 1 Department of Experimental and Clinical Medicine, University Magna Graecia, Catanzaro, Italy 2 Department of Medical and Surgical Sciences, University Magna Graecia, Catanzaro, Italy 3 Department of Health Sciences, University Magna Graecia, Catanzaro, Italy 4 UOC Chirurgia Generale, Azienda Ospedaliera dei Colli, Napoli, Italy * These authors have contributed equally to this work Correspondence to: Giuseppe Viglietto, email: viglietto@unicz.it Donatella Malanga, email: malanga@unicz.it Keywords: colon cancer; ion torrent; colon segments Received: March 29, 2017 Accepted: April 06, 2018 Published: May 08, 2018 ABSTRACT The objective of this study was to investigate the mutational profiles of cancers arising in different colon segments. To this aim, we have analyzed 37 colon cancer samples by use of the Ion AmpliSeq™ Comprehensive Cancer Panel. Overall, we have found 307 mutated genes, most of which already implicated in the development of colon cancer. Among these, 15 genes were mutated in tumors originating in all six colon segments and were defined “common genes” (i.e. APC, PIK3CA, TP53) whereas 13 genes were preferentially mutated in tumors originating only in specific colon segments and were defined “site-associated genes” (i.e. BLNK, PTPRD). In addition, the presence of mutations in 10 of the 307 identified mutated genes (NBN, SMUG1, ERBB2, PTPRT, EPHB1, ALK, PTPRD, AURKB, KDR and GPR124) were found to be of clinical relevance. Among clinically relevant genes, NBN and SMUG1 were identified as independent prognostic factors that predicted poor survival in colon cancer patients. In conclusion, the findings reported here indicate that tumors arising in different colon segments present differences in the type and/or frequency of genetic variants, with two of them being independent prognostic factors that predict poor survival in colon cancer patients.

Background Improvement in genetic characterization of Colon Cancer (CC) patients is required to propose new potential targets, since surgical resection coupled to chemotherapy, presents several limits such as cancer recurrence and drug resistance. Targeted therapies have more efficacy and less toxicity than standard treatments. One of the most relevant cancer-specific actionable targets are receptor tyrosine kinases (RTKs) whose role in CC need to be better investigated. Methods We have analysed 37 CC patients using the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP). We have confirmed the somatic nature of RET variants through Sanger sequencing and assessed RET activation status and protein expression by immunofluorescence and western-blot analyses. We have used RET mutant expression vectors to evaluate the effect of selected mutations in HEK293 cells by performing proliferation, migration and clonogenic assays. Results Among the 409 cancer-related genes included in the CCP we have focused on the RTKs. Overall, we have observed 101 different potentially damaging variants distributed across 31 RTK genes in 28 patients. The most frequently mutated RTKs were FLT4, ROS1, EPH7, ERBB2, EGFR, RET, FGFR3 and FGFR4. In particular, we have identified 4 different somatic variants in 10% of CC patients in RET proto-oncogene. Among them, we have demonstrated that the G533C variant was able to activate RET by promoting dimer formation and enhancing Y1062 phosphorylation. Moreover, we have demonstrated that RET G533C variant was able to stimulate anchorage-dependent proliferation, migration and clonogenic cell survival. Notably, the effects induced by the RET G533C variant were abolished by vandetanib. Conclusions The discovery of pathogenic variants across RTK genes in 75% of the CC patients under analysis, suggests a previously underestimated role for RTKs in CC development. The identification of a gain-of-function RET mutation in CC highlights the potential use of RET in targeted therapy. Electronic supplementary material The online version of this article (10.1186/s13046-018-0746-y) contains supplementary material, which is available to authorized users.

The objective of this study was to determine the feasibility to detect copy number alterations in colon cancer samples using Next Generation Sequencing data and to elucidate the association between copy number alterations in specific genes and the development of cancer in different colon segments. We report the successful detection of somatic changes in gene copy number in 37 colon cancer patients by analysis of sequencing data through Amplicon CNA Algorithm. Overall, we have found a total of 748 significant copy number alterations in 230 significant genes, of which 143 showed CN losses and 87 showed CN gains. Validation of results was performed on 20 representative genes by quantitative qPCR and/or immunostaining. By this analysis, we have identified 4 genes that were subjected to copy number alterations in tumors arising in all colon segments (defined "common genes") and the presence of copy number alterations in 14 genes that were significantly associated to one specific site (defined "site-associated genes"). Finally, copy number alterations in ASXL1, TSC1 and IL7R turned out to be clinically relevant since the loss of TSC1 and IL7R was associated with advanced stages and/or reduced survival whereas copy number gain of ASXL1 was associated with good prognosis.

