Nintedanib selectively inhibits the activation and tumour-promoting effects of fibroblasts from lung adenocarcinoma patients

Background: Nintedanib is a clinically approved multikinase receptor inhibitor to treat non-small cell lung cancer with adenocarcinoma (ADC) histology in combination with docetaxel, based on the clinical benefits reported on ADC but not on squamous cell carcinoma (SCC), which are the two most common histologic lung cancer subtypes.Methods: We examined the potential role of tumour-associated fibroblasts (TAFs) in the differential effects of nintedanib in ADC and SCC. Because TAFs are largely quiescent and activated in histologic sections, we focused on the antifibrotic effects of nintedanib on TAFs stimulated with the potent fibroblast activator TGF-beta 1, which is upregulated in lung cancer.Results: Nintedanib dose-dependently inhibited the TGF-beta 1-induced expression of a panel of pro-fibrotic activation markers in both ADC-TAFs and control fibroblasts derived from uninvolved lung parenchyma, whereas such inhibition was very modest in SCC-TAFs. Remarkably, nintedanib abrogated the stimulation of growth and invasion in a panel of carcinoma cell lines induced by secreted factors from activated TAFs in ADC but not SCC, thereby supporting that TGF-beta signalling and aberrant TAF-carcinoma cross-talk is regulated by different mechanisms in ADC and SCC.Conclusions: These results reveal that nintedanib is an effective inhibitor of fibrosis and its associated tumour-promoting effects in ADC, and that the poor antifibrotic response of SCC-TAFs to nintedanib may contribute to the differential clinical benefit observed in both subtypes. Our findings also support that preclinical models based on carcinoma-TAF interactions may help defining the mechanisms of the poor antifibrotic response of SCC-TAFs to nintedanib and testing new combined therapies to further expand the therapeutic effects of this drug in solid tumours

Dysregulated collagen homeostasis by matrix stiffening and TGF-β1 in fibroblasts from idiopathic pulmonary fibrosis patients: role of FAK/Akt

Idiopathic pulmonary fibrosis (IPF) is an aggressive disease in which normal lung parenchyma is replaced by a stiff dysfunctional scar rich in activated fibroblasts and collagen-I. We examined how the mechanochemical pro-fibrotic microenvironment provided by matrix stiffening and TGF-β1 cooperates in the transcriptional control of collagen homeostasis in normal and fibrotic conditions. For this purpose we cultured fibroblasts from IPF patients or control donors on hydrogels with tunable elasticity, including 3D collagen-I gels and 2D polyacrylamide (PAA) gels. We found that TGF-β1 consistently increased COL1A1 while decreasing MMP1 mRNA levels in hydrogels exhibiting pre-fibrotic or fibrotic-like rigidities concomitantly with an enhanced activation of the FAK/Akt pathway, whereas FAK depletion was sufficient to abrogate these effects. We also demonstrate a synergy between matrix stiffening and TGF-β1 that was positive for COL1A1 and negative for MMP1. Remarkably, the COL1A1 expression upregulation elicited by TGF-β1 alone or synergistically with matrix stiffening were higher in IPF-fibroblasts compared to control fibroblasts in association with larger FAK and Akt activities in the former cells. These findings provide new insights on how matrix stiffening and TGF-β1 cooperate to elicit excessive collagen-I deposition in IPF, and support a major role of the FAK/Akt pathway in this cooperation

Interleukin-1β Modulation of the Mechanobiology of Primary Human Pulmonary Fibroblasts: Potential Implications in Lung Repair

Pro-inflammatory cytokines like interleukin-1β (IL-1β) are upregulated during early responses to tissue damage and are expected to transiently compromise the mechanical microenvironment. Fibroblasts are key regulators of tissue mechanics in the lungs and other organs. However, the effects of IL-1β on fibroblast mechanics and functions remain unclear. Here we treated human pulmonary fibroblasts from control donors with IL-1β and used Atomic Force Microscopy to unveil that IL-1β significantly reduces the stiffness of fibroblasts concomitantly with a downregulation of filamentous actin (F-actin) and alpha-smooth muscle (α-SMA). Likewise, COL1A1 mRNA was reduced, whereas that of collagenases MMP1 and MMP2 were upregulated, favoring a reduction of type-I collagen. These mechanobiology changes were functionally associated with reduced proliferation and enhanced migration upon IL-1β stimulation, which could facilitate lung repair by drawing fibroblasts to sites of tissue damage. Our observations reveal that IL-1β may reduce local tissue rigidity by acting both intracellularly and extracellularly through the downregulation of fibroblast contractility and type I collagen deposition, respectively. These IL-1β-dependent mechanical effects may enhance lung repair further by locally increasing pulmonary tissue compliance to preserve normal lung distension and function. Moreover, our results support that IL-1β provides innate anti-fibrotic protection that may be relevant during the early stages of lung repair

