Endoscopic follow-up of low-grade precancerous bronchial lesions in high-risk patients: long-term results of the SELEPREBB randomised multicentre trial.

BACKGROUND: 3-9% of low-grade preinvasive bronchial lesions progress to cancer. This study assessed the usefulness of an intensive bronchoscopy surveillance strategy in patients with bronchial lesions up to moderate squamous dysplasia. METHODS: SELEPREBB (ClinicalTrials.gov NCT00213603) was a randomised study conducted in 17 French centres. After baseline lung computed tomography (CT) and autofluorescence bronchoscopy (AFB) to exclude lung cancer and bronchial severe squamous dysplasia or carcinoma in situ (CIS), patients were assigned to standard surveillance (arm A) with CT and AFB at 36 months or to intensive surveillance (arm B) with AFB every 6 months. Further long-term data were obtained with a median follow-up of 4.7 years. RESULTS: 364 patients were randomised (A: 180, B: 184). 27 patients developed invasive lung cancer and two developed persistent CIS during the study, with no difference between arms (OR 0.63, 95% CI 0.20-1.96, p=0.42). Mild or moderate dysplasia at baseline bronchoscopy was a significant lung cancer risk factor both at 3 years (8 of 74 patients, OR 6.9, 95% CI 2.5-18.9, p<0.001) and at maximum follow-up (16 of 74 patients, OR 5.9, 95% CI 2.9-12.0, p<0.001). Smoking cessation was significantly associated with clearance of bronchial dysplasia on follow-up (OR 0.12, 95% CI 0.01-0.66, p=0.005) and with a reduced risk of lung cancer at 5 years (OR 0.15, 95% CI 0.003-0.99, p=0.04). CONCLUSION: Patients with mild or moderate dysplasia are at very high risk for lung cancer at 5 years, with smoking cessation significantly reducing the risk. Whereas intensive bronchoscopy surveillance does not improve patient outcomes, the identification of bronchial dysplasia using initial bronchoscopy maybe useful for risk stratification strategies in lung cancer screening programmes.

FHITlow /pHER2high signature in non-small cell lung cancer is predictive of anti-HER2 molecule efficacy.

Despite its efficacy in solid tumours, in particular HER2+ breast cancer, HER2-targeted therapy has given rise to disappointing results in non-small cell lung cancer (NSCLC). With the aim of refining the target population for anti-HER2 therapies in NSCLC, we investigated the relationships between HER2 and the tumour suppressor fragile histidine triad (FHIT) in lung tumour cells. First, we observed a negative correlation between FHIT expression and the activated form of HER2 (pHER2) in NSCLC samples and in lung tumour cell lines. Moreover, the silencing or overexpression of FHIT in lung cell lines led to an increase or decrease of HER2 activity, respectively. We also demonstrated that two anti-HER2 drugs, irbinitinib and trastuzumab, restore a more epithelial phenotype and counteract cell invasiveness and growth of FHIT-silenced tumour cell lines. Finally, we showed that the FHITlow /pHER2high phenotype predicts sensitivity to an anti-HER2 therapy in primary tumour cells from NSCLC patients. Our results show that FHIT regulates the activity of HER2 in lung tumour cells and that FHIT-inactivated tumour cells are sensitive to HER2 inhibitors. A new subclass of patients with NSCLC may be eligible for an anti-HER2 therapy. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Sonic hedgehog signalling as a potential endobronchial biomarker in COPD.

