New roles for corticosteroid binding globulin and opposite expression profiles in lung and liver.

Corticosteroid-binding globulin (CBG) is the specific plasma transport glycoprotein for glu- cocorticoids. Circulating CBG is mainly synthesized in liver but, its synthesis has been located also in other organs as placenta, kidney and adipose tissue with unknown role. Using an experimental model of acute pancreatitis in cbg -/- mice we investigated whether changes in CBG affect the progression of the disease as well as the metabolism of gluco- corticoids in the lung. Lack of CBG does not modify the progression of inflammation associ- ated to pancreatitis but resulted in the loss of gender differences in corticosterone serum levels. In the lung, CBG expression and protein level were detected, and it is noteworthy that these showed a sexual dimorphism opposite to the liver, i.e. with higher levels in males. Reduced expression of 11 β -HSD2, the enzyme involved in the deactivation of corticoste- rone, was also observed. Our results indicate that, in addition to glucocorticoids transporter, CBG is involved in the gender differences observed in corticosteroids circulating levels and plays a role in the local regulation of corticosteroids availability in organs like lung

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air-liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and 'benchmarked' against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017

Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis:a randomised, double-blind, placebo-controlled, phase 2b trial

Background: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. Methods: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50–90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene–liposome complex or 0·9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. Findings: Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1–7·3; p=0·046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. Interpretation: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials

Caracterització dels tipus d'activació de diferents poblacions de macròfags durant la pancreatitis aguda experimental i la seva relació amb la severitat del procés

Malgrat els esforços dedicats a investigar la pancreatitis aguda (PA), segueixen sense resposta la major part de les preguntes plantejades fa anys. En particular, encara no es coneixen tots els mecanismes pels que una lesió localitzada en el pàncrees pot induir una afecció en òrgans distants, com el pulmó. Durant els últims anys s’ha pogut establir que l’activació de les cèl•lules inflamatòries juga un paper central en el desenvolupament d’aquesta malaltia. Per una altra banda, els macròfags poden presentar diferents fenotips en funció del tipus d’activació a la que es veuen sotmesos, destacant l’activació clàssica (induïda per INFγ) i l’alternativa (induïda per citocines com IL4, IL13, IL10, glucocorticoids..). Aquests tipus d’activació podrien condicionar la progressió de la pancreatitis cap a formes lleus o severes. En aquesta tesi s’ha avaluat el tipus d’activació que presenten diferents poblacions de macròfags i la seva relació amb la severitat del procés (lleu, moderada o severa). El treball s’ha realitzat a nivell experimental in vivo, utilitzant un model de PA per infusió de sals biliars. També s’ha analitzat la resposta de les diferents poblacions de macròfags als estímuls generats durant la pancreatitis i s’ha treballat sobre aquests macròfags amb la finalitat de modificar la via d’activació que presenten i ,amb això, modular el procés inflamatori sistèmic associat a la PA. La hipòtesi de partida és que el tipus d’activació de les diferents poblacions de macròfags implicats en la pancreatitis aguda condiciona l’evolució i la severitat de la malaltia. Actuant sobre el tipus d’activació de diferents poblacions de macròfags es podria modificar el curs de la malaltia de severa a lleu. Així doncs els objectius concrets d’aquest treball serien: 1. Obtenir i caracteritzar el tipus d’activació presentat per els macròfags peritoneals i pulmonars durant el desenvolupament de la pancreatitis aguda. Correlacionar el tipus d’activació de les diferents poblacions de macròfags amb el grau de severitat de la patologia. 2. Avaluar el paper de les diferents vies de senyalització intracel•lular implicades en el fenotip dels macròfags. 3. Determinar els possibles mediadors implicats en l’activació dels macròfags durant la pancreatitis aguda

New roles for corticosteroid binding globulin and opposite expression profiles in lung and liver.

Corticosteroid-binding globulin (CBG) is the specific plasma transport glycoprotein for glu- cocorticoids. Circulating CBG is mainly synthesized in liver but, its synthesis has been located also in other organs as placenta, kidney and adipose tissue with unknown role. Using an experimental model of acute pancreatitis in cbg -/- mice we investigated whether changes in CBG affect the progression of the disease as well as the metabolism of gluco- corticoids in the lung. Lack of CBG does not modify the progression of inflammation associ- ated to pancreatitis but resulted in the loss of gender differences in corticosterone serum levels. In the lung, CBG expression and protein level were detected, and it is noteworthy that these showed a sexual dimorphism opposite to the liver, i.e. with higher levels in males. Reduced expression of 11 β -HSD2, the enzyme involved in the deactivation of corticoste- rone, was also observed. Our results indicate that, in addition to glucocorticoids transporter, CBG is involved in the gender differences observed in corticosteroids circulating levels and plays a role in the local regulation of corticosteroids availability in organs like lung