Canine atopic dermatitis (AD) is a very common inflammatory skin disease, but limited data are available on the genetic characterization (somatic mutations, microarrays, and genome-wide association study (GWAS)) of skin lesions in affected dogs. microRNAs are good biomarkers in inflammatory and neoplastic diseases in people. The aim of this study was to evaluate microRNA expression in the skin of atopic beagles, before and after exposure to Dermatophagoides farinae. Four atopic and four unrelated age-matched healthy beagle dogs were enrolled. Total RNA was extracted from flash-frozen skin biopsies of healthy and atopic dogs. For the atopic dogs, skin biopsies were taken from non-lesional (day 0) and lesional skin (day 28 of weekly environmental challenge with Dermatophagoides farinae). Small RNA libraries were constructed and sequenced. The microRNA sequences were aligned to CanFam3.1 genome. Differential expressed microRNAs were selected on the basis of fold-change and statistical significance (fold-change ≥ 1.5 and p ≤ 0.05 as thresholds. A total of 277 microRNAs were sequenced. One hundred and twenty-one differentially regulated microRNAs were identified between non-lesional and healthy skin. Among these, two were increased amount and 119 were decreased amount. A total of 45 differentially regulated microRNAs between lesional and healthy skin were identified, 44 were decreased amount and one was increased amount. Finally, only two increased amount microRNAs were present in lesional skin when compared with that of non-lesional skin. This is the first study in which dysregulation of microRNAs has been associated with lesional and non-lesional canine AD. Larger studies are needed to understand the role of microRNA in canine AD.

// Duarte Mendes Oliveira 1, 6 , Teresa Mirante 1, 6 , Chiara Mignogna 2 , Marianna Scrima 1, 8 , Simona Migliozzi 1 , Gaetano Rocco 3 , Renato Franco 4 , Francesco Corcione 7 , Giuseppe Viglietto 1, 6 , Donatella Malanga 1, 6 and Antonia Rizzuto 5 1 Department of Experimental and Clinical Medicine, University “Magna Graecia”, Catanzaro, Italy 2 Department of Health Sciences, University “Magna Graecia”, Catanzaro, Italy 3 Pathology Unit, Istituto Nazionale Tumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 4 Pathology Unit, Second University of Naples, Italy 5 Department of Medical and Surgical Sciences, University “Magna Graecia”, Catanzaro, Italy 6 Interdepartmental Center of Services (CIS), University “Magna Graecia”, Catanzaro, Italy 7 UOC Chirurgia Generale, Azienda Ospedaliera dei Colli, Napoli, Italy 8 Biogem scarl, Institute of Genetic Research, Ariano Irpino (AV), Italy Correspondence to: Giuseppe Viglietto, email: viglietto@unicz.it Donatella Malanga, email: malanga@unicz.it Keywords: solid tumors; oncomine focus assay; copy number variation; gene fusions Received: December 09, 2017 Accepted: April 05, 2018 Published: April 27, 2018 ABSTRACT In this study, we have set-up a routine pipeline to evaluate the clinical application of Oncomine™ Focus Assay, a panel that allows the simultaneous detection of single nucleotide hotspot mutations in 35 genes, copy number alterations (CNAs) in 19 genes and gene fusions involving 23 genes in cancer samples. For this study we retrospectively selected 106 patients that were submitted to surgical resection for lung, gastric, colon or rectal cancer. We found that 56 patients out of 106 showed at least one alteration (53%), with 47 patients carrying at least one relevant nucleotide variant, 10 patients carrying at least one CNA and 3 patients carrying one gene fusion. On the basis of the mutational profiles obtained, we have identified 22 patients (20.7%) that were potentially eligible for targeted therapy. The most frequently mutated genes across all tumor types included KRAS (30 patients), PIK3CA (16 patients), BRAF (6 patients), EGFR (5 patients), NRAS (4 patients) and ERBB2 (3 patients) whereas CCND1, ERBB2, EGFR and MYC were the genes most frequently subjected to copy number gain. Finally, gene fusions were identified only in lung cancer patients and involved MET [MET(13)–MET(15) fusion] and FGFR3 [FGFR3(chr 17)–TACC3(chr 11)]. In conclusion, we demonstrate that the analysis with a multi-biomarker panel of cancer patients after surgery, may present several potential advantages in clinical daily practice, including the simultaneous detection of different potentially druggable alterations, reasonable costs, short time of testing and automated interpretation of results.

