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.

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.

An innate contribution of human nicotinic receptor polymorphisms to COPD-like lesions.

Chronic Obstructive Pulmonary Disease is a generally smoking-linked major cause of morbidity and mortality. Genome-wide Association Studies identified a locus including a non-synonymous single nucleotide polymorphism in CHRNA5, rs16969968, encoding the nicotinic acetylcholine receptor α5 subunit, predisposing to both smoking and Chronic Obstructive Pulmonary Disease. Here we report that nasal polyps from rs16969968 non-smoking carriers exhibit airway epithelium remodeling and inflammation. These hallmarks of Chronic Obstructive Pulmonary Disease occur spontaneously in mice expressing human rs16969968. They are significantly amplified after exposure to porcine pancreatic elastase, an emphysema model, and to oxidative stress with a polymorphism-dependent alteration of lung function. Targeted rs16969968 expression in epithelial cells leads to airway remodeling in vivo, increased proliferation and production of pro-inflammatory cytokines through decreased calcium entry and increased adenylyl-cyclase activity. We show that rs16969968 directly contributes to Chronic Obstructive Pulmonary Disease-like lesions, sensitizing the lung to the action of oxidative stress and injury, and represents a therapeutic target.

Identification of Neoadjuvant Chemotherapy Response in Muscle-Invasive Bladder Cancer by Fourier-Transform Infrared Micro-Imaging.

BACKGROUND: Neoadjuvant chemotherapy (NAC) improves survival in responder patients. However, for non-responders, the treatment represents an ineffective exposure to chemotherapy and its potential adverse events. Predicting the response to treatment is a major issue in the therapeutic management of patients, particularly for patients with muscle-invasive bladder cancer. METHODS: Tissue samples of trans-urethral resection of bladder tumor collected at the diagnosis time, were analyzed by mid-infrared imaging. A sequence of spectral data processing was implemented for automatic recognition of informative pixels and scoring each pixel according to a continuous scale (from 0 to 10) associated with the response to NAC. The ground truth status of the responder or non-responder was based on histopathological examination of the samples. RESULTS: Although the TMA spots of tumors appeared histologically homogeneous, the infrared approach highlighted spectral heterogeneity. Both the quantification of this heterogeneity and the scoring of the NAC response at the pixel level were used to construct sensitivity and specificity maps from which decision criteria can be extracted to classify cancerous samples. CONCLUSIONS: This proof-of-concept appears as the first to evaluate the potential of the mid-infrared approach for the prediction of response to neoadjuvant chemotherapy in MIBC tissues.

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.

S100A4 is a Biomarker of Tumorigenesis, EMT, Invasion, and Colonization of Host Organs in Experimental Malignant Mesothelioma.

Recent findings suggest that S100A4, a protein involved in communication between stromal cells and cancer cells, could be more involved than previously expected in cancer invasiveness. To investigate its cumulative value in the multistep process of the pathogenesis of malignant mesothelioma (MM), SWATH-MS (sequential window acquisition of all theoretical fragmentation spectra), an advanced and robust technique of quantitative proteomics, was used to analyze a collection of 26 preneoplastic and neoplastic rat mesothelial cell lines and models of MM with increasing invasiveness. Secondly, proteomic and histological analyses were conducted on formalin-fixed paraffin-embedded sections of liver metastases vs. primary tumor, and spleen from tumor-bearing rats vs. controls in the most invasive MM model. We found that S100A4, along with 12 other biomarkers, differentiated neoplastic from preneoplastic mesothelial cell lines, and invasive vs. non-invasive tumor cells in vitro, and MM tumors in vivo. Additionally, S100A4 was the only protein differentiating preneoplastic mesothelial cell lines with sarcomatoid vs. epithelioid morphology in relation to EMT (epithelial-to-mesenchymal transition). Finally, S100A4 was the most significantly increased biomarker in liver metastases vs. primary tumor, and in the spleen colonized by MM cells. Overall, we showed that S100A4 was the only protein that showed increased abundance in all situations, highlighting its crucial role in all stages of MM pathogenesis.

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.

Vimentin prevents a miR-dependent negative regulation of tissue factor mRNA during epithelial-mesenchymal transitions and facilitates early metastasis.

Epithelial-mesenchymal transitions (EMTs) are high-profile in the field of circulating tumor cells (CTCs). EMT-shifted CTCs are considered to encompass pre-metastatic subpopulations though underlying molecular mechanisms remain elusive. Our previous work identified tissue factor (TF) as an EMT-induced gene providing tumor cells with coagulant properties and supporting metastatic colonization by CTCs. We here report that vimentin, the type III intermediate filament considered a canonical EMT marker, contributes to TF regulation and positively supports coagulant properties and early metastasis. Different evidence further pointed to a new post-transcriptional regulatory mechanism of TF mRNA by vimentin: (1) vimentin silencing accelerated TF mRNA decay after actinomycin D treatment, reflecting TF mRNA stabilization, (2) RNA immunoprecipitation revealed enriched levels of TF mRNA in vimentin immunoprecipitate, (3) TF 3'-UTR-luciferase reporter vector assays implicated the 3'-UTR of TF mRNA in vimentin-dependent TF regulation, and (4) using different TF 3'UTR-luciferase reporter vectors mutated for potential miR binding sites and specific Target Site Blockers identified a key miR binding site in vimentin-dependent TF mRNA regulation. All together, these data support a novel mechanism by which vimentin interferes with a miR-dependent negative regulation of TF mRNA, thereby promoting coagulant activity and early metastasis of vimentin-expressing CTCs.