Aberrant TIMP-1 overexpression in tumor-associated fibroblasts drives tumor progression through CD63 in lung adenocarcinoma

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important regulator of extracellular matrix turnover that has been traditionally regarded as a potential tumor suppressor owing to its inhibitory effects of matrix metalloproteinases. Intriguingly, this interpretation has been challenged by the consistent observation that increased expression of TIMP-1 is associated with poor prognosis in virtually all cancer types including lung cancer, supporting a tumor-promoting function. However, how TIMP-1 is dysregulated within the tumor microenvironment and how it drives tumor progression in lung cancer is poorly understood. We analyzed the expression of TIMP-1 and its cell surface receptor CD63 in two major lung cancer subtypes: lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), and defined the tumor-promoting effects of their interaction. We found that TIMP-1 is aberrantly overexpressed in tumor-associated fibroblasts (TAFs) in ADC compared to SCC. Mechanistically, TIMP-1 overexpression was mediated by the selective hyperactivity of the pro-fibrotic TGF-β1/SMAD3 pathway in ADC-TAFs. Likewise, CD63 was upregulated in ADC compared to SCC cells. Genetic analyses revealed that TIMP-1 secreted by TGF-β1-activated ADC-TAFs is both necessary and sufficient to enhance growth and invasion of ADC cancer cells in culture, and that tumor cell expression of CD63 was required for these effects. Consistently, in vivo analyses revealed that ADC cells co-injected with fibroblasts with reduced SMAD3 or TIMP-1 expression into immunocompromised mice attenuated tumor aggressiveness compared to tumors bearing parental fibroblasts. We also found that high TIMP1 and CD63 mRNA levels combined define a stronger prognostic biomarker than TIMP1 alone. Our results identify an excessive stromal TIMP-1 within the tumor microenvironment selectively in lung ADC, and implicate it in a novel tumor-promoting TAF-carcinoma crosstalk, thereby pointing to TIMP-1/CD63 interaction as a novel therapeutic target in lung cancer

Epigenetic SMAD3 Repression in Tumor-Associated Fibroblasts Impairs Fibrosis and Response to the Antifibrotic Drug Nintedanib in Lung Squamous Cell Carcinoma

The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in non– small cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs in vitro and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs was associated with increased promoter methylation of the profibrotic TGFb transcription factor SMAD3 compared with ADC-TAFs, which elicited a compensatory increase in TGFb1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFb1/SMAD3 activation, whereas genetic downregulation of SMAD3 in ADCTAFs and control fibroblasts increased TGFb1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib in vitro and in vivo. Our results also support that smoking and/or the anatomic location of SCC in the proximal airways, which are more exposed to cigarette smoke particles, may prime SCC-TAFs to stronger SMAD3 epigenetic repression, because cigarette smoke condensate selectively increased SMAD3 promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential SMAD3 promoter methylation in TAFs and provide new mechanistic insights on the selective poor response of SCCTAFs to nintedanib. Moreover, our findings support that patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFb1/SMAD3 activation. Significance: This study implicates the selective epigenetic repression of SMAD3 in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFb1/ SMAD3

Epigenetic SMAD3 Repression in Tumor-Associated Fibroblasts Impairs Fibrosis and Response to the Antifibrotic Drug Nintedanib in Lung Squamous Cell Carcinoma

The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in non– small cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs in vitro and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs was associated with increased promoter methylation of the profibrotic TGFb transcription factor SMAD3 compared with ADC-TAFs, which elicited a compensatory increase in TGFb1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFb1/SMAD3 activation, whereas genetic downregulation of SMAD3 in ADCTAFs and control fibroblasts increased TGFb1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib in vitro and in vivo. Our results also support that smoking and/or the anatomic location of SCC in the proximal airways, which are more exposed to cigarette smoke particles, may prime SCC-TAFs to stronger SMAD3 epigenetic repression, because cigarette smoke condensate selectively increased SMAD3 promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential SMAD3 promoter methylation in TAFs and provide new mechanistic insights on the selective poor response of SCCTAFs to nintedanib. Moreover, our findings support that patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFb1/SMAD3 activation. Significance: This study implicates the selective epigenetic repression of SMAD3 in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFb1/ SMAD3