Lung Myofibroblasts Are Characterized by DownRegulated Cyclooxygenase-2 and Its Main Metabolite, Prostaglandin E2

Background: Prostaglandin E2 (PGE(2)), the main metabolite of cyclooxygenase (COX), is a well-known anti-fibrotic agent. Moreover, myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA), fibroblast expansion and epithelial-mesenchymal transition (EMT) are critical to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our aim was to investigate the expression of COX-2 and PGE(2) in human lung myofibroblasts and establish whether fibroblast-myofibroblast transition (FMT) and EMT are associated with COX-2 and PGE(2) down-regulation. Methods: Fibroblasts obtained from IPF patients (n = 6) and patients undergoing spontaneous pneumothorax (control, n = 6) and alveolar epithelial cell line A549 were incubated with TGF-beta 1 and FMT and EMT markers were evaluated. COX-2 and alpha-SMA expression, PGE(2) secretion and cell proliferation were measured after IL-1 beta and PGE(2) incubation. Results: Myofibroblasts from both control and IPF fibroblast cultures stimulated with IL-1 beta showed no COX-2 expression. IPF fibroblasts showed increased myofibroblast population and reduced COX-2 expression in response to IL-1 beta. TGF-beta 1 increased the number of myofibroblasts in a time-dependent manner. In contrast, TGF-beta 1 induced slight COX-2 expression at 4 h (without increase in myofibroblasts) and 24 h, but not at 72 h. Both IPF and control cultures incubated with TGF-beta 1 for 72 h showed diminished COX-2 induction, PGE(2) secretion and alpha-SMA expression after IL-1 beta addition. The latter decreased proliferation in fibroblasts but not in myofibroblasts. A549 cells incubated with TGF-beta 1 for 72 h showed down-regulated COX-2 expression and low basal PGE(2) secretion in response to IL-1 beta. Immuno-histochemical analysis of IPF lung tissue showed no COX-2 immuno-reactivity in myofibroblast foci. Conclusions: Myofibroblasts are associated with COX-2 down-regulation and reduced PGE(2) production, which could be crucial in IPF development and progression

Epithelial contribution to the profibrotic stiff microenvironment and myofibroblast population in lung fibrosis

The contribution of epithelial-to-mesenchymal transition (EMT) to the profibrotic stiff microenvironment and myofibroblast accumulation in pulmonary fibrosis remains unclear. We examined EMT-competent lung epithelial cells and lung fibroblasts from control (fibrosis-free) donors or patients with idiopathic pulmonary fibrosis (IPF), which is a very aggressive fibrotic disorder. Cells were cultured on profibrotic conditions including stiff substrata and TGF-beta 1, and analyzed in terms of morphology, stiffness, and expression of EMT/myofibroblast markers and fibrillar collagens. All fibroblasts acquired a robust myofibroblast phenotype on TGF-beta 1 stimulation. Yet IPF myofibroblasts exhibited higher stiffness and expression of fibrillar collagens than control fibroblasts, concomitantly with enhanced FAK(Y397) activity. FAK inhibition was sufficient to decrease fibroblast stiffness and collagen expression, supporting that FAK(Y397) hyperactivation may underlie the aberrant mechanobiology of IPF fibroblasts. In contrast, cells undergoing EMT failed to reach the values exhibited by IPF myofibroblasts in all parameters examined. Likewise, EMT could be distinguished from nonactivated control fibroblasts, suggesting that EMT does not elicit myofibroblast precursors either. Our data suggest that EMT does not contribute directly to the myofibroblast population, and may contribute to the stiff fibrotic microenvironment through their own stiffness but not their collagen expression. Our results also support that targeting FAK(Y397) may rescue normal mechanobiology in IPF

Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung

Senescence in cancer cells acts as a tumor suppressor, whereas in fibroblasts enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of cancer subtypes. However, the presence of senescent TAFs in lung cancer remains undefined. We examined senescence in TAFs from primary lung cancer and paired control fibroblasts from unaffected tissue in three major histologic subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Three independent senescence markers (senescence-associated beta-galactosidase, permanent growth arrest and spreading) were consistently observed in cultured LCC-TAFs only, revealing a selective premature senescence. Intriguingly, SCC-TAFs exhibited a poor growth response in the absence of senescence markers, indicating a dysfunctional phenotype rather than senescence. Co-culturing normal fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to render fibroblasts senescent through oxidative stress, indicating that senescence in LCC-TAFs is driven by heterotypic signaling. In addition, senescent fibroblasts provided selective growth and invasive advantages to LCC cells in culture compared to normal fibroblasts. Likewise, senescent fibroblasts enhanced tumor growth and lung dissemination of tumor cells when co-injected with LCC cells in nude mice beyond the effects induced by control fibroblasts. These results define the subtype-specific aberrant phenotypes of lung TAFs, thereby challenging the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless of their subtype. Importantly, because LCC often distinguishes itself in the clinic by its aggressive nature, we argue that senescent TAFs may contribute to the selective aggressive behavior of LCC tumors

Aberrant DNA methylation in non-small cell lung cancer-associated fibroblasts

Epigenetic changes through altered DNA methylation have been implicated in critical aspects of tumor progression, and have been extensively studied in a variety of cancer types. In contrast, our current knowledge of the aberrant genomic DNA methylation in tumor-associated fibroblasts (TAFs) or other stromal cells that act as critical coconspirators of tumor progression is very scarce. To address this gap of knowledge, we conducted genome-wide DNA methylation profiling on lung TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer patients using the HumanMethylation450 microarray. We found widespread DNA hypomethylation concomitant with focal gain of DNA methylation in TAFs compared to CFs. The aberrant DNA methylation landscape of TAFs had a global impact on gene expression and a selective impact on the TGF-β pathway. The latter included promoter hypermethylation-associated SMAD3 silencing, which was associated with hyperresponsiveness to exogenous TGF-β1 in terms of contractility and extracellular matrix deposition. In turn, activation of CFs with exogenous TGF-β1 partially mimicked the epigenetic alterations observed in TAFs, suggesting that TGF-β1 may be necessary but not sufficient to elicit such alterations. Moreover, integrated pathway-enrichment analyses of the DNA methylation alterations revealed that a fraction of TAFs may be bone marrow-derived fibrocytes. Finally, survival analyses using DNA methylation and gene expression datasets identified aberrant DNA methylation on the EDARADD promoter sequence as a prognostic factor in non-small cell lung cancer patients. Our findings shed light on the unique origin and molecular alterations underlying the aberrant phenotype of lung TAFs, and identify a stromal biomarker with potential clinical relevance

Estudi del fenotip miofibroblast i la seva implicació en l'expressió de ciclooxigenasa-2 i en la secreció de prostaglandina E2 en la fibrosi pulmonar idiopàtica

