RESUMO
Glioblastoma (GBM) is a deadly and the most common primary brain tumor in adults. Due to their regulation of a high number of mRNA transcripts, microRNAs (miRNAs) are key molecules in the control of biological processes and are thereby promising therapeutic targets for GBM patients. In this regard, we recently reported miRNAs as strong modulators of GBM aggressiveness. Here, using an integrative and comprehensive analysis of the TCGA database and the transcriptome of GBM biopsies, we identified three critical and clinically relevant miRNAs for GBM, miR-17-3p, miR-222, and miR-340. In addition, we showed that the combinatorial modulation of three of these miRNAs efficiently inhibited several biological processes in patient-derived GBM cells of all these three GBM subtypes (Mesenchymal, Proneural, Classical), induced cell death, and delayed tumor growth in a mouse tumor model. Finally, in a doxycycline-inducible model, we observed a significant inhibition of GBM stem cell viability and a significant delay of orthotopic tumor growth. Collectively, our results reveal, for the first time, the potential of miR-17-3p, miR-222 and miR-340 multi-targeting as a promising therapeutic strategy for GBM patients.
Assuntos
Glioblastoma , MicroRNAs , Adulto , Humanos , Animais , Camundongos , MicroRNAs/genética , Glioblastoma/genética , Agressão , Biópsia , Morte Celular , Modelos Animais de DoençasRESUMO
PURPOSE: Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract, with mutant succinate dehydrogenase (SDH) subunits (A-D) comprising less than 7.5% (i.e., 150-200/year) of new cases annually in the United States. Contrary to GISTs harboring KIT or PDGFRA mutations, SDH-mutant GISTs affect adolescents/young adults, often metastasize, and are frequently resistant to tyrosine kinase inhibitors (TKI). Lack of human models for any SDH-mutant tumors, including GIST, has limited molecular characterization and drug discovery. EXPERIMENTAL DESIGN: We describe methods for establishing novel patient-derived SDH-mutant (mSDH) GIST models and interrogated the efficacy of temozolomide on these tumor models in vitro and in clinical trials of patients with mSDH GIST. RESULTS: Molecular and metabolic characterization of our patient-derived mSDH GIST models revealed that these models recapitulate the transcriptional and metabolic hallmarks of parent tumors and SDH deficiency. We further demonstrate that temozolomide elicits DNA damage and apoptosis in our mSDH GIST models. Translating our in vitro discovery to the clinic, a cohort of patients with SDH-mutant GIST treated with temozolomide (n = 5) demonstrated a 40% objective response rate and 100% disease control rate, suggesting that temozolomide represents a promising therapy for this subset of GIST. CONCLUSIONS: We report the first methods to establish patient-derived mSDH tumor models, which can be readily employed for understanding patient-specific tumor biology and treatment strategies. We also demonstrate that temozolomide is effective in patients with mSDH GIST who are refractory to existing chemotherapeutic drugs (namely, TKIs) in clinic for GISTs, bringing a promising treatment option for these patients to clinic.See related commentary by Blakely et al., p. 3.
Assuntos
Neoplasias Gastrointestinais , Tumores do Estroma Gastrointestinal , Adolescente , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Humanos , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Succinato Desidrogenase/metabolismo , Adulto JovemRESUMO
Gastrointestinal stromal tumor (GIST) is commonly driven by oncogenic KIT mutations that are effectively targeted by imatinib (IM), a tyrosine kinase inhibitor (TKI). However, IM does not cure GIST, and adjuvant therapy only delays recurrence in high-risk tumors. We hypothesized that GIST contains cells with primary IM resistance that may represent a reservoir for disease persistence. Here, we report a subpopulation of CD34+KITlow human GIST cells that have intrinsic IM resistance. These cells possess cancer stem cell-like expression profiles and behavior, including self-renewal and differentiation into CD34+KIThigh progeny that are sensitive to IM treatment. We also found that TKI treatment of GIST cell lines led to induction of stem cell-associated transcription factors (OCT4 and NANOG) and concomitant enrichment of the CD34+KITlow cell population. Using a data-driven approach, we constructed a transcriptomic-oncogenic map (Onco-GPS) based on the gene expression of 134 GIST samples to define pathway activation during GIST tumorigenesis. Tumors with low KIT expression had overexpression of cancer stem cell gene signatures consistent with our in vitro findings. Additionally, these tumors had activation of the Gas6/AXL pathway and NF-κB signaling gene signatures. We evaluated these targets in vitro and found that primary IM-resistant GIST cells were effectively targeted with either single-agent bemcentinib (AXL inhibitor) or bardoxolone (NF-κB inhibitor), as well as with either agent in combination with IM. Collectively, these findings suggest that CD34+KITlow cells represent a distinct, but targetable, subpopulation in human GIST that may represent a novel mechanism of primary TKI resistance, as well as a target for overcoming disease persistence following TKI therapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Gastrointestinais/tratamento farmacológico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and ß1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles' heel for a significant and unique subset of GBM patients.
Assuntos
Proteínas Sanguíneas/metabolismo , Neoplasias Encefálicas/metabolismo , Galectinas/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Proteínas Sanguíneas/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Galectinas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Pinocitose , Mapas de Interação de Proteínas , Transcriptoma , Células Tumorais CultivadasRESUMO
Cancer-associated fibroblasts (CAFs) are the most abundant cells in the tumor microenvironment. Crosstalk between tumor cells and CAFs contributes to tumor survival in most epithelial cancers. Recently, utilizing gastrointestinal stromal tumor (GIST) as a model for sarcomas, we identified paracrine networks by which CAFs promote tumor progression and metastasis. However, the mechanisms by which CAFs arise in sarcomas remain unclear. Here, RNA sequencing analysis revealed that transforming growth factor-ß1 (TGF-ß1) is highly expressed in both tumor cells and CAFs. To determine the functional role of TGF-ß1, we treated normal gastric fibroblasts (GFs) with recombinant TGF-ß1, which caused the GFs to adopt a more stellate morphology, as well as increased the mRNA expression of CAF-mediated genes (CCL2, RAB3B, and TNC) and genes encoding fibroblast growth factors (FGFs). Moreover, while either GIST or CAF conditioned media enhanced the transition from GFs to CAFs, a TGF-ß1-blocking antibody attenuated this effect. Transwell migration assays revealed that the TGF-ß1-mediated transition from GFs to CAFs enhanced tumor cell migration. This migratory effect was abrogated by an anti-TGF-ß1 antibody, suggesting that TGF-ß1 secreted from GIST cells or CAFs is associated with GIST migration via GF-to-CAF transition. In addition, the murine spleen-to-liver metastasis model showed that GF pre-treated with TGF-ß1 promoted GIST metastasis. Collectively, these findings reveal unappreciated crosstalk among tumor cells, CAFs, and normal resident fibroblasts in the stroma of sarcomas, which enhances a GF-to-CAF transition associated with tumor migration and metastasis.
RESUMO
Targeted therapies for gastrointestinal stromal tumor (GIST) are modestly effective, but GIST cannot be cured with single agent tyrosine kinase inhibitors. In this study, we sought to identify new therapeutic targets in GIST by investigating the tumor microenvironment. Here, we identified a paracrine signaling network by which cancer-associated fibroblasts (CAFs) drive GIST growth and metastasis. Specifically, CAFs isolated from human tumors were found to produce high levels of platelet-derived growth factor C (PDGFC), which activated PDGFC-PDGFRA signal transduction in GIST cells that regulated the expression of SLUG, an epithelial-mesenchymal transition (EMT) transcription factor and downstream target of PDGFRA signaling. Together, this paracrine induce signal transduction cascade promoted tumor growth and metastasis in vivo. Moreover, in metastatic GIST patients, SLUG expression positively correlated with tumor size and mitotic index. Given that CAF paracrine signaling modulated GIST biology, we directly targeted CAFs with a dual PI3K/mTOR inhibitor, which synergized with imatinib to increase tumor cell killing and in vivo disease response. Taken together, we identified a previously unappreciated cellular target for GIST therapy in order to improve disease control and cure rates.
Assuntos
Tumores do Estroma Gastrointestinal/genética , Linfocinas/genética , Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Transcrição da Família Snail/genética , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Humanos , Metástase Neoplásica , Comunicação Parácrina/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Microambiente Tumoral/efeitos dos fármacosRESUMO
Importance: Gastrointestinal stromal tumor (GIST) is frequently driven by oncogenic KIT variations. Imatinib targeting of KIT marked a new era in GIST treatment and ushered in precision oncological treatment for all solid malignant neoplasms. However, studies on the molecular biological traits of GIST have found that tumors respond differentially to imatinib dosage based on the KIT exon with variation. Despite this knowledge, few patients undergo genetic testing at diagnosis, and empirical imatinib therapy remains routine. Barriers to genetic profiling include concerns about the cost and utility of testing. Objective: To determine whether targeted gene testing (TGT) is a cost-effective diagnostic for patients with metastatic GIST from the US payer perspective. Design, Setting, and Participants: This economic evaluation developed a Markov model to compare the cost-effectiveness of TGT and tailored first-line therapy compared with empirical imatinib therapy among patients with a new diagnosis of metastatic GIST. The main health outcome, quality-adjusted life years (QALYs), and costs were obtained from the literature, and transitional probabilities were modeled from disease progression and survival estimates from randomized clinical trials of patients with metastatic GIST. Data analyses were conducted October 2019 to January 2020. Exposure: TGT and tailored first-line therapy. Main Outcomes and Measures: The primary outcome was QALYs and cost. Cost-effectiveness was defined using an incremental cost-effectiveness ratio, with an incremental cost-effectiveness ratio less than $100â¯000/QALY considered cost-effective. One-way and probabilistic sensitivity analyses were conducted to assess model stability. Results: Therapy directed by TGT was associated with an increase of 0.10 QALYs at a cost of $9513 compared with the empirical imatinib approach, leading to an incremental cost-effectiveness ratio of $92â¯100. These findings were sensitive to the costs of TGT, drugs, and health utility model inputs. Therapy directed by TGT remained cost-effective for genetic testing costs up to $3730. Probabilistic sensitivity analysis found that TGT-directed therapy was considered cost-effective 70% of the time. Conclusions and Relevance: These findings suggest that using genetic testing to match treatment of KIT variations to imatinib dosing is a cost-effective approach compared with empirical imatinib.
Assuntos
Tumores do Estroma Gastrointestinal , Testes Genéticos , Mesilato de Imatinib , Proteínas Proto-Oncogênicas c-kit/genética , Antineoplásicos/economia , Antineoplásicos/farmacologia , Análise Custo-Benefício , Custos de Medicamentos , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/economia , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Testes Genéticos/economia , Testes Genéticos/métodos , Humanos , Mesilato de Imatinib/economia , Mesilato de Imatinib/farmacologia , Cadeias de Markov , Metástase Neoplásica , Estadiamento de Neoplasias , Farmacogenética/métodos , Anos de Vida Ajustados por Qualidade de VidaRESUMO
Gastrointestinal stromal tumor (GIST), the most common sarcoma, is characterized by KIT protein overexpression, and tumors are frequently driven by oncogenic KIT mutations. Targeted inhibition of KIT revolutionized GIST therapy and ushered in the era of precision medicine for the treatment of solid malignancies. Here, we present the first use of a KIT-specific DNA aptamer for targeted labeling of GIST. We found that an anti-KIT DNA aptamer bound cells in a KIT-dependent manner and was highly specific for GIST cell labeling in vitro Functionally, the KIT aptamer bound extracellular KIT in a manner similar to KIT mAb staining, and was trafficked intracellularly in vitro The KIT aptamer bound dissociated primary human GIST cells in a mutation agnostic manner such that tumors with KIT and PDGFRA mutations were labeled. In addition, the KIT aptamer specifically labeled intact human GIST tissue ex vivo, as well as peritoneal xenografts in mice with high sensitivity. These results represent the first use of an aptamer-based method for targeted detection of GIST in vitro and in vivo.