BACKGROUND: The hedgehog (HH) pathway has been associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies and recent studies suggest that HH signalling could be altered in COPD. We therefore used minimally invasive endobronchial procedures to assess activation of the HH pathway including the main transcription factor, Gli2, and the ligand, Sonic HH (Shh). METHODS: Thirty non-COPD patients and 28 COPD patients were included. Bronchial brushings, bronchoalveolar lavage fluid (BALF) and bronchial biopsies were obtained from fiberoptic bronchoscopy. Characterization of cell populations and subcellular localization were evaluated by immunostaining. ELISA and RNAseq analysis were performed to identify Shh proteins in BAL and transcripts on lung tissues from non-COPD and COPD patients with validation in an external and independent cohort. RESULTS: Compared to non-COPD patients, COPD patients exhibited a larger proportion of basal cells in bronchial brushings (26 ± 11% vs 13 ± 6%; p < 0.0001). Airway basal cells of COPD subjects presented less intense nuclear staining for Gli2 in bronchial brushings and biopsies (p < 0.05). Bronchial BALF from COPD patients contained lower Shh concentrations than non-COPD BALF (12.5 vs 40.9 pg/mL; p = 0.002); SHH transcripts were also reduced in COPD lungs in the validation cohort (p = 0.0001). CONCLUSION: This study demonstrates the feasibility of assessing HH pathway activation in respiratory samples collected by bronchoscopy and identifies impaired bronchial epithelial HH signalling in COPD.

A new mouse model to study the role of ectopic Nanos3 expression in cancer.

BACKGROUND: NANOS3 is a gene conserved throughout evolution. Despite the quite low conservation of Nanos sequences between different organisms and even between Nanos paralogs, their role in germ cell development is remarkably universal. Human Nanos3 expression is normally restricted to the gonads and the brain. However, ectopic activation of this gene has been detected in various human cancers. Until now, Nanos3 and other Nanos proteins have been studied almost exclusively in germ cell development. METHODS: Transgenic mice were generated by targeted insertion of a human Nanos3 cDNA into the ROSA26 locus. The transgene could be spatiotemporally induced by Cre recombinase activity removing an upstream floxed STOP cassette. A lung tumor model with ectopic Nanos3 expression was based on the lung-specific activation of the reverse tetracycline transactivator gene, in combination with a tetO-CMV promoter controlling Cre expression. When doxycycline was provided to the mice, Cre was activated leading to deletion of TP53 alleles and activation of both oncogenic KRasG12D and Nanos3. Appropriate controls were foreseen. Tumors and tumor-derived cell cultures were analyzed in various ways. RESULTS: We describe the successful generation of Nanos3LSL/- and Nanos3LSL/LSL mice in which an exogenous human NANOS3 gene can be activated in vivo upon Cre expression. These mice, in combination with different conditional and doxycycline-inducible Cre lines, allow the study of the role of ectopic Nanos3 expression in several cancer types. The Nanos3LSL mice were crossed with a non-small cell lung cancer (NSCLC) mouse model based on conditional expression of oncogenic KRas and homozygous loss of p53. This experiment demonstrated that ectopic expression of Nanos3 in the lungs has a significant negative effect on survival. Enhanced bronchiolar dysplasia was observed when Nanos3-expressing NSCLC mice were compared with control NSCLC mice. An allograft experiment, performed with cell cultures derived from primary lung tumors of control and Nanos3-expressing NSCLC mice, revealed lymph node metastasis in mice injected with Nanos3-expressing NSCLC cells. CONCLUSIONS: A new mouse model was generated allowing examination of Nanos3-associated pathways and investigation of the influence of ectopic Nanos3 expression in various cancer types. This model might identify Nanos3 as an interesting target in cancer therapeutics.

Zonula occludens-1/NF-κB/CXCL8: a new regulatory axis for tumor angiogenesis.

Zonula occludens-1 (ZO-1) is a submembrane scaffolding protein that may display proinvasive functions when it relocates from tight junctions into the cytonuclear compartment. This article examines the functional involvement of ZO-1 in CXCL8/IL-8 chemokine expression in lung and breast tumor cells. ZO-1 small interfering RNA and cDNA transfection experiments emphasized regulation of CXCL8/IL-8 expression via a cytonuclear pool of ZO-1. Luciferase reporter assays highlighted a 173-bp region of CXCL8/IL-8 promoter that responded to ZO-1. Moreover, by using mutated promoter constructs, we identified a NF-κB site as critical in this activation. Furthermore, NF-κB pathway signaling analysis revealed both IκBα and p65 phosphorylation in ZO-1-overexpressing cells, and subsequent p65 silencing validated its requirement for CXCL8/IL-8 induction. Investigation of the functional implication of this regulatory axis next showed the proangiogenic activity of ZO-1 in both ex vivo and in vivo angiogenesis assays. Finally, we found that non-small-cell lung carcinoma that presented a cytonuclear ZO-1 pattern was significantly more angiogenic that that without detectable cytonuclear ZO-1 expression. Taken together, our results demonstrate that ZO-1 regulates CXCL8/IL-8 expression via the NF-κB signaling pathway and its p65 subunit, which subsequently modulates the transcription of IL-8. We also provide evidence of a newly identified regulatory pathway that could promote angiogenesis. Thus, our results support the concept that the ZO-1 shuttle from the cell junction to the cytonuclear compartment may affect both the intrinsic invasive properties of tumor cells and the establishment of the protumoral microenvironment.-Lesage, J., Suarez-Carmona, M., Neyrinck-Leglantier, D., Grelet, S., Blacher, S., Hunziker, W., Birembaut, P., Noël, A., Nawrocki-Raby, B., Gilles, C., Polette, M. Zonula occludens-1/NF-κB/CXCL8: a new regulatory axis for tumor angiogenesis.

RNA sequencing and transcriptome arrays analyses show opposing results for alternative splicing in patient derived samples.

BACKGROUND: RNA sequencing (RNA-seq) and microarrays are two transcriptomics techniques aimed at the quantification of transcribed genes and their isoforms. Here we compare the latest Affymetrix HTA 2.0 microarray with Illumina 2000 RNA-seq for the analysis of patient samples - normal lung epithelium tissue and squamous cell carcinoma lung tumours. Protein coding mRNAs and long non-coding RNAs (lncRNAs) were included in the study. RESULTS: Both platforms performed equally well for protein-coding RNAs, however the stochastic variability was higher for the sequencing data than for microarrays. This reduced the number of differentially expressed genes and genes with predictive potential for RNA-seq compared to microarray data. Analysis of this variability revealed a lack of reads for short and low abundant genes; lncRNAs, being shorter and less abundant RNAs, were found especially susceptible to this issue. A major difference between the two platforms was uncovered by analysis of alternatively spliced genes. Investigation of differential exon abundance showed insufficient reads for many exons and exon junctions in RNA-seq while the detection on the array platform was more stable. Nevertheless, we identified 207 genes which undergo alternative splicing and were consistently detected by both techniques. CONCLUSIONS: Despite the fact that the results of gene expression analysis were highly consistent between Human Transcriptome Arrays and RNA-seq platforms, the analysis of alternative splicing produced discordant results. We concluded that modern microarrays can still outperform sequencing for standard analysis of gene expression in terms of reproducibility and cost.

Epithelial to mesenchymal transition and HPV infection in squamous cell oropharyngeal carcinomas: the papillophar study.

BACKGROUND: Human Papillomavirus (HPV) infection is recognised as aetiological factor of carcinogenesis in oropharyngeal squamous cell carcinomas (OPC). HPV-related OPC respond better to treatments and have a significantly favourable outcome. Epithelial to mesenchymal transition (EMT) implicated in tumour invasion, is a hallmark of a poor prognosis in carcinomas. METHODS: We have studied the relationship of EMT markers (E-cadherin, ß-catenin and vimentin) with HPV infection (DNA and E6/E7 mRNA detection), p16INK4a expression and survival outcomes in a cohort of 296 patients with OPC. RESULTS: Among the 296 OPSSC, 26% were HPV positive, 20.3% had overt EMT (>25% of vimentin positive tumour cells). Lower E-cadherin expression was associated with a higher risk of distant metastasis in univariate (P=0.0110) and multivariate analyses (hazard ratios (HR)=6.86 (1.98; 23.84)). Vimentin expression tends towards worse metastasis-free survival (MFS; HR=2.53 (1.00; 6.41)) and was an independent prognostic factor of progression-free survival (HR=1.55 (1.03; 2.34)). CONCLUSIONS: There was a non significant association of EMT with HPV status. This may be explained by a mixed subpopulation of patients HPV positive with associated risk factors (HPV, tobacco and alcohol). Thus, the detection of EMT in OPC represents another reliable approach in the prognosis and the management of OPC whatever their HPV status.

Renal Graft Fibrosis and Inflammation Quantification by an Automated Fourier-Transform Infrared Imaging Technique.

Renal interstitial fibrosis and interstitial active inflammation are the main histologic features of renal allograft biopsy specimens. Fibrosis is currently assessed by semiquantitative subjective analysis, and color image analysis has been developed to improve the reliability and repeatability of this evaluation. However, these techniques fail to distinguish fibrosis from constitutive collagen or active inflammation. We developed an automatic, reproducible Fourier-transform infrared (FTIR) imaging-based technique for simultaneous quantification of fibrosis and inflammation in renal allograft biopsy specimens. We generated and validated a classification model using 49 renal biopsy specimens and subsequently tested the robustness of this classification algorithm on 166 renal grafts. Finally, we explored the clinical relevance of fibrosis quantification using FTIR imaging by comparing results with renal function at 3 months after transplantation (M3) and the variation of renal function between M3 and M12. We showed excellent robustness for fibrosis and inflammation classification, with >90% of renal biopsy specimens adequately classified by FTIR imaging. Finally, fibrosis quantification by FTIR imaging correlated with renal function at M3, and the variation in fibrosis between M3 and M12 correlated well with the variation in renal function over the same period. This study shows that FTIR-based analysis of renal graft biopsy specimens is a reproducible and reliable label-free technique for quantifying fibrosis and active inflammation. This technique seems to be more relevant than digital image analysis and promising for both research studies and routine clinical practice.

Vibrational Analysis of Lung Tumor Cell Lines: Implementation of an Invasiveness Scale Based on the Cell Infrared Signatures.

Assessing the tumor invasiveness is a paramount diagnostic step to improve the patients care. Infrared spectroscopy access the chemical composition of samples; and in combination with statistical multivariate processing, presents the capacity to highlight subtle molecular alterations associated with malignancy development. Our investigation demonstrated that infrared signatures of cell lines presenting various invasiveness phenotypes contain discriminant spectral features, which are useful informative signals to implement an objective invasiveness scale. This last development reflects the interest of vibrational approach as a candidate biophotonic label-free technique, usable in routine clinics, to characterize quantitatively tumor aggressiveness. In addition, the methodology can reveal the heterogeneity of cancer cells, opening the way to further researches in cancer science.

The human NANOS3 gene contributes to lung tumour invasion by inducing epithelial-mesenchymal transition.

We have explored the role of the human NANOS3 gene in lung tumour progression. We show that NANOS3 is over-expressed by invasive lung cancer cells and is a prognostic marker for non-small cell lung carcinomas (NSCLCs). NANOS3 gene expression is restricted in testis and brain and is regulated by epigenetic events. It is up-regulated in cultured cells undergoing epithelial - mesenchymal transition (EMT). NANOS3 over-expression in human NSCLC cell lines enhances their invasiveness by up-regulating EMT, whereas its silencing induces mesenchymal - epithelial transition. NANOS3 represses E-cadherin at the transcriptional level and up-regulates vimentin post-transcriptionally. Also, we show that NANOS3 binds mRNAs encoding vimentin and regulates the length of their poly(A) tail. Finally, NANOS3 can also protect vimentin mRNA from microRNA-mediated repression. We thus demonstrate a role for NANOS3 in the acquisition of invasiveness by human lung tumour cells and propose a new mechanism of post-transcriptional regulation of EMT.

Soluble factors regulated by epithelial-mesenchymal transition mediate tumour angiogenesis and myeloid cell recruitment.

Epithelial-mesenchymal transition (EMT) programmes provide cancer cells with invasive and survival capacities that might favour metastatic dissemination. Whilst signalling cascades triggering EMT have been extensively studied, the impact of EMT on the crosstalk between tumour cells and the tumour microenvironment remains elusive. We aimed to identify EMT-regulated soluble factors that facilitate the recruitment of host cells in the tumour. Our findings indicate that EMT phenotypes relate to the induction of a panel of secreted mediators, namely IL-8, IL-6, sICAM-1, PAI-1 and GM-CSF, and implicate the EMT-transcription factor Snail as a regulator of this process. We further show that EMT-derived soluble factors are pro-angiogenic in vivo (in the mouse ear sponge assay), ex vivo (in the rat aortic ring assay) and in vitro (in a chemotaxis assay). Additionally, conditioned medium from EMT-positive cells stimulates the recruitment of myeloid cells. In a bank of 40 triple-negative breast cancers, tumours presenting features of EMT were significantly more angiogenic and infiltrated by a higher quantity of myeloid cells compared to tumours with little or no EMT. Taken together, our results show that EMT programmes trigger the expression of soluble mediators in cancer cells that stimulate angiogenesis and recruit myeloid cells in vivo, which might in turn favour cancer spread.

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure.

Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na(+) absorption, a defective cAMP-dependent Cl(-) conductance, higher concentrations of Na(+), Cl(-), K(+), and Ca(2+) in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca(2+) entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.

Deep brush-based cytology in tonsils resected for benign diseases.

A fraction of oropharyngeal cancer (OPC), especially in the tonsil, is caused by human papillomavirus (HPV), mainly HPV16. Noninvasive diagnostic methods to detect precancerous lesions in the tonsil would be useful, e.g., liquid-based cytology (LBC). However, ill-characterized precancerous lesions may be hidden in the depth of the tonsillar crypts. We therefore conducted a study on HPV and tonsillar precancerous lesions to evaluate, among other things, the utility of LBC obtained by deep brushing of the resected tonsils. Two hundred non-paediatric patients (mean age: 30.3 years) who underwent tonsillectomy for infection-related conditions (69%) or other conditions (mainly obstructive sleep apnoea, 31%) were included. An ultra-sensitive Luminex bead-based platform was used to test for the DNA of 21 mucosal HPV types; 56% of slides were unsatisfactory due to low number of squamous epithelial cells or the masking effect of a large number of lymphocytes. Three patients (1.5%; 95% CI: 0.5-4.3) showed suspicious cytological findings (atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion, ASC-H) while 3 others were HPV-positive (2 for HPV16 and 1 for HPV39). None of the ASC-H patients and HPV-positive patients showed dysplasia at histological examination. The rarity of HPV infection in the tonsil conflicts with the relatively frequent detection of the virus in the mouth. In conclusion, aggressive deep brushing of tonsils, while hardly applicable in vivo, is unlikely to be a reliable method to detect precancerous lesions. The absence of OPC screening modalities places the priority on multi-purpose primary prevention strategies, i.e., HPV vaccination and reduction of smoking and drinking.

Imprinting of the COPD airway epithelium for dedifferentiation and mesenchymal transition.

In chronic obstructive pulmonary disease (COPD), epithelial changes and subepithelial fibrosis are salient features in conducting airways. Epithelial-to-mesenchymal transition (EMT) has been recently suggested in COPD, but the mechanisms and relationship to peribronchial fibrosis remain unclear. We hypothesised that de-differentiation of the COPD respiratory epithelium through EMT could participate in airway fibrosis and thereby, in airway obstruction. Surgical lung tissue and primary broncho-epithelial cultures (in air-liquid interface (ALI)) from 104 patients were assessed for EMT markers. Cell cultures were also assayed for mesenchymal features and for the role of transforming growth factor (TGF)-ß1. The bronchial epithelium from COPD patients showed increased vimentin and decreased ZO-1 and E-cadherin expression. Increased vimentin expression correlated with basement membrane thickening and airflow limitation. ALI broncho-epithelial cells from COPD patients also displayed EMT phenotype in up to 2 weeks of culture, were more spindle shaped and released more fibronectin. Targeting TGF-ß1 during ALI differentiation prevented vimentin induction and fibronectin release. In COPD, the airway epithelium displays features of de-differentiation towards mesenchymal cells, which correlate with peribronchial fibrosis and airflow limitation, and which are partly due to a TGF-ß1-driven epithelial reprogramming.

Delay of airway epithelial wound repair in COPD is associated with airflow obstruction severity.