[cat] Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with bad prognosis and mean survival from 2 to 5 years from the diagnosis. The most accepted hypothesis of its cause is that a repeated epithelial lung damage would cause epithelial cells apoptosis and the release of pro-fibrotic factors, like TGF-b1 and the diminishing of anti-fibrotic factors, like prostaglandin E2 (PGE2). In this profibrotic environment fibroblast foci are formed by fibroblasts, myofibroblasts and extracellular matrix deposits. PGE2 is synthesized via cyclooxygenase enzimes, COX-1 and COX-2. COX-2 is inducible by several pro-inflammatory stimuli, for example IL-1b. We obtained an enriched myofibroblast culture treating primary fibroblasts obtained from control and IPF lung tissue with TGF-b1 during 3 days. This treatment induced an upregulation of aSMA expression, a myofibroblast associated protein, and collagen synthesis. It also induced epithelial to mesenchymal transition (EMT) in A549 epithelial lung cell line. Myofibroblasts obtained from FPI cultures showed increased migratory capacity in comparison with control ones. Proliferation associated with this phenotype was very low. We also measured the closure of an in vitro wound by these cell types and we observed that fibroblasts are actually the most important phenotype associated to wound healing, and not myofibroblasts. Moreover, we analized aSMA and COX-2 expression and PGE2 secretion in those cells. IPF cultures showed increased aSMA levels but decreased COX-2 induced expression by IL-1b. Myofibroblast enriched cultures also showed these features, also associated to a suppressed PGE2 secretion. Both in cell cultures and in tissue inmunohisochemistry no coincidence was observed between aSMA and COX-2 positive cells, associating myofibroblasts to an altered COX-2 and PGE2 metabolism. A549-derived myofibroblasts shared the ssame alterations. We analyzed IL-1 specific receptor IL-1RI in order to stablish if it was an stimuli-dependent alteration, and we observed that both IPF and myofibroblasts showed diminished IL-1R expression. IL-1b failed to upregulate its own receptor expression specifically in myofibroblasts. In the profibrotic environment, miofibroblast transition is chronically induced by TGF-b. Moreover this phenotype is associated to a downregulated IL-1RI expression and therefore IL-1b is uncapable of inducing COX-2 expression and PGE2 secretion, worsening migratory and proliferative features of these cells and contributing to fibrotic progression.[eng] La fibrosi pulmonar idiopàtica (FPI) és una pneumònia intersticial de causa desconeguda i de mal pronòstic, amb una supervivència mitjana de 2 a 5 anys des del moment del diagnòstic. La hipòtesi fisiopatològica més acceptada en l’actualitat és que una lesió reiterada a l’epiteli alveolar causaria l’apoptosi massiva de les cèl·lules epitelials, l’alliberament de factors solubles pro-fibròtics com el TGF-β1 i el descens de la producció de factors anti-fibròtics com la prostaglandina E2 (PGE2). En aquestes circumstàncies s’induiria la formació de focus fibroblàstics, formats per fibroblastes, miofibroblastes i matriu extracel·lular excessiva. La PGE2 prové de la via de l’àcid araquidònic a través dels enzims ciclooxigenasa (COX). La COX-2 és induïble per estímuls pro-inflamatoris com la interleuquina-1b (IL-1b). En primer lloc, vam obtenir un cultiu enriquit de miofibroblastes in vitro tractant fibroblastes primaris de pulmó humà control i amb FPI amb TGF-b1 durant 72h. En aquestes condicions es va induir la transició fibroblast a miofibroblast i també la transició epiteli-mesenquimal (EMT) en cèl·lules de la línia A549. Els cultius de miofibroblastes van presentar un increment en l’expressió d’alfa actina del múscul llis (α-SMA), proteïna característica d’aquest fenotip, i en la síntesi de col·lagen. Analitzant la capacitat de migració i proliferació d’aquestes cèl·lules realitzant una ferida al cultiu in vitro, vam observar que els cultius de miofibroblastes procedents de pacients amb FPI presentaven una capacitat invasiva superior als controls. Aquesta no era deguda a la proliferació, ja que la seva inhibició no comportava diferències. La proliferació dels miofibroblastes era baixa tant en cultius control com en FPI. Vam avaluar la capacitat de tancament d’una ferida in vitro per par d’aquestes cèl·lules i vam observar que els fibroblastes són el fenotip principal encarregat del tancament d’una ferida in vitro i que la proliferació és una component important en aquest tancament. Vam analitzar l’expressió de α-SMA, COX-2 i secreció de PGE2 en cultius control i FPI i en cultius enriquits de miofibroblastes. Els cultius FPI van presentar una major expressió d’α-SMA respecte els controls i alhora una menor capacitat d’inducció de l’expressió de COX-2 per part d’IL-1β. En els cultius enriquits de miofibroblastes, aquest efecte va ser encara més marcat, fins al punt de no haver-hi coincidència entre l’expressió de COX-2 i la d’α-SMA. La deficiència en la inducció de COX-2 va implicar una disminució de l’expressió de PGE2. Tot plegat va afectar a la capacitat de migració i proliferació d’aquestes cèl·lules. Els mateixos resultats es van observar en el fenotip tipus-miofibroblast procedent de la EMT de les cèl·lules epitelials A549. En tincions immunohistoquímiques de teixit normal i de FPI no vam observar coincidència entre el marcatge de COX-2 i d’α-SMA en els focus fibroblàstics. Així doncs, els resultats observats in vitro es corroborarien in vivo. Finalment, vam analitzar l’expressió del receptor d’interleuquina-1 tipus 1 (IL-R1) com a possible causa de la deficient inducció de COX-2 en aquestes cèl·lules. IL-1RI és el receptor específic pel qual la IL-1β realitza les seves accions pro-inflamatòries. Els fibroblastes FPI van presentar una menor expressió basal de IL-1RI en comparació amb els control. La IL-1β va ser capaç d’induir l’expressió del seu propi receptor. En miofibroblastes en canvi, el TGF-β va inhibir-ne l’expressió i va eliminar la capacitat d’inducció per part de la IL-1β. Així doncs, en un ambient pro-fibròtic, el TGF-β induiria de manera permanent la transició de fibroblastes i cèl·lules epitelials del pulmó cap a miofibroblastes. El fenotip miofibroblast estaria associat a una deficiència del receptor IL-1RI que impediria a la IL-1β induir l’expressió de COX-2 i la secreció de PGE2. La baixa PGE2 afavoriria la migració i proliferació d’aquestes cèl·lules i per tant la progressió de la fibrosi pulmonar. Aquest pot ser un mecanisme destacat en el manteniment de la FPI i presenta una possible diana terapèutica per aturar i arribar a reverir el procés fibròtic

