RESUMO
The fibrotic tumor microenvironment is a pivotal therapeutic target. Nintedanib, a clinically approved multikinase antifibrotic inhibitor, is effective against lung adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). Previous studies have implicated the secretome of tumor-associated fibroblasts (TAFs) in the selective effects of nintedanib in ADC, but the driving factor(s) remained unidentified. Here we examined the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a tumor-promoting cytokine overproduced in ADC-TAFs. To this aim, we combined genetic approaches with in vitro and in vivo preclinical models based on patient-derived TAFs. Nintedanib reduced TIMP-1 production more efficiently in ADC-TAFs than SCC-TAFs through a SMAD3-dependent mechanism. Cell culture experiments indicated that silencing TIMP1 in ADC-TAFs abolished the therapeutic effects of nintedanib on cancer cell growth and invasion, which were otherwise enhanced by the TAF secretome. Consistently, co-injecting ADC cells with TIMP1-knockdown ADC-TAFs into immunocompromised mice elicited a less effective reduction of tumor growth and invasion under nintedanib treatment compared to tumors bearing unmodified fibroblasts. Our results unveil a key mechanism underlying the selective mode of action of nintedanib in ADC based on the excessive production of TIMP-1 in ADC-TAFs. We further pinpoint reduced SMAD3 expression and consequent limited TIMP-1 production in SCC-TAFs as key for the resistance of SCC to nintedanib. These observations strongly support the emerging role of TIMP-1 as a critical regulator of therapy response in solid tumors.
Assuntos
Adenocarcinoma de Pulmão , Fibroblastos Associados a Câncer , Indóis , Neoplasias Pulmonares , Proteína Smad3 , Inibidor Tecidual de Metaloproteinase-1 , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Humanos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Indóis/farmacologia , Indóis/uso terapêutico , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Proteína Smad3/metabolismo , Linhagem Celular Tumoral , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , FemininoRESUMO
BACKGROUND: The TGF-ß1 transcription factor SMAD3 is epigenetically repressed in tumour-associated fibroblasts (TAFs) from lung squamous cell carcinoma (SCC) but not adenocarcinoma (ADC) patients, which elicits a compensatory increase in SMAD2 that renders SCC-TAFs less fibrotic. Here we examined the effects of altered SMAD2/3 in fibroblast migration and its impact on the desmoplastic stroma formation in lung cancer. METHODS: We used a microfluidic device to examine descriptors of early protrusions and subsequent migration in 3D collagen gels upon knocking down SMAD2 or SMAD3 by shRNA in control fibroblasts and TAFs. RESULTS: High SMAD3 conditions as in shSMAD2 fibroblasts and ADC-TAFs exhibited a migratory advantage in terms of protrusions (fewer and longer) and migration (faster and more directional) selectively without TGF-ß1 along with Erk1/2 hyperactivation. This enhanced migration was abrogated by TGF-ß1 as well as low glucose medium and the MEK inhibitor Trametinib. In contrast, high SMAD2 fibroblasts were poorly responsive to TGF-ß1, high glucose and Trametinib, exhibiting impaired migration in all conditions. CONCLUSIONS: The basal migration advantage of high SMAD3 fibroblasts provides a straightforward mechanism underlying the larger accumulation of TAFs previously reported in ADC compared to SCC. Moreover, our results encourage using MEK inhibitors in ADC-TAFs but not SCC-TAFs.
Assuntos
Adenocarcinoma de Pulmão , Adenocarcinoma , Fibroblastos Associados a Câncer , Neoplasias Pulmonares , Humanos , Adenocarcinoma/patologia , Fibroblastos Associados a Câncer/metabolismo , Colágeno , Fibroblastos/metabolismo , Glucose/farmacologia , Neoplasias Pulmonares/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Fibrillar collagens are the most abundant extracellular matrix components in non-small cell lung cancer (NSCLC). However, the potential of collagen fiber descriptors as a source of clinically relevant biomarkers in NSCLC is largely unknown. Similarly, our understanding of the aberrant collagen organization and associated tumor-promoting effects is very scarce. To address these limitations, we identified a digital pathology approach that can be easily implemented in pathology units based on CT-FIRE software (version 2; https://loci.wisc.edu/software/ctfire) analysis of Picrosirius red (PSR) stains of fibrillar collagens imaged with polarized light (PL). CT-FIRE settings were pre-optimized to assess a panel of collagen fiber descriptors in PSR-PL images of tissue microarrays from surgical NSCLC patients (106 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SCC]). Using this approach, we identified straightness as the single high-accuracy diagnostic collagen fiber descriptor (average area under the curve = 0.92) and fiber density as the single descriptor consistently associated with a poor prognosis in both ADC and SCC independently of the gold standard based on the TNM staging (hazard ratio, 2.69; 95% CI, 1.55-4.66; P < .001). Moreover, we found that collagen fibers were markedly straighter, longer, and more aligned in tumor samples compared to paired samples from uninvolved pulmonary tissue, particularly in ADC, which is indicative of increased tumor stiffening. Consistently, we observed an increase in a panel of stiffness-associated processes in the high collagen fiber density patient group selectively in ADC, including venous/lymphatic invasion, fibroblast activation (α-smooth muscle actin), and immune evasion (programmed death-ligand 1). Similarly, a transcriptional correlation analysis supported the potential involvement of the major YAP/TAZ pathway in ADC. Our results provide a proof-of-principle to use CT-FIRE analysis of PSR-PL images to assess new collagen fiber-based diagnostic and prognostic biomarkers in pathology units, which may improve the clinical management of patients with surgical NSCLC. Our findings also unveil an aberrant stiff microenvironment in lung ADC that may foster immune evasion and dissemination, encouraging future work to identify therapeutic opportunities.
Assuntos
Adenocarcinoma , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Prognóstico , Colágenos Fibrilares/análise , Colágenos Fibrilares/uso terapêutico , Adenocarcinoma/patologia , Colágeno , Carcinoma de Células Escamosas/patologia , Microambiente TumoralRESUMO
BACKGROUND: Pulmonary vascular abnormalities are a characteristic feature of chronic obstructive pulmonary disease (COPD). Cigarette smoking is the most important risk factor for COPD. It is believed that its constant exposure triggers endothelial cell damage and vascular remodelling. Under pathological conditions, progenitor cells (PCs) are mobilized from the bone marrow and recruited to sites of vascular injury. The aim of the study was to investigate whether in COPD the number of circulating PCs is related to the presence of bone marrow-derived cells in pulmonary arteries and the association of these phenomena to both systemic and pulmonary endothelial dysfunction. METHODS: Thirty-nine subjects, 25 with COPD, undergoing pulmonary resection because of a localized carcinoma, were included. The number of circulating PCs was assessed by flow cytometry using a triple combination of antibodies against CD45, CD133 and CD34. Infiltrating CD45+ cells were identified by immunohistochemistry in pulmonary arteries. Endothelial function in systemic and pulmonary arteries was measured by flow-mediated dilation and adenosine diphosphate-induced vasodilation, respectively. RESULTS: COPD patients had reduced numbers of circulating PCs (p < 0.05) and increased numbers of CD45+ cells (< 0.05) in the pulmonary arterial wall than non-COPD subjects, being both findings inversely correlated (r = - 0.35, p < 0.05). In pulmonary arteries, the number of CD45+ cells correlated with the severity of vascular remodelling (r = 0.4, p = 0.01) and the endothelium-dependent vasodilation (r = - 0.3, p = 0.05). Systemic endothelial function was unrelated to the number of circulating PCs and changes in pulmonary vessels. CONCLUSION: In COPD, the decrease of circulating PCs is associated with their recruitment in pulmonary arteries, which in turn is associated with endothelial dysfunction and vessel remodelling, suggesting a mechanistic link between these phenomena. Our findings are consistent with the notion of an imbalance between endothelial damage and repair capacity in the pathogenesis of pulmonary vascular abnormalities in COPD.
Assuntos
Movimento Celular/fisiologia , Endotélio Vascular/metabolismo , Artéria Pulmonar/metabolismo , Doença Pulmonar Obstrutiva Crônica/sangue , Células-Tronco/metabolismo , Idoso , Endotélio Vascular/patologia , Feminino , Volume Expiratório Forçado/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Artéria Pulmonar/patologia , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Células-Tronco/patologiaRESUMO
BACKGROUND: c-Kit + lung stem cells have been described in the human healthy lung. Their potential relation with smoking and/or chronic obstructive pulmonary disease (COPD) is unknown. METHODS: We characterized and compared c-Kit+ cells in lung tissue of 12 never smokers (NS), 15 smokers with normal spirometry (S) and 44 COPD patients who required lung resectional surgery. Flow cytometry (FACS) was used to characterize c-Kit+ cells in fresh lung tissue disaggregates, and immunofluorescence (IF) for further characterization and to determine their location in OCT- embedded lung tissue. RESULTS: We identified 4 c-Kit+ cell populations, with similar proportions in NS, S and COPD: (1) By FACS, c-Kithigh/CD45+ cells (4.03 ± 2.97% (NS), 3.96 ± 5.30% (S), and 5.20 ± 3.44% (COPD)). By IF, these cells were tryptase+ (hence, mast cells) and located around the airways; (2) By IF, c-Kitlow/CD45+/triptase- (0.07 ± 0.06 (NS), 0.03 ± 0.02 (S), and 0.06 ± 0.07 (COPD) cells/field), which likely correspond to innate lymphoid cells; (3) By FACS, c-Kitlow/CD45-/CD34+ (0.95 ± 0.84% (NS), 1.14 ± 0.94% (S) and 0.95 ± 1.38% (COPD)). By IF these cells were c-Kitlow/CD45-/CD31+, suggesting an endothelial lineage, and were predominantly located in the alveolar wall; and, (4) by FACS, an infrequent c-Kitlow/CD45-/CD34- population (0.09 ± 0.14% (NS), 0.08 ± 0.09% (S) and 0.08 ± 0.11% (COPD)) compatible with a putative lung stem cell population. Yet, IF failed to detect them and we could not isolate or grow them, thus questioning the existence of c-Kit+ lung stem-cells. CONCLUSIONS: The adult human lung contains a mixture of c-Kit+ cells, unlikely to be lung stem cells, which are independent of smoking status and/or presence of COPD.
Assuntos
Pulmão/patologia , Proteínas Proto-Oncogênicas c-kit/genética , Doença Pulmonar Obstrutiva Crônica/genética , Fumar , Células-Tronco/citologia , Idoso , Feminino , Citometria de Fluxo , Humanos , Pulmão/citologia , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
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-ß pathway. The latter included promoter hypermethylation-associated SMAD3 silencing, which was associated with hyperresponsiveness to exogenous TGF-ß1 in terms of contractility and extracellular matrix deposition. In turn, activation of CFs with exogenous TGF-ß1 partially mimicked the epigenetic alterations observed in TAFs, suggesting that TGF-ß1 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.
Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Metilação de DNA , Fibroblastos/metabolismo , Neoplasias Pulmonares/genética , Adenocarcinoma/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Epigênese Genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Receptores de IgG/genética , Receptores de IgG/metabolismo , Análise de Sequência de DNA , Proteína Smad3/genética , Proteína Smad3/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta1/fisiologia , Células Tumorais CultivadasRESUMO
Cigarette smoke (CS) induces an inflammatory process in the lung that may underlie the development of chronic obstructive pulmonary disease (COPD). The nature and characteristics of this process have not been fully established in animal models. We aimed to evaluate the pulmonary inflammatory reaction and its involvement in structural changes in guinea pigs chronically exposed to CS. 19 Hartley guinea pigs were exposed to 7 cigarettes/day, during 3 or 6 months. 18 control guinea pigs were sham-exposed. Numbers of neutrophils, macrophages and eosinophils and lymphoid follicles were assessed in different lung structures. Airway and vessel morphometry, alveolar space size and collagen deposition were also quantified. After 6 months of exposure, CS-exposed guinea pigs showed increased numbers of neutrophils, macrophages and eosinophils in the airways, intrapulmonary vessels and alveolar septa, as well as lymphoid follicles. Increased numbers of muscularized intrapulmonary vessels were apparent at 3 months. After 6 months of exposure, the airway wall thickened and the alveolar space size increased. Collagen deposition was also apparent in airway walls and alveolar septa after 6 months' exposure. The magnitude of airway wall-thickening correlated with the number of infiltrating inflammatory cells, and the extension of collagen deposition correlated with alveolar space size. We conclude that in the guinea pig, 6 months of CS exposure induces inflammatory cell infiltrate in lung structures, at an intensity that correlates with airway remodelling. These changes resemble those observed in COPD, thus endorsing the pathogenic role of CS and the usefulness of this animal model for its study.
Assuntos
Remodelação das Vias Aéreas , Inflamação , Pulmão , Doença Pulmonar Obstrutiva Crônica , Fumar , Animais , Vasos Sanguíneos/patologia , Modelos Animais de Doenças , Granulócitos/citologia , Granulócitos/patologia , Cobaias , Inflamação/imunologia , Inflamação/patologia , Pulmão/citologia , Pulmão/imunologia , Pulmão/patologia , Masculino , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Fumaça/efeitos adversos , Fumar/imunologia , Fumar/patologia , Nicotiana/efeitos adversosRESUMO
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-ß1/SMAD3 pathway in ADC-TAFs. Likewise, CD63 was upregulated in ADC compared to SCC cells. Genetic analyses revealed that TIMP-1 secreted by TGF-ß1-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.
Assuntos
Adenocarcinoma de Pulmão , Fibroblastos Associados a Câncer , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Tetraspanina 30 , Inibidor Tecidual de Metaloproteinase-1 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Fibroblastos Associados a Câncer/metabolismo , Carcinoma de Células Escamosas/metabolismo , Fibroblastos/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Tetraspanina 30/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Microambiente TumoralRESUMO
Large cell carcinoma (LCC) is a rare and aggressive lung cancer subtype with poor prognosis and no targeted therapies. Tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we identify MMP1 as overexpressed specifically in LCC cell lines, and we show that expression of MMP1 by LCC cells is necessary for induction of fibroblast senescence and consequent tumor promotion in both cell culture and mouse models. We also show that MMP1, in combination with TGF-ß1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-ß1-induced TAF senescence. Our results establish an entirely new role for MMP1 in cancer, and support a novel therapeutic strategy in LCC based on targeting senescent TAFs.
Assuntos
Fibroblastos Associados a Câncer/enzimologia , Carcinoma de Células Grandes/enzimologia , Proliferação de Células , Senescência Celular , Neoplasias Pulmonares/enzimologia , Metaloproteinase 1 da Matriz/metabolismo , Animais , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Metaloproteinase 1 da Matriz/genética , Camundongos Nus , Estresse Oxidativo , Comunicação Parácrina , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Carga TumoralAssuntos
Leiomiossarcoma/cirurgia , Neoplasias Vasculares/cirurgia , Veia Cava Inferior/cirurgia , Idoso , Feminino , Humanos , Leiomiossarcoma/diagnóstico por imagem , Leiomiossarcoma/patologia , Pessoa de Meia-Idade , Neoplasias Vasculares/diagnóstico por imagem , Neoplasias Vasculares/patologia , Veia Cava Inferior/diagnóstico por imagem , Veia Cava Inferior/patologiaRESUMO
OBJECTIVES: Tumor associated fibroblasts (TAFs) are essential contributors of the progression of non-small cell lung cancer (NSCLC). Most lung TAFs exhibit an activated phenotype characterized by the expression of α-SMA and fibrillar collagens. However, the prognostic value of these activation markers in NSCLC remains unclear. MATERIAL AND METHODS: We conducted a quantitative image analysis of α-SMA immunostaining and picrosirius red staining of fibrillar collagens imaged by bright-field and polarized microscopy, respectively, using tissue microarrays with samples from 220 surgical patients, which elicited a percentage of positive staining area for each marker and patient. RESULTS: Kaplan-Meier curves showed that all TAF activation markers were significantly associated with poor survival, and their prognostic value was independent of TNM staging as revealed by multivariate analysis, which elicited an adjusted increased risk of death after 3â¯years of 129% and 94% for fibrillar collagens imaged with bright-field (pâ¯=â¯0.004) and polarized light (pâ¯=â¯0.003), respectively, and of 89% for α-SMA (pâ¯=â¯0.009). We also found a significant association between all TAF activation markers and tumor necrosis, which is often indicative of hypoxia, supporting a pathologic link between tumor desmoplasia and necrosis/hypoxia. CONCLUSIONS: Our findings identify patients with large histologic coverage of fibrillar collagens and α-SMAâ¯+â¯TAFs to be at higher risk of recurrence and death, supporting that they could be considered for adjuvant therapy.
Assuntos
Biomarcadores Tumorais , Fibroblastos Associados a Câncer/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Idoso , Idoso de 80 Anos ou mais , Fibroblastos Associados a Câncer/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , PrognósticoRESUMO
BACKGROUND: Potassium channels are important in pulmonary circulation because they have been closely related to hypoxic pulmonary vasoconstriction (HPV). The objective of the study was to determine whether structural changes in pulmonary arteries (PA), such as those observed in patients with chronic obstructive pulmonary disease (COPD), might be associated with changes in the mRNA expression of both BK(Ca) and K(V) channels and their potential relationship with HPV. METHODS: PA (about 1.5 mm in diameter) were obtained from 16 patients who underwent resective lung surgery. Intimal thickening was evaluated morphometrically. mRNA expression of BK(Ca), K(V)1.2, K(V)1.5, K(V)2.1 and K(V)3.1 was evaluated by RT-PCR in PA homogenates. Endothelial function and HPV were assessed in vitro in isolated PA using an organ bath. RESULTS: Intimal enlargement was closely associated with an increase in the expression of BK(Ca) channel (r=0.57, p<0.05). Pulmonary arteries incubated with charybdotoxin, a BK(Ca) channel blocking agent, showed lower response to endothelium-dependent vasodilators indicating its contribution to reduce vascular tone. Pulmonary arteries with more pronounced responses to hypoxia were those with greater gene expression of BK(Ca) channels, suggesting a potential role in attenuating HPV (r=0.52, p<0.05). No changes in the expression of K(V) channels were found in remodeled arteries. CONCLUSIONS: Structural changes of PA in COPD could alter the response to hypoxia due to changes in BK(Ca) potassium channel distribution. Since BK(Ca) channels contribute to diminish vascular tone, their increased expression in remodeled PA might play a role in attenuating HPV.
Assuntos
Endotélio Vascular/fisiopatologia , Canais de Potássio Cálcio-Ativados/fisiologia , Artéria Pulmonar/fisiopatologia , Vasoconstrição/fisiologia , Idoso , Western Blotting , Charibdotoxina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Histamina/farmacologia , Humanos , Hipóxia/fisiopatologia , Imunoquímica , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Norepinefrina/farmacologia , Canais de Potássio Cálcio-Ativados/genética , Canais de Potássio Cálcio-Ativados/metabolismo , Cloreto de Potássio/farmacologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Vasoconstrição/efeitos dos fármacosRESUMO
BACKGROUND: Cancer-derived exosomes are involved in metastasis. YKT6 is a SNARE protein that participates in the regulation of exosome production and release, but its role in non-small cell lung cancer (NSCLC) has not been examined. MATERIALS AND METHODS: Ultracentrifugation-purified exosomes from the A549 cell line were studied by CRYO-TEM, nanoparticle tracking analysis and western blot (TSG101 marker). YKT6 was inhibited using a DsiRNA and selected pre-microRNAs. MicroRNAs targeting YKT6 were validated by Renilla/Luciferase assay and western blot. YKT6 expression and its prognostic impact were analyzed in 98 tissue specimens from resected NSCLC patients. RESULTS: Membranous nanosized vesicles (mode size: 128nm) with TSG101 protein were purified from A549 cells. YKT6 inhibition reduced exosome release by 80.9%. We validated miR-134 and miR-135b as miRNAs targeting YKT6, and transfection with the pre-miRNAs also produced a significant reduction in exosome release. The analysis of YKT6 in tumor samples showed that patients with high levels had shorter disease-free and overall survival. CONCLUSIONS: YKT6 is a key molecule in the regulation of exosome release in lung cancer cells and is in turn precisely regulated by miR-134 and miR-135b. Moreover, YKT6 levels impact prognosis of resected NSCLC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Exossomos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas R-SNARE/genética , Células A549 , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , PrognósticoRESUMO
INTRODUCTION: Long intergenic noncoding RNA-p21 (lincRNA-p21) is a long noncoding RNA transcriptionally activated by tumor protein p53 (TP53) and hypoxia inducible factor 1 alpha subunit (HIF1A). It is involved in the regulation of TP53-dependent apoptosis and the Warburg effect. We have investigated the role of lincRNA-p21 in NSCLC. METHODS: LincRNA-p21 expression was assessed in tumor and normal tissue from 128 patients with NSCLC and correlated with time to relapse and cancer-specific survival (CSS). H23, H1299, and HCC-44 cell lines were cultured in hypoxic conditions after silencing of lincRNA-p21. The TaqMan human angiogenesis array was used to explore angiogenesis-related gene expression. Levels of the protein vascular endothelial growth factor A were measured by enzyme-linked immunosorbent assay in the cell supernatants. Angiogenic capability was measured by human umbilical vein endothelial cell tube formation assay. Microvascular density in tumor samples was analyzed by immunohistochemistry. RESULTS: LincRNA-p21 was down-regulated in tumor tissue, but no association was observed with TP53 mutational status. High lincRNA-p21 levels were associated with poor CSS in all patients (p = 0.032). When patients were classified according to histological subtypes, the impact of lincRNA-p21 was confined to patients with adenocarcinoma in both time to relapse (p = 0.006) and CSS (p < 0.001). To explain the poor outcome of patients with high lincRNA-p21 expression, we studied the role of lincRNA-p21 in angiogenesis in vitro and observed a global downregulation in the expression of angiogenesis-related genes when lincRNA-p21 was inhibited. Moreover, supernatants from lincRNA-p21-inhibited cells were significantly less angiogenic and had lower levels of secreted vascular endothelial growth factor A than controls did. Finally, tumor samples with high lincRNA-p21 levels had higher microvascular density. CONCLUSIONS: Our findings suggest that lincRNA-p21 affects outcome in patients with NSCLC adenocarcinoma through the regulation of angiogenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Longo não Codificante/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Longo não Codificante/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Carcinoma de Células Grandes/metabolismo , Senescência Celular , Neoplasias Pulmonares/metabolismo , Miofibroblastos/metabolismo , Comunicação Parácrina , Animais , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Grandes/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Forma Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Progressão da Doença , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Miofibroblastos/patologia , Invasividade Neoplásica , Estresse Oxidativo , Fenótipo , Transdução de Sinais , Fatores de Tempo , Microambiente Tumoral , beta-Galactosidase/metabolismoRESUMO
The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1-positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) (p = 0.006) and overall survival (OS) (p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue (p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR (p = 0.048) and OS (p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression.
Assuntos
Proteínas Argonautas/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Idoso , Idoso de 80 Anos ou mais , Proteínas Argonautas/genética , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Linhagem Celular Tumoral , Metilação de DNA , Progressão da Doença , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pulmão/embriologia , Pulmão/cirurgia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia , Modelos de Riscos Proporcionais , Proteínas de Ligação a RNA , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: Little evidence is available concerning the impact of virtual microscopy (VM) in the undergraduate teaching of pathology. We aimed: (1) to determine the impact in student scores when moving from conventional microscopy (CM) to VM; (2) to assess the students' impressions and changes in study habits regarding the impact of this tool. METHODS: We evaluated two groups taking the discipline of pathology in the same course, one using CM and the other VM. The same set of slides used in the CM classes was digitized in a VENTANA iScan HT (Roche Diagnostics, Sant Cugat, Spain) at ×20 and observed by the students using the Virtuoso viewer (Roche Diagnostics). We evaluated the skill level reached by the students with an online test. A voluntary survey was undertaken by the VM group to assess the students' impressions regarding the resource. The day and time of any accession to the viewer were registered. RESULTS: There were no differences between the two groups in their marks in the online test (mean marks for the CM and the VM groups: 9.87 ± 0.34 and 9.86 ± 0.53, respectively; P = 0.880). 86.6% of the students found the software friendly, easy-to-use and effective. 71.6% of the students considered navigation easier with VM than with CM. The most appreciated feature of VM was the possibility to access the images anywhere and at any time (93.3%). 57.5% of the accesses were made on holidays and 41.9% later than 6:00 pm. CONCLUSIONS: Virtual microscopy can effectively replace the traditional methods of learning pathology, providing mobility and convenience to medical students.
RESUMO
UNLABELLED: The crucial role of tumor-associated fibroblasts (TAF) in cancer progression is now clear in non-small cell lung cancer (NSCLC). However, therapies against TAFs are limited due to a lack of understanding in the subtype-specific mechanisms underlying their accumulation. Here, the mechanical (i.e., matrix rigidity) and soluble mitogenic cues that drive the accumulation of TAFs from major NSCLC subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were dissected. Fibroblasts were cultured on substrata engineered to exhibit normal- or tumor-like stiffnesses at different serum concentrations, and critical regulatory processes were elucidated. In control fibroblasts from nonmalignant tissue, matrix stiffening alone increased fibroblast accumulation, and this mechanical effect was dominant or comparable with that of soluble growth factors up to 0.5% serum. The stimulatory cues of matrix rigidity were driven by ß1 integrin mechano-sensing through FAK (pY397), and were associated with a posttranscriptionally driven rise in ß1 integrin expression. The latter mechano-regulatory circuit was also observed in TAFs but in a subtype-specific fashion, because SCC-TAFs exhibited higher FAK (pY397), ß1 expression, and ERK1/2 (pT202/Y204) than ADC-TAFs. Moreover, matrix stiffening induced a larger TAF accumulation in SCC-TAFs (>50%) compared with ADC-TAFs (10%-20%). In contrast, SCC-TAFs were largely serum desensitized, whereas ADC-TAFs responded to high serum concentration only. These findings provide the first evidence of subtype-specific regulation of NSCLC-TAF accumulation. Furthermore, these data support that therapies aiming to restore normal lung elasticity and/or ß1 integrin-dependent mechano regulation may be effective against SCC-TAFs, whereas inhibiting stromal growth factor signaling may be effective against ADC-TAFs. IMPLICATIONS: This study reveals distinct mechanisms underlying the abnormal accumulation of tumor-supporting fibroblasts in two major subtypes of lung cancer, which will assist the development of personalized therapies against these cells.
Assuntos
Adenocarcinoma/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Integrina beta1/biossíntese , Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Técnicas de Cultura de Células , Meios de Cultura/farmacologia , Fibroblastos/efeitos dos fármacos , Quinase 1 de Adesão Focal/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina beta1/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacosRESUMO
BACKGROUND AND AIM OF THE WORK: In vitro studies have suggested that fibroblasts from idiopathic pulmonary fibrosis (IPF) may have an impaired induction of cyclooxygenase (Cox)-2. We have investigated Cox-1 and Cox-2 expression in lung tissue from IPF. METHODS: Cox-1 and Cox-2 expression were determined using RT-competitive PCR and immunohistochemistry in pulmonary biopsies from IPF (n = 22), chronic obstructive pulmonary disease (COPD) (n = 13), and lung tissue from subjects undergoing pleurodesis for spontaneous pneumothorax (control group, n = 17). RESULTS: Immunohistochemical analysis showed that the score of Cox-2 positive cells was higher in COPD (1 +/- 0) with respect to fibrosis (0.37 +/- 0.1, p < 0.05) and controls (0.57 +/- 0.2). There were no differences between fibrosis and controls in Cox-2 positive cells. The expression of Cox-2 mRNA was significantly higher in COPD (3.26 +/- 0.72 x 10(6) molecules cDNA/microg total RNA) in comparison to IPF (0.57 +/- 0.17) and controls (0.54 +/- 0.16) (p < 0.001). After IL-1beta stimulation (1-10 ng/ml) Cox-2 mRNA basal expression increased significantly in controls (from 35 +/- 12 to 94 +/- 4 x10(6) molecules cDNA/microg total RNA, p < 0.01) and in COPD (from 38 +/- 8 to 92 +/- 3, p < 0.01). In contrast, no significant changes in Cox-2 mRNA expression were found in IPF (from 30 +/- 12 to 43 +/- 16). CONCLUSIONS: Our results suggest that differences in Cox-2 expression may play a role in the regulation of inflammatory responses in lung diseases. Excessive activity is associated with the development of chronic obstructive lung disease, while a limited activation following pro-inflammatory stimulation might contribute to fibrogenic responses.