Airway epithelial cell differentiation relies on deficient Hedgehog signalling in COPD.

BACKGROUND: Hedgehog (HH) pathway is constantly under scrutiny in the context of organ development. Lung morphogenesis requires HH signalling which participates thereafter to the pulmonary homeostasis by regulating epithelial cell quiescence and repair. Since epithelial remodelling is a hallmark of Chronic Obstructive Pulmonary Disease (COPD), we investigated whether the main molecular actors of HH pathway participate to airway epithelial cell differentiation and we analysed their alterations in COPD patients. METHODS: Sonic HH (Shh) secretion was assessed by ELISA in airway epithelial cell (AEC) air-liquid interface culture supernatants. HH pathway activation was evaluated by RT-qPCR, western blot and immunostaining. Inhibition of HH signalling was achieved upon Shh chelation during epithelial cell differentiation. HH pathway core components localization was investigated in lung tissues from non-COPD and COPD patients. FINDINGS: We demonstrate that progenitors of AEC produced Shh responsible for the activation of HH signalling during the process of differentiation. Preventing the ligand-induced HH activation led to the establishment of a remodelled epithelium with increased number of basal cells and reduced ciliogenesis. Gli2 activating transcription factor was demonstrated as a key-element in the regulation of AEC differentiation. More importantly, Gli2 and Smo were lost in AEC from COPD patients. INTERPRETATION: Our data suggest that HH pathway is crucial for airway epithelial cell differentiation and highlight its role in COPD-associated epithelial remodelling.

Programmed Death-Ligand 1 and Vimentin: A Tandem Marker as Prognostic Factor in NSCLC.

In non-metastatic non-small-cell lung cancer (NSCLC), outcomes remain poor. Adjuvant chemotherapies provide a limited improvement in disease-free survival. Recent exploratory studies on early-stage NSCLC show that immunotherapy given according to Programmed Death-Ligand 1 expression generates variable results, emphasizing a need to improve tumor characterization. We aimed to conjointly assess NSCLC, the expression of PD-L1, and epithelial-mesenchymal transition, frequently involved in tumor aggressiveness. 188 resected NSCLCs were analyzed. Among 188 patients with curatively resected NSCLC, 127 adenocarcinomas and 61 squamous cell carcinomas were stained for PD-L1 and vimentin expression. Overall survival has been compared regarding PD-L1 and vimentin statuses both separately and conjointly in Tumor Cancer Genome Atlas databases. PD-L1 and vimentin higher expressions were strongly associated (OR = 4.682, p < 0.0001). This co-expression occurred preferentially in tumors with lymph node invasion (p = 0.033). PD-L1 was significantly associated with high EMT features. NSCLC harboring both PD-L1high/vimentinhigh expressions were significantly associated with poor overall survival (p = 0.019). A higher co-expression of vimentin and PD-L1 was able to identify patients with worse outcomes. Similar to an important prognostic marker in NSCLC, this tandem marker needs to be further presented to anti-PD-L1 immunotherapies to improve outcome.

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.

New insights into spectral histopathology: infrared-based scoring of tumour aggressiveness of squamous cell lung carcinomas.

Spectral histopathology, based on infrared interrogation of tissue sections, proved a promising tool for helping pathologists in characterizing histological structures in a quantitative and automatic manner. In cancer diagnosis, the use of chemometric methods permits establishing numerical models able to detect cancer cells and to characterize their tissular environment. In this study, we focused on exploiting multivariate infrared data to score the tumour aggressiveness in preneoplastic lesions and squamous cell lung carcinomas. These lesions present a wide range of aggressive phenotypes; it is also possible to encounter cases with various degrees of aggressiveness within the same lesion. Implementing an infrared-based approach for a more precise histological determination of the tumour aggressiveness should arouse interest among pathologists with direct benefits for patient care. In this study, the methodology was developed from a set of samples including all degrees of tumour aggressiveness and by constructing a chain of data processing steps for an automated analysis of tissues currently manipulated in routine histopathology.

Evaluation of intracavitary administration of curcumin for the treatment of sarcomatoid mesothelioma.