Nintedanib selectively inhibits the activation and tumour-promoting effects of fibroblasts from lung adenocarcinoma patients

Background: Nintedanib is a clinically approved multikinase receptor inhibitor to treat non-small cell lung cancer with adenocarcinoma (ADC) histology in combination with docetaxel, based on the clinical benefits reported on ADC but not on squamous cell carcinoma (SCC), which are the two most common histologic lung cancer subtypes.Methods: We examined the potential role of tumour-associated fibroblasts (TAFs) in the differential effects of nintedanib in ADC and SCC. Because TAFs are largely quiescent and activated in histologic sections, we focused on the antifibrotic effects of nintedanib on TAFs stimulated with the potent fibroblast activator TGF-beta 1, which is upregulated in lung cancer.Results: Nintedanib dose-dependently inhibited the TGF-beta 1-induced expression of a panel of pro-fibrotic activation markers in both ADC-TAFs and control fibroblasts derived from uninvolved lung parenchyma, whereas such inhibition was very modest in SCC-TAFs. Remarkably, nintedanib abrogated the stimulation of growth and invasion in a panel of carcinoma cell lines induced by secreted factors from activated TAFs in ADC but not SCC, thereby supporting that TGF-beta signalling and aberrant TAF-carcinoma cross-talk is regulated by different mechanisms in ADC and SCC.Conclusions: These results reveal that nintedanib is an effective inhibitor of fibrosis and its associated tumour-promoting effects in ADC, and that the poor antifibrotic response of SCC-TAFs to nintedanib may contribute to the differential clinical benefit observed in both subtypes. Our findings also support that preclinical models based on carcinoma-TAF interactions may help defining the mechanisms of the poor antifibrotic response of SCC-TAFs to nintedanib and testing new combined therapies to further expand the therapeutic effects of this drug in solid tumours

Dysregulated collagen homeostasis by matrix stiffening and TGF-β1 in fibroblasts from idiopathic pulmonary fibrosis patients: role of FAK/Akt

Idiopathic pulmonary fibrosis (IPF) is an aggressive disease in which normal lung parenchyma is replaced by a stiff dysfunctional scar rich in activated fibroblasts and collagen-I. We examined how the mechanochemical pro-fibrotic microenvironment provided by matrix stiffening and TGF-β1 cooperates in the transcriptional control of collagen homeostasis in normal and fibrotic conditions. For this purpose we cultured fibroblasts from IPF patients or control donors on hydrogels with tunable elasticity, including 3D collagen-I gels and 2D polyacrylamide (PAA) gels. We found that TGF-β1 consistently increased COL1A1 while decreasing MMP1 mRNA levels in hydrogels exhibiting pre-fibrotic or fibrotic-like rigidities concomitantly with an enhanced activation of the FAK/Akt pathway, whereas FAK depletion was sufficient to abrogate these effects. We also demonstrate a synergy between matrix stiffening and TGF-β1 that was positive for COL1A1 and negative for MMP1. Remarkably, the COL1A1 expression upregulation elicited by TGF-β1 alone or synergistically with matrix stiffening were higher in IPF-fibroblasts compared to control fibroblasts in association with larger FAK and Akt activities in the former cells. These findings provide new insights on how matrix stiffening and TGF-β1 cooperate to elicit excessive collagen-I deposition in IPF, and support a major role of the FAK/Akt pathway in this cooperation