// Ilaria Guerriero 1 , Daniela D’Angelo 2 , Pierlorenzo Pallante 2 , Mafalda Santos 1 , Marianna Scrima 1 , Donatella Malanga 3 , Carmela De Marco 3 ,Maria Ravo 4 , Alessandro Weisz 4 , Carmelo Laudanna 3 , Michele Ceccarelli 1, 5 , Geppino Falco 1, 6 , Antonia Rizzuto 7 , Giuseppe Viglietto 1, 3 1 Biogems.c.ar.l., Istituto di Ricerche Genetiche “Gaetano Salvatore”, Ariano Irpino, Avellino, Italy 2 Istituto per l’Endocrinologia e l’Oncologia Sperimentale, IEOS-CNR, c/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita “Federico II” of Napoli, Napoli, Italy 3 Dipartimento di Medicina Sperimentale e Clinica, Universita Magna Graecia of Catanzaro, Italy 4 Laboratorio di Medicina Molecolare e Genomica, Dipartimentodi Medicina e Chirurgia, Universita di Salerno, Baronissi, Italy 5 Department of Biological and Environmental Studies, Universita del Sannio, Benevento, Italy 6 Dipartimento di Biologia, Universita degli Studi di Napoli Federico II, Complesso Universitario Monte S.Angelo, Napoli, Italy 7 Dipartimento di Scienze Mediche e Chirurgiche, Universita Magna Graecia of Catanzaro, Italy Correspondence to: Giuseppe Viglietto, email: viglietto@unicz.it Keywords: NSCLC, PI3K/AKT, microRNA, miR-196a Received: January 11, 2016 Accepted: November 02, 2016 Published: November 17, 2016 ABSTRACT Hyperactivation of the PI3K/AKT pathway is observed in most human cancer including lung carcinomas. Here we have investigated the role of miRNAs as downstream targets of activated PI3K/AKT signaling in Non Small Cell Lung Cancer (NSCLC). To this aim, miRNA profiling was performed in human lung epithelial cells (BEAS-2B) expressing active AKT1 (BEAS-AKT1-E17K), active PI3KCA (BEAS-PIK3CA-E545K) or with silenced PTEN (BEAS-shPTEN). Twenty-four differentially expressed miRNAs common to BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells were identified through this analysis, with miR-196a being the most consistently up-regulated miRNA. Interestingly, miR-196a was significantly overexpressed also in human NSCLC-derived cell lines (n=11) and primary lung cancer samples (n=28). By manipulating the expression of miR-196a in BEAS-2B and NCI-H460 cells, we obtained compelling evidence that this miRNA acts downstream the PI3K/AKT pathway, mediating some of the proliferative, pro-migratory and tumorigenic activity that this pathway exerts in lung epithelial cells, possibly through the regulation of FoxO1, CDKN1B (hereafter p27) and HOXA9.

The hotspot AKT1(E17K) mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6-2% of human lung cancers. Recently, we have demonstrated that AKT1(E17K) transforms immortalized human bronchial cells. Here by use of a transgenic Cre-inducible murine strain in the wild type Rosa26 (R26) locus (R26-AKT1(E17)K mice) we demonstrate that AKT1(E17K) is a bona-fide oncogene and plays a role in the development of lung cancer in vivo. In fact, we report that mutant AKT1(E17K) induces bronchial and/or bronchiolar hyperplastic lesions in murine lung epithelium, which progress to frank carcinoma at very low frequency, and accelerates tumor formation induced by chemical carcinogens. In conclusion, AKT1(E17K) induces hyperplasia of mouse lung epithelium in vivo and cooperates with urethane to induce the fully malignant phenotype.