A rat model of sarcomatoid mesothelioma, mimicking some of the worst clinical conditions encountered, was established to evaluate the therapeutic potential of intracavitary curcumin administration. The M5-T1 cell line, selected from a collection established from F344 rats induced with asbestos, produces tumors within three weeks, with extended metastasis in normal tissues, after intraperitoneal inoculation in syngeneic rats. The optimal concentration/time conditions for killing M5-T1 cells with curcumin were first determined in vitro. Secondly, the potential of intraperitoneal curcumin administration to kill tumor cells in vivo was evaluated in tumor-bearing rats, in comparison with a reference epigenetic drug, SAHA. Both agents administered at days 21 and 26 after tumor challenge produced necrosis within the solid tumors at day 28. However, tumor tissue necrosis induced with curcumin was much more extensive than with SAHA, and was characterized by infiltration with mononuclear phagocytic cells. In contrast, tumor tissue treated with SAHA contained foci of resistant cells and was infiltrated by many isolated CD8+ cells. The treatment of tumor-bearing rats with 1.5 mg/kg curcumin on days 7, 9, 11 and 14 after tumor challenge dramatically reduced the mean total tumor mass at day 16. Clusters of CD8+ T lymphocytes were observed at the periphery of small residual tumor masses in the peritoneal cavity, which presented a significant reduction in mitotic index, IL6 and vimentin expression compared with tumors in untreated rats. These data open up interesting new prospects for the therapy of sarcomatoid mesothelioma with curcumin and its derivatives.

Nuclear protein in testis carcinoma of the mediastinum: a case report.

BACKGROUND: Nuclear protein in testis carcinoma is a rare and very aggressive undifferentiated cancer which characteristically arises in the midline of the head, neck, and mediastinum. CASE PRESENTATION: We describe the case of a 46-year-old white woman admitted for superior vena cava syndrome revealing a mediastinal tumor. Pathological examination of specimens obtained by mediastinoscopy revealed an undifferentiated tumor with solid growth and positive immunoreactivity for p40 and negative immunoreactivity for cytokeratin markers. Immunohistochemical staining was positive for nuclear protein in testis, allowing the diagnosis of nuclear protein in testis midline carcinoma of the mediastinum. CONCLUSIONS: We present a rare case of mediastinal nuclear protein in testis carcinoma with diagnosis based on nuclear protein in testis protein positivity and atypical immunohistochemical features including p40 positivity and anti-cytokeratin negativity. Physicians must remain aware of the possibility of nuclear protein in testis carcinoma especially in young patients with thoracic symptoms and suspicion of neoplasm.

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.

Oropharyngeal cancer prognosis by tumour HPV status in France: The multicentric Papillophar study.

AIMS: To evaluate the impact of human papillomavirus (HPV) status, tobacco smoking and initial treatment approach on progression-free survival (PFS) and overall survival (OS) for oropharyngeal cancer (OPC) in France, a country where smoking declines started late (1990s). METHODS: 340 OPC patients (median age: 60years) from 14 French hospitals were followed up (median 26.7months). PCR-based positivity for both HPV DNA and E6/E7 mRNA was used to distinguish HPV-positive OPC (27.1%). Hospital-stratified hazard ratios (HR) and corresponding 95% confidence intervals (CI) were used to compare PFS and OS according to HPV and other prognostic factors in hospital-stratified unadjusted and multivariate models. The combined effect of HPV status with either smoking, stage, or initial treatment on PFS was also evaluated. RESULTS: PFS in multivariate analysis was better in HPV-positive patients (HR=0.42; 95% CI: 0.24-0.73) and worse in older patients (HR for 5-year age increase=1.12) and those having had firstly radiotherapy (HR=1.86; 95% CI: 1.19-2.92) or induction chemotherapy (HR=1.73; 95% CI: 1.08-2.79) instead of upfront surgery. Findings for OS were similar. Loco-regional recurrences were less frequent in HPV-positive (10.5%) than HPV-negative patients (26.0%) but distant recurrences were similarly frequent. HPV status did not modify the influence of smoking or stage on PFS but the impossibility to perform upfront surgery may be more relevant for HPV-negative patients. CONCLUSIONS: HPV-positive OPC patients fare better than HPV-negative OPC and may benefit from toxicity-sparing. Whether HPV-negative patients responded less well to radiation and chemotherapy because of more severe genomic damage or bulkier tumours is unclear.

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.

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.

Raman-based detection of hydroxyethyl starch in kidney allograft biopsies as a potential marker of allograft quality in kidney transplant recipients.

In brain-dead donor resuscitation, hydroxyethyl starch (HES) use has been associated with presence of osmotic-nephrosis-like lesions in kidney transplant recipients. Our aim was to determine whether the presence of HES in protocol renal graft biopsies at three months (M3) after transplantation is associated with renal graft quality. According to the HES administered to the donor during the procurement procedure, two groups of patients were defined according graft exposition to HES: HES group, (N = 20) and control group (N = 6). Detection and relative quantification of HES was performed by Raman spectroscopy microimaging on M3 protocol renal graft biopsies. Statistical analyses were used to investigate the association between Raman data and graft characteristics. HES spectral signal was revealed negative in the control group, whereas it was positive in 40% of biopsies from the HES group. In the HES group, a stronger HES signal was associated with a lower risk of graft failure measured by the Kidney Donor Risk Index (KDRI) and was correlated with the allograft kidney function. Thus, HES accumulation in donor kidney, as probed by Raman biophotonic technique, is correlated with the quality of donor kidney and consequently the graft renal function and graft survival.