Assuntos
Anticorpos Monoclonais/farmacologia , Aptâmeros de Nucleotídeos/administração & dosagem , Neoplasias Gastrointestinais/tratamento farmacológico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Animais , Apoptose , Aptâmeros de Nucleotídeos/genética , Proliferação de Células , Feminino , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Humanos , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-kit/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Plexiform fibromyxoma (PF) is a rare gastric tumor often confused with gastrointestinal stromal tumor. These so-called "benign" tumors often present with upper GI bleeding and gastric outlet obstruction. It was recently demonstrated that approximately one-third of PF have activation of the GLI1 oncogene, a transcription factor in the hedgehog (Hh) pathway, via a MALAT1-GLI1 fusion protein or GLI1 up-regulation. Despite this discovery, the biology of most PFs remains unknown. METHODS: Next generation sequencing (NGS) was performed on formalin-fixed paraffin-embedded (FFPE) samples of PF specimens collected from three institutions (UCSD, NCI and OHSU). Fresh frozen tissue from one tumor was utilized for in vitro assays, including quantitative RT-PCR and cell viability assays following drug treatment. RESULTS: Eight patients with PF were identified and 5 patients' tumors were analyzed by NGS. An index case had a mono-allelic PTCH1 deletion of exons 15-24 and a second case, identified in a validation cohort, also had a PTCH1 gene loss associated with a suspected long-range chromosome 9 deletion. Building on the role of Hh signaling in PF, PTCH1, a tumor suppressor protein, functions upstream of GLI1. Loss of PTCH1 induces GLI1 activation and downstream gene transcription. Utilizing fresh tissue from the index PF case, RT-qPCR analysis demonstrated expression of Hh pathway components, SMO and GLI1, as well as GLI1 transcriptional targets, CCND1 and HHIP. In turn, short-term in vitro treatment with a Hh pathway inhibitor, sonidegib, resulted in dose-dependent cell killing. CONCLUSIONS: For the first time, we report a novel association between PTCH1 inactivation and the development of plexiform fibromyxoma. Hh pathway inhibition with SMO antagonists may represent a target to study for treating a subset of plexiform fibromyxomas.
Assuntos
Fibroma/genética , Genes Supressores de Tumor , Receptor Patched-1/genética , RNA Longo não Codificante/genética , Adolescente , Adulto , Idoso , Proteínas de Transporte/genética , Deleção Cromossômica , Ciclina D1/genética , Éxons , Feminino , Proteínas Hedgehog/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Estudos Retrospectivos , Receptor Smoothened/genética , Adulto Jovem , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Although integrin αvß3 is linked to cancer progression, its role in epithelial development is unclear. Here, we show that αvß3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. Whereas αvß3 is a luminal progenitor marker in the virgin gland, we noted increased αvß3 expression in MaSCs at midpregnancy. Accordingly, mice lacking αvß3 or expressing a signaling-deficient receptor showed defective mammary gland morphogenesis during pregnancy. This was associated with decreased MaSC expansion, clonogenicity, and expression of Slug, a master regulator of MaSCs. Surprisingly, αvß3-deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. Transforming growth factor ß2 (TGF-ß2) induced αvß3 expression, enhancing Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells, αvß3 was necessary and sufficient for Slug activation, tumorsphere formation, and tumor initiation. Thus, pregnancy-associated MaSCs require a TGF-ß2/αvß3/Slug pathway, which may contribute to breast cancer progression and stemness.
Assuntos
Transformação Celular Neoplásica/metabolismo , Integrina alfaVbeta3/metabolismo , Glândulas Mamárias Animais/citologia , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Animais , Neoplasias da Mama/metabolismo , Diferenciação Celular , Células Epiteliais/citologia , Feminino , Humanos , Integrina alfaVbeta3/deficiência , Camundongos , Gravidez , Fatores de Transcrição da Família Snail , Fator de Crescimento Transformador beta2/metabolismoRESUMO
Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin α(v)ß3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, α(v)ß3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, α(v)ß3 expression and the resulting KRAS-RalB-NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify α(v)ß3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Integrina beta3/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos Fase II como Assunto , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Feminino , Humanos , Integrina alfaVbeta3/metabolismo , Integrina beta3/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Quinazolinas/uso terapêutico , Interferência de RNA , Ensaios Clínicos Controlados Aleatórios como Assunto , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ral de Ligação ao GTP/genética , Proteínas ras/genéticaRESUMO
As a therapy for type I diabetes, islet