BACKGROUND: Airway epithelium integrity is essential to maintain its role of mechanical and functional barrier. Recurrent epithelial injuries require a complex mechanism of repair to restore its integrity. In chronic obstructive pulmonary disease (COPD), an abnormal airway epithelial repair may participate in airway remodeling. The objective was to determine if airway epithelial wound repair of airway epithelium is abnormal in COPD. METHODS: Patients scheduled for lung resection were prospectively recruited. Demographic, clinical data and pulmonary function tests results were recorded. Emphysema was visually scored and histological remodeling features were noted. Primary bronchial epithelial cells (BEC) were extracted and cultured for wound closure assay. We determined the mean speed of wound closure (MSWC) and cell proliferation index, matrix metalloprotease (MMP)-2, MMP-9 and cytokines levels in supernatants of BEC 18 hours after cell wounding. In a subset of patients, bronchiolar epithelial cells were also cultured for wound closure assay for MSWC analyze. RESULTS: 13 COPD and 7 non COPD patients were included. The severity of airflow obstruction and the severity of emphysema were associated with a lower MSWC in BEC (p = 0.01, 95% CI [0.15-0.80]; p = 0.04, 95% CI [-0.77;-0.03] respectively). Cell proliferation index was decreased in COPD patients (19 ± 6% in COPD vs 27 ± 3% in non COPD, p = 0.04). The severity of COPD was associated with a lower level of MMP-2 (7.8 ± 2 10(5) AU in COPD GOLD D vs 12.8 ± 0.13 10(5) AU in COPD GOLD A, p = 0.04) and a lower level of IL-4 (p = 0.03, 95% CI [0.09;0.87]). Moreover, higher levels of IL-4 and IL-2 were associated with a higher MSWC (p = 0.01, 95% CI [0.17;0.89] and p = 0.02, 95% CI [0.09;0.87] respectively). Clinical characteristics and smoking history were not associated with MSWC, cell proliferation index or MMP and cytokines levels. Finally, we showed an association of the MSWC of bronchial and corresponding bronchiolar epithelial cells obtained from the same patients (p = 0.02, 95% CI [0.12;0.89]). CONCLUSION: Our results showed an abnormal bronchial epithelial wound closure process in severe COPD. Further studies are needed to elucidate the contribution and the regulation of this mechanism in the complex pathophysiology of COPD.

High levels of HPV16-L1 antibody but not HPV16 DNA load or integration predict oropharyngeal patient outcome: The Papillophar study.

The incidence of oropharyngeal cancers (OPC) is increasing in the world. Among OPC, those induced by human papillomaviruses have a better prognosis than non-HPV-associated OPC. The objective of this study was to highlight the relevance of HPV16 load, HPV16 DNA integration and HPV16-L1 serology on progression-free survival and overall survival of OPC patients. The PAPILLOPHAR cohort consists of 362 patients with oropharyngeal squamous cell carcinomas prospectively followed up for 5 years after treatment. Tumor biopsies and sera were collected at inclusion to investigate tumor HPV DNA/RNA characteristics and HPV16 L1 serology, respectively. Twenty-seven percent of tumor biopsies were HPV DNA- and RNA-positive and HPV16 represented 93% of HPV-positive cases. Among them, neither HPV16 viral load nor HPV16 DNA integration was associated with overall survival (OS) or progression-free survival (PFS). In contrast, high anti-HPV16 L1 antibody titers were significantly associated with a better OS and PFS. This study reveals that HPV16 load and integration are not relevant prognosis biomarkers in OPC patients.Clinical Relevance: High levels of HPV16 L1 antibodies may be useful to predict OPC patient outcome following treatment.ClinicalTrials.gov Identifier: NCT00918710, May 2017.

Sirolimus-based regimen is associated with decreased expression of glomerular vascular endothelial growth factor.

BACKGROUND: Sirolimus (SRL) is a potent immunosuppressant used in organ transplantation. It is known to decrease vascular endothelial growth factor (VEGF) synthesis, making it an interesting treatment option for transplant patients who develop Kaposi sarcoma or other malignant diseases. Because VEGF plays a key role in glomerular function and vascular remodelling, we determined the effect of SRL on renal VEGF expression. METHODS: Using immunohistochemistry and quantitative image analysis, we examined renal VEGF expression in routine kidney biopsies performed at 1 year post-transplant in the CONCEPT study, a prospective randomized study comparing a cyclosporine (CsA)-based regimen to a SRL-based regimen in association with mycophenolate mofetil (MMF). RESULTS: A total of 74 patients were included in this substudy; 35 were randomized to the CsA group and 39 to the SRL group. Using continuous variables, the mean percentage of glomerular VEGF expression at Week 52 was significantly lower in the SRL group (14.7 ± 13%) compared to CsA group (21.2 ± 14%: P = 0.02). The percentage of glomerular VEGF expression at Week 52 was not influenced by recipient or donor age, gender, renal function, CsA dose, CsA blood level, SRL dose or SRL blood level. It was significantly lower in patients with a proteinuria over versus below 0.5 g/day (11.58 ± 7.9 versus 19.45 ± 15.53; P = 0.036). CONCLUSIONS: There is emerging evidence that the VEGF system can play either a beneficial or a detrimental role depending on the specific pathologic situations. Therefore, modulating the renal VEGF axis by using an SRL-based regimen may influence the evolution of kidney injury associated with renal transplantation.

CHRNA5 as negative regulator of nicotine signaling in normal and cancer bronchial cells: effects on motility, migration and p63 expression.

Genome-wide association studies have linked lung cancer risk with a region of chromosome 15q25.1 containing CHRNA3, CHRNA5 and CHRNB4 encoding α3, α5 and ß4 subunits of nicotinic acetylcholine receptors (nAChR), respectively. One of the strongest associations was observed for a non-silent single-nucleotide polymorphism at codon 398 in CHRNA5. Here, we have used pharmacological (antagonists) or genetic (RNA interference) interventions to modulate the activity of CHRNA5 in non-transformed bronchial cells and in lung cancer cell lines. In both cell types, silencing CHRNA5 or inhibiting receptors containing nAChR α5 with α-conotoxin MII exerted a nicotine-like effect, with increased motility and invasiveness in vitro and increasing calcium influx. The effects on motility were enhanced by addition of nicotine but blocked by inhibiting CHRNA7, which encodes the homopentameric receptor α7 subunit. Silencing CHRNA5 also decreased the expression of cell adhesion molecules P120 and ZO-1 in lung cancer cells as well as the expression of DeltaNp63α in squamous cell carcinoma cell lines. These results demonstrate a role for CHRNA5 in modulating adhesion and motility in bronchial cells, as well as in regulating p63, a potential oncogene in squamous cell carcinoma.

Nicotinic acetylcholine receptors and predisposition to lung cancer.

PURPOSE OF REVIEW: To highlight the current body of knowledge regarding the role of nicotinic acetylcholine receptors (nAChRs) in lung cancer predisposition. RECENT FINDINGS: Smoking is a documented risk factor for cancer, especially for lung carcinomas. Nicotine and its derived carcinogenic nitrosamines contribute to lung cancer development and progression through the binding to nAChRs, which then activate proliferation, apoptosis, angiogenesis and tumour invasion. Recent genome-wide association studies have associated single nucleotide polymorphisms spanning the nAChR encoding genes cluster CHRNA3/A5/B4 with both nicotine dependence and lung cancer incidence and susceptibility. The α7 nAChR has also been implicated in the regulation of inflammation and immunity and, as a repressor of airway basal cell proliferation, α7 nAChR plays a role in the remodelling of the airway epithelium. Its decreased function may lead to squamous metaplasia and possibly the emergence of preneoplastic lesions. SUMMARY: nAChRs participate in the predisposition for preneoplastic lesions and the further emergence of lung carcinomas. More studies are needed to determine the influence of gene polymorphisms on nAChRs function and of nAChRs activation/desensitization on lung diseases, which represents a new stimulating approach in the understanding of lung tumorigenesis with potential clinical applications.