// Carmela De Marco 1, 2 , Donatella Malanga 1, 2 , Nicola Rinaldo 2 , Fernanda De Vita 2 , Marianna Scrima 2 , Sara Lovisa 3 , Linda Fabris 3 , Maria Vincenza Carriero 4 , Renato Franco 4 , Antonia Rizzuto 5 , Gustavo Baldassarre 3 , Giuseppe Viglietto 1, 2 1 Department of Experimental and Clinical Medicine, University “Magna Graecia”, Catanzaro, Italy 2 BIOGEM-Institute of Genetic Research, Ariano Irpino (AV), Italy 3 Experimental Oncology 2, Centro di Riferimento Oncologico, Aviano (PN), Italy 4 Experimental Oncology, IRCCS Fondazione Pascale, Napoli, Italy 5 Department of Medical and Surgical Sciences, University “Magna Graecia” Medical School, Catanzaro, Italy Correspondence to: Giuseppe Viglietto, e-mail: viglietto@unicz.it Keywords: lung cancer, AKT1-E17K, human lung epithelial cells, p27 Received: April 03, 2015 Accepted: May 13, 2015 Published: May 25, 2015 ABSTRACT The hotspot E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6–2% of human lung cancers. In this manuscript, we sought to determine whether this AKT1 variant is a bona-fide activating mutation and plays a role in the development of lung cancer. Here we report that in immortalized human bronchial epithelial cells (BEAS-2B cells) mutant AKT1-E17K promotes anchorage-dependent and -independent proliferation, increases the ability to migrate, invade as well as to survive and duplicate in stressful conditions, leading to the emergency of cells endowed with the capability to form aggressive tumours at high efficiency. We provide also evidence that the molecular mechanism whereby AKT1-E17K is oncogenic in lung epithelial cells involves phosphorylation and consequent cytoplasmic delocalization of the cyclin-dependent kinase (cdk) inhibitor p27. In agreement with these results, cytoplasmic p27 is preferentially observed in primary NSCLCs with activated AKT and predicts poor survival.

// Donatella Malanga 1 , Carmela De Marco 1, 2 , Ilaria Guerriero 2 , Fabiana Colelli 2 , Nicola Rinaldo 2 , Marianna Scrima 1, 2 , Teresa Mirante 3 , Claudia De Vitis 7 , Pietro Zoppoli 2 , Michele Ceccarelli 1, 4 , Miriam Riccardi 1, 2 , Maria Ravo 5 , Alessandro Weisz 5 , Antonella Federico 6 , Renato Franco 7 , Gaetano Rocco 7 , Rita Mancini 8 , Antonia Rizzuto 3 , Elio Gulletta 9 , Gennaro Ciliberto 7 , Giuseppe Viglietto 1, 2 1 Dipartimento di Medicina Sperimentale e Clinica, Universita Magna Graecia, Catanzaro, Italy 2 Biogem scarl, Istituto di Ricerche Genetiche, Ariano Irpino (Avellino), Italy 3 Dipartimento di Scienze Mediche e Chirurgiche, Universita Magna Graecia, Catanzaro, Italy 4 Dipartimento di Scienze e Tecnologie, Universita del Sannio, Benevento, Italy 5 Dipartimento di Medicina e Chirurgia, Universita di Salerno, Baronissi, Italy 6 Dipartimento di Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita Federico II, Napoli, Italy 7 IRCCS Istituto Nazionale Tumori Fondazione G. Pascale, Napoli, Italy 8 Dipartimento di Medicina Clinica e Molecolare, Universita di Roma “La Sapienza” Ospedale S. Andrea, Roma, Italy 9 Dipartimento di Scienze della Salute, Universita Magna Graecia, Catanzaro, Italy Correspondence to: Giuseppe Viglietto, e-mail: viglietto@unicz.it Keywords: NSCLC, tumor initiating cells, Akt1, IL-6, STAT3 Received: June 15, 2015 Accepted: September 09, 2015 Published: October 13, 2015 ABSTRACT Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation ( n = 94; p < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC.