RESUMEN
Beckwith-Wiedemann Syndrome (BWS) is an imprinting disorder characterized by overgrowth, stemming from various genetic and epigenetic changes. This study delves into the role of IGF2 upregulation in BWS, focusing on insulin-like growth factor pathways, which are poorly known in this syndrome. We examined the IGF2R, the primary receptor of IGF2, WNT, and autophagy/lysosomal pathways in BWS patient-derived lymphoblastoid cell lines, showing different genetic and epigenetic defects. The findings reveal a decreased expression and mislocalization of IGF2R protein, suggesting receptor dysfunction. Additionally, our results point to a dysregulation in the AKT/GSK-3/mTOR pathway, along with imbalances in autophagy and the WNT pathway. In conclusion, BWS cells, regardless of the genetic/epigenetic profiles, are characterized by alteration of the IGF2R pathway that is associated with the perturbation of the autophagy and lysosome processes. These alterations seem to be a key point of the molecular pathogenesis of BWS and potentially contribute to BWS's characteristic overgrowth and cancer susceptibility. Our study also uncovers alterations in the WNT pathway across all BWS cell lines, consistent with its role in growth regulation and cancer development.
Asunto(s)
Síndrome de Beckwith-Wiedemann , Neoplasias , Humanos , Autofagia/genética , Síndrome de Beckwith-Wiedemann/genética , Línea Celular , Glucógeno Sintasa Quinasa 3RESUMEN
Three model hydantoin-based universal peptidomimetics were designed and synthetized. Their preferred amphiphilic ß-turn conformation was assessed using molecular modeling and NMR experiments, and their antibacterial activity was tested against Gram-positive and Gram-negative bacteria strains, which demonstrated that these compounds could be a captivating class of antibiotics to fight emergent drug resistance.
RESUMEN
Chromatinopathies are defined as genetic disorders caused by mutations in genes coding for protein involved in the chromatin state balance. So far 82 human conditions have been described belonging to this group of congenital disorders, sharing some molecular features and clinical signs. For almost all of these conditions, no specific treatment is available. For better understanding the molecular cascade caused by chromatin imbalance and for envisaging possible therapeutic strategies it is fundamental to combine clinical and basic research studies. To this end, animal modelling systems represent an invaluable tool to study chromatinopathies. In this review, we focused on available data in the literature of animal models mimicking the human genetic conditions. Importantly, affected organs and abnormalities are shared in the different animal models and most of these abnormalities are reported as clinical manifestation, underlying the parallelism between clinics and translational research.
RESUMEN
Cutaneous lesions are one of the hallmarks of tuberous sclerosis complex (TSC), a genetic disease in which mTOR is hyperactivated due to the lack of hamartin or tuberin. To date, novel pharmacological treatments for TSC cutaneous lesions that are benign but still have an impact on a patient's life are needed, because neither surgery nor rapamycin administration prevents their recurrence. Here, we demonstrated that primary TSC2-/meth cells that do not express tuberin for an epigenetic event caused cutaneous lesions and follicular neogenesis when they were subcutaneously injected in nude mice. Tuberin-null cells localized in the hair bulbs and alongside mature hairs, where high phosphorylation of S6 and Erk indicated mTOR hyperactivation. Interestingly, 5-azacytidine treatment reduced hair follicles, indicating that chromatin remodeling agents might be effective on TSC lesions in which cells lack tuberin for an epigenetic event. Moreover, we demonstrated that the primary TSC2-/meth cells had metastatic capability: when subcutaneously injected, they reached the bloodstream and lymphatics and invaded the lungs, causing the enlargement of the alveolar walls. The capability of TSC2-/meth cells to survive and migrate in vivo makes our mouse model ideal to follow the progression of the disease and test potential pharmacological treatments in a time-dependent manner.
Asunto(s)
Esclerosis Tuberosa , Animales , Ratones , Ratones Desnudos , Serina-Treonina Quinasas TOR , Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Background: Saliva molecular tests have shown a similar sensitivity and specificity compared to nasopharyngeal test for SARS-CoV-2 diagnosis in both symptomatic and asymptomatic individuals. The SARS-CoV-2 pandemic affected Lombardy prisons, generating the need for extensive contact tracing activities and for detecting asymptomatic carriers. The availability of a less invasive test in a setting that hosts a high-risk and often hard-to-reach population, suggests its possible use in prisons. Methods: The study was carried out on a population of new incomers in Milan San Vittore pre-trial prison. All the new incomers were submitted to quarantine and to saliva test and nasopharyngeal swab (NPS) for SARS-CoV-2 detection at the entry and at the end of quarantine before their admission in community (Protocol 1-February 2nd to March 5th, 2021). Starting from March 8th to July 30th, 2021, the screening protocol was adjusted to avoid biases in sample collection (Protocol II), and saliva testing was performed at entrance. Results: 12/1,120 enrolled subjects were excluded from the study. Among the 1,080 processed samples, 1 tested positive, 5 weakly positive, 1,069 negative, 3 were invalid, and 2 samples tested positive for the viral gene N2 only, with Ct value above 38. During Protocol I, 6/156 coupled saliva/NPS tests were discordant due to food ingestion prior saliva collection, prompting us to establishing Protocol II. Conclusions: Saliva molecular testing is feasible in prison setting, being less invasive and easier to use, and reliable. Acceptability was very high even in a complex context as that of newly incarcerated persons.
Asunto(s)
COVID-19 , Prueba de COVID-19 , Estudios de Factibilidad , Humanos , Prisiones , SARS-CoV-2 , SalivaRESUMEN
Senescence is a stress-response process characterized by the irreversible inhibition of cell proliferation, associated to the acquisition of a senescence-associated secretory phenotype (SASP), that may drive pathological conditions. Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells, featuring the hyperactivation of the mammalian Target of Rapamycin Complex 1 (mTORC1) for the absence of tuberin expression, cause the disruption of the lung parenchyma. Considering that LAM cells secrete SASP factors and that mTOR is also a driver of senescence, we deepened the contribution of senescence in LAM cell phenotype. We firstly demonstrated that human primary tuberin-deficient LAM cells (LAM/TSC cells) have senescent features depending on mTOR hyperactivation, since their high positivity to SA-ß galactosidase and to phospho-histone H2A.X are reduced by inducing tuberin expression and by inhibiting mTOR with rapamycin. Then, we demonstrated the capability of LAM/TSC cells to induce senescence. Indeed, primary lung fibroblasts (PLFs) grown in LAM/TSC conditioned medium increased the positivity to SA-ß galactosidase and to phospho-histone H2A.X, as well as p21WAF1/CIP1 expression, and enhanced the mRNA expression and the secretion of the SASP component IL-8. Taken together, these data make senescence a novel field of study to understand LAM development and progression.
Asunto(s)
Linfangioleiomiomatosis , Humanos , beta-Galactosidasa/metabolismo , Senescencia Celular/genética , Histonas , Linfangioleiomiomatosis/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Microambiente TumoralRESUMEN
KMT2A (Lysine methyltransferase 2A) is a member of the epigenetic machinery, encoding a lysine methyltransferase responsible for the transcriptional activation through lysine 4 of histone 3 (H3K4) methylation. KMT2A has a crucial role in gene expression, thus it is associated to pathological conditions when found mutated. KMT2A germinal mutations are associated to Wiedemann-Steiner syndrome and also in patients with initial clinical diagnosis of several other chromatinopathies (i.e., Coffin-Siris syndromes, Kabuki syndrome, Cornelia De Lange syndrome, Rubinstein-Taybi syndrome), sharing an overlapping phenotype. On the other hand, KMT2A somatic mutations have been reported in several tumors, mainly blood malignancies. Due to its evolutionary conservation, the role of KMT2A in embryonic development, hematopoiesis and neurodevelopment has been explored in different animal models, and in recent decades, epigenetic treatments for disorders linked to KMT2A dysfunction have been extensively investigated. To note, pharmaceutical compounds acting on tumors characterized by KMT2A mutations have been formulated, and even nutritional interventions for chromatinopathies have become the object of study due to the role of microbiota in epigenetic regulation.
Asunto(s)
Anomalías Múltiples , Discapacidad Intelectual , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Animales , Epigénesis Genética , Humanos , Discapacidad Intelectual/genética , Lisina , FenotipoRESUMEN
Multiple myeloma (MM) is an incurable hematologic neoplasm, whose poor prognosis is deeply affected by the propensity of tumor cells to localize in the bone marrow (BM) and induce the protumorigenic activity of normal BM cells, leading to events associated with tumor progression, including tumor angiogenesis, osteoclastogenesis, and the spread of osteolytic bone lesions. The interplay between MM cells and the BM niche does not only rely on direct cell-cell interaction, but a crucial role is also played by MM-derived extracellular vesicles (MM-EV). Here, we demonstrated that the oncogenic NOTCH receptors are part of MM-EV cargo and play a key role in EV protumorigenic ability. We used in vitro and in vivo models to investigate the role of EV-derived NOTCH2 in stimulating the protumorigenic behavior of endothelial cells and osteoclast progenitors. Importantly, MM-EV can transfer NOTCH2 between distant cells and increase NOTCH signaling in target cells. MM-EV stimulation increases endothelial cell angiogenic ability and osteoclast differentiation in a NOTCH2-dependent way. Indeed, interfering with NOTCH2 expression in MM cells may decrease the amount of NOTCH2 also in MM-EV and affect their angiogenic and osteoclastogenic potential. Finally, we demonstrated that the pharmacologic blockade of NOTCH activation by γ-secretase inhibitors may hamper the biological effect of EV derived by MM cell lines and by the BM of MM patients. These results provide the first evidence that targeting the NOTCH pathway may be a valid therapeutic strategy to hamper the protumorigenic role of EV in MM as well as other tumors.
Asunto(s)
Vesículas Extracelulares , Mieloma Múltiple , Médula Ósea/patología , Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Mieloma Múltiple/patología , Microambiente TumoralRESUMEN
Matrix metalloproteinase (MMP) dysregulation is implicated in several diseases, given their involvement in extracellular matrix degradation and cell motility. In lymphangioleiomyomatosis (LAM), a pulmonary rare disease, MMP-2 and MMP-9 have been detected at high levels in serum and urine. LAM cells, characterized by a mutation in the tuberous sclerosis complex (TSC)1 or TSC2, promote cystic lung destruction. The role of MMPs in invasive and destructive LAM cell capability has not yet been fully understood. We evaluated MMP-2 and MMP-7 expression, secretion, and activity in primary LAM/TSC cells that bear a TSC2 germline mutation and an epigenetic modification and depend on epidermal growth factor (EGF) for survival. 5-azacytidine restored tuberin expression with a reduction of MMP-2 and MMP-7 levels and inhibits motility, similarly to rapamycin and anti-EGFR antibody. Both drugs reduced MMP-2 and MMP-7 secretion and activity during wound healing and decreased their expression in lung nodules of a LAM mouse model. In LAM/TSC cells, MMP-2 and MMP-7 are dependent on tuberin expression, cellular adhesion, and migration. MMPs appears sensitive to rapamycin and anti-EGFR antibody only during cellular migration. Our data indicate a complex and differential modulation of MMP-2 and MMP-7 in LAM/TSC cells, likely critical for lung parenchyma remodeling during LAM progression.
RESUMEN
Background: Lymphangioleiomyomatosis can develop in a sporadic form (S-LAM) or in women with tuberous sclerosis complex (TSC). The matrix metalloproteinases (MMPs) are extracellular matrix-degrading enzymes potentially involved in cystic lung destruction, and in the process of migration of LAM cells. The aim of the study was to explore the role of MMP-2 and MMP-7, such as vascular endothelial growth factor (VEGF) -C and -D in women with LAM, including patients with minor pulmonary disease (i.e., <10 lung cysts), and TSC with or without LAM. Methods: We evaluated 50 patients: 13 individuals affected by S-LAM, 20 with TSC-LAM, of whom six with minor pulmonary disease, and 17 with TSC without pulmonary involvement. Sixteen healthy women were used as controls. Results: MMP-2 resulted higher in LAM compared to healthy volunteers, and TSC patients (p = 0.040). MMP-7 was higher in TSC-LAM patient, with even greater values in patients with TSC-LAM minor pulmonary disease, than in S-LAM patients, and in controls (p = 0.001). VEGF-D level was lower than 800 pg/mL in all healthy controls and resulted higher in S-LAM and TSC-LAM than in TSC patients and controls (p < 0.001). VEGF-C values were not statistically different in the study population (p = 0.354). The area under ROC curves (AUCs) of MMP-2, and MMP-7 for predicting LAM diagnosis were of 0.756 ± 0.079 (p = 0.004), and 0.828 ± 0.060 (p < 0.001), respectively. Considering only patients with TSC, the AUCs for MMP-2, and MMP-7 in predicting LAM were 0.694 ± 0.088 (p = 0.044), and 0.713 ± 0.090 (p = 0.027), respectively. Conclusions: Our data suggest that MMP-2 and MMP-7 could be promising biomarkers for LAM diagnosis.
RESUMEN
Multiple myeloma is still incurable due to an intrinsic aggressiveness or, more frequently, to the interactions of malignant plasma cells with the bone marrow (BM) microenvironment. Myeloma cells educate BM cells to support neoplastic cell growth, survival, acquisition of drug resistance resulting in disease relapse. Myeloma microenvironment is characterized by Notch signaling hyperactivation due to the increased expression of Notch1 and 2 and the ligands Jagged1 and 2 in tumor cells. Notch activation influences myeloma cell biology and promotes the reprogramming of BM stromal cells. In this work we demonstrate, in vitro, ex vivo and by using a zebrafish multiple myeloma model, that Jagged inhibition causes a decrease in both myeloma-intrinsic and stromal cell-induced resistance to currently used drugs, i.e. bortezomib, lenalidomide and melphalan. The molecular mechanism of drug resistance involves the chemokine system CXCR4/SDF1α. Myeloma cell-derived Jagged ligands trigger Notch activity in BM stromal cells. These, in turn, secrete higher levels of SDF1α in the BM microenvironment increasing CXCR4 activation in myeloma cells, which is further potentiated by the concomitant increased expression of this receptor induced by Notch activation. Consistently with the augmented pharmacological resistance, SDF1α boosts the expression of BCL2, Survivin and ABCC1. These results indicate that a Jagged-tailored approach may contribute to disrupting the pharmacological resistance due to intrinsic myeloma cell features or to the pathological interplay with BM stromal cells and, conceivably, improve patients' response to standard-of-care therapies.
Asunto(s)
Proteína Jagged-1/genética , Proteína Jagged-2/genética , Mieloma Múltiple , Animales , Médula Ósea , Línea Celular Tumoral , Resistencia a Medicamentos , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Receptores Notch , Microambiente Tumoral , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.
Asunto(s)
Linfangioleiomiomatosis/metabolismo , Receptores de Prolactina/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Linfangioleiomiomatosis/genética , Ratones , Receptores de Prolactina/genética , Factor de Transcripción STAT3/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Tuberous Sclerosis Complex (TSC) is characterized by the presence of benign tumors in the brain, kidneys, heart, eyes, lungs, and skin. The typical brain lesions are cortical tubers, subependimal nodules and subependymal giant-cell astrocytomas. The occurrence of malignant astrocytomas such as glioblastoma is rare. We report on a child with a clinical diagnosis of TSC and a rapidly evolving glioblastoma multiforme. Genetic analysis identified a de novo mutation in TSC2. Molecular characterization of the tumor was performed and discussed, as well as a review of the literature where cases of TSC and glioblastoma multiforme are described. Although the co-occurrence of TSC and glioblastoma multiforme seems to be rare, this possible association should be kept in mind, and proper clinical and radiological follow up should be recommended in these patients.
Asunto(s)
Neoplasias Encefálicas/genética , Glioblastoma/genética , Mutación , Esclerosis Tuberosa/genética , Proteínas Supresoras de Tumor/genética , Antineoplásicos/uso terapéutico , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/efectos de la radiación , Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Niño , Resultado Fatal , Rayos gamma/uso terapéutico , Expresión Génica , Glioblastoma/complicaciones , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Masculino , Esclerosis Tuberosa/complicaciones , Esclerosis Tuberosa/patología , Esclerosis Tuberosa/terapia , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
EGFR belongs to the HER/ErbB family of tyrosine kinase receptors and its activation in cancer cells has been linked with increased proliferation, angiogenesis, and metastasis. Lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm that occurs sporadically or in association with tuberous sclerosis complex (TSC), a genetic, multisystem disorder characterized by hamartomas in several organs. From chylous of a LAM/TSC patient, we previously isolated smooth muscle-like LAM/TSC cells whose proliferation depends on EGF and monoclonal anti-EGFR antibodies reduced proliferation and caused cell death. We demonstrated that the dependency from EGF was caused by the absence of tuberin. To study the role of EGFR pathway in vivo, we developed a mouse model by administration of LAM/TSC cells to female nude mice. LAM/TSC cells caused pulmonary airspace enlargement and, after 30 weeks, nodule formation which express EGFR. Anti-EGFR antibody decreased the number and dimension of lung nodules likely for the inhibition of Erk and S6 signaling, reversed the pulmonary alterations, and reduced lymphatic and blood vessels. Moreover, in pulmonary nodules anti-EGFR antibody reduced the positivity to estrogen and progesterone receptors which enhance survival of LAM cells and Snail expression. These results suggest that the inhibition of EGFR signalling has a potential in treatment of LAM/TSC lung alterations.
Asunto(s)
Anticuerpos Antiidiotipos/administración & dosificación , Receptores ErbB/metabolismo , Linfangioleiomiomatosis/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/patología , Ratones , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 or TSC2 genes. Lymphangioleiomyomatosis (LAM) can be sporadic or associated with TSC and is characterized by widespread pulmonary proliferation of abnormal α-smooth muscle (ASM)-like cells. We investigated the features of ASM cells isolated from chylous thorax of a patient affected by LAM associated with TSC, named LAM/TSC cells, bearing a germline TSC2 mutation and an epigenetic defect causing the absence of tuberin. Proliferation of LAM/TSC cells is epidermal growth factor (EGF)-dependent and blockade of EGF receptor causes cell death as we previously showed in cells lacking tuberin. LAM/TSC cells spontaneously detach probably for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway and display the ability to survive independently from adhesion. Non-adherent LAM/TSC cells show an extremely low proliferation rate consistent with tumour stem-cell characteristics. Moreover, LAM/TSC cells bear characteristics of stemness and secrete high amount of interleukin (IL)-6 and IL-8. Anti-EGF receptor antibodies and rapamycin affect proliferation and viability of non-adherent cells. In conclusion, the understanding of LAM/TSC cell features is important in the assessment of cell invasiveness in LAM and TSC and should provide a useful model to test therapeutic approaches aimed at controlling their migratory ability.
Asunto(s)
Epigénesis Genética , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/patología , Proteínas Supresoras de Tumor/genética , Anticuerpos/farmacología , Secuencia de Bases , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Epigénesis Genética/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/inmunología , Femenino , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Datos de Secuencia Molecular , Sirolimus/farmacología , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/patología , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
Tuberous sclerosis complex (TSC) is a multi-systemic syndrome caused by mutations in TSC1 or TSC2 gene. In TSC2-null cells, Rheb, a member of the Ras family of GTPases, is constitutively activated. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase and block the synthesis of isoprenoid lipids with inhibition of Rheb farnesylation and RhoA geranylgeranylation. The effects of rosuvastatin on the function of human TSC2(-/-) and TSC2(-/meth) α-actin smooth muscle (ASM) cells have been investigated. The TSC2(-/-) and TSC2(-/meth) ASM cells, previously isolated in our laboratory from the renal angiomyolipoma of two TSC patients, do not express tuberin and bear loss of heterozigosity caused by a double hit on TSC2 and methylation of TSC2 promoter, respectively. Exposure to rosuvastatin affected TSC2(-/meth) ASM cell growth and promoted tuberin expression by acting as a demethylating agent. This occurred without changes in interleukin release. Rosuvastatin also reduced RhoA activation in TSC2(-/meth) ASM cells, and it required coadministration with the specific mTOR (mammalian target of rapamycin) inhibitor rapamycin to be effective in TSC2(-/-) ASM cells. Rapamycin enhanced rosuvastatin effect in inhibiting cell proliferation in TSC2(-/-) and TSC2(-/meth) ASM cells. Rosuvastatin alone did not alter phosphorylation of S6 and extracellular signal-regulated kinase (ERK), and at the higher concentration, rosuvastatin and rapamycin slightly decreased ERK phosphorylation. These results suggest that rosuvastatin may potentially represent a treatment adjunct to the therapy with mTOR inhibitors now in clinical development for TSC. In particular, rosuvastatin appears useful when the disease is originated by epigenetic defects.
Asunto(s)
Antiinflamatorios , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Fluorobencenos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Pirimidinas/farmacología , Sulfonamidas/farmacología , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Actinas/metabolismo , Adulto , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Guanosina Trifosfato/metabolismo , Humanos , Masculino , Ácido Mevalónico/metabolismo , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Rosuvastatina Cálcica , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Lymphangioleiomyomatosis (LAM) is an interstitial lung disease characterized by invasion and proliferation of abnormal smooth muscle (ASM) cells in lung parenchyma and axial lymphatics. LAM cells bear mutations in tuberous sclerosis (TSC) genes. TSC2(-/-) ASM cells, derived from a human renal angiomyolipoma, require epidermal growth factor (EGF) for proliferation. Blockade of EGF receptors (EGFR) causes cell death. TSC2(-/-) ASM cells, previously labeled with PKH26-GL dye, were endonasally administered to 5-week-old immunodeficient female nude mice, and 4 or 26 weeks later anti-EGFR antibody or rapamycin was administered twice a week for 4 consecutive weeks. TSC2(-/-) ASM cells infiltrated lymph nodes and alveolar lung walls, causing progressive destruction of parenchyma. Parenchymal destruction was efficiently reversed by anti-EGFR treatment and partially by rapamycin treatment. Following TSC2(-/-) ASM cell administration, lymphangiogenesis increased in lungs as indicated by more diffuse LYVE1 expression and high murine VEGF levels. Anti-EGFR antibody and rapamycin blocked the increase in lymphatic vessels. This study shows that TSC2(-/-) ASM cells can migrate and invade lungs and lymph nodes, and anti-EGFR antibody is more effective than rapamycin in promoting lung repair and reducing lymphangiogenesis. The development of a model to study metastasis by TSC cells will also help to explain how they invade different tissues and metastasize to the lung.
Asunto(s)
Modelos Animales de Enfermedad , Linfangioleiomiomatosis/patología , Miocitos del Músculo Liso/trasplante , Proteínas Supresoras de Tumor/deficiencia , Administración Intranasal , Animales , Anticuerpos/farmacología , Recuento de Células , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Ratones , Ratones Desnudos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Fosforilación/efectos de los fármacos , Condicionamiento Físico Animal , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Sirolimus/farmacología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that is caused by mutations in either the TSC1 or TSC2 gene. Smooth muscle-like cells (ASMs) were isolated from an angiomyolipoma of a patient with TSC. These cells lacked tuberin, were labeled by both HMB45 and CD44v6 antibodies, and had constitutive S6 phosphorylation. The cells bear a germline TSC2 intron 8-exon 9 junction mutation, but DNA analysis and polymerase chain reaction amplification failed to demonstrate loss of heterozygosity. Testing for an epigenetic alteration, we detected methylation of the TSC2 promoter. Its biological relevance was confirmed by tuberin expression and a reduction in HMB45 labeling and S6 constitutive phosphorylation after exposure to the chromatin-remodeling agents, trichostatin A and 5-azacytidine. These cells were named TSC2(-/meth) ASMs. Their proliferation required epidermal growth factor in the medium as previously described for TSC2(-/-) ASMs. Blockade of epidermal growth factor with monoclonal antibodies caused the death of TSC2(-/meth) ASMs. In addition, rapamycin effectively blocked the proliferation of these cells. Our data show for the first time that methylation of the TSC2 promoter might cause a complete loss of tuberin in TSC2 cells, and that the pathogenesis of angiomyolipomas might also originate from epigenetic defects in smooth muscle cells. Additionally, the effect of chromatin-remodeling agents in these cells suggests a further avenue for the treatment of TSC as well as lymphangioleiomyomatosis.
Asunto(s)
Angiomiolipoma/genética , Angiomiolipoma/patología , Metilación de ADN/genética , Miocitos del Músculo Liso/patología , Proteínas Supresoras de Tumor/genética , Adulto , Angiomiolipoma/etiología , Antibióticos Antineoplásicos/farmacología , Antígenos de Neoplasias , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Análisis Mutacional de ADN , Mutación de Línea Germinal , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Antígenos Específicos del Melanoma , Microscopía Fluorescente , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas de Neoplasias , Regiones Promotoras Genéticas/genética , Sirolimus/farmacología , Esclerosis Tuberosa/complicaciones , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/patología , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
BACKGROUND: Tuberous sclerosis complex (TSC), a tumor syndrome caused by mutations in TSC1 or TSC2 genes, is characterized by the development of hamartomas. We previously isolated, from an angiomyolipoma of a TSC2 patient, a homogenous population of smooth muscle-like cells (TSC2(-/-) ASM cells) that have a mutation in the TSC2 gene as well as TSC2 loss of heterozygosity (LOH) and consequently, do not produce the TSC2 gene product, tuberin. TSC2(-/-) ASM cell proliferation is EGF-dependent. METHODS AND FINDINGS: Effects of EGF on proliferation of TSC2(-/-) ASM cells and TSC2(-/-) ASM cells transfected with TSC2 gene were determined. In contrast to TSC2(-/-) ASM cells, growth of TSC2-transfected cells was not dependent on EGF. Moreover, phosphorylation of Akt, PTEN, Erk and S6 was significantly decreased. EGF is a proliferative factor of TSC2(-/-) ASM cells. Exposure of TSC2(-/-) ASM cells to anti-EGFR antibodies significantly inhibited their proliferation, reverted reactivity to HMB45 antibody, a marker of TSC2(-/-) cell phenotype, and inhibited constitutive phosphorylation of S6 and ERK. Exposure of TSC2(-/-) ASM cells to rapamycin reduced the proliferation rate, but only when added at plating time. Although rapamycin efficiently inhibited S6 phosphorylation, it was less efficient than anti-EGFR antibody in reverting HMB45 reactivity and blocking ERK phosphorylation. In TSC2(-/-) ASM cells specific PI3K inhibitors (e.g. LY294002, wortmannin) and Akt1 siRNA had little effect on S6 and ERK phosphorylation. Following TSC2-gene transfection, Akt inhibitor sensitivity was observed. CONCLUSION: Our results show that an EGF independent pathway is more important than that involving IGF-I for growth and survival of TSC(-/-) ASM cells, and such EGF-dependency is the result of the lack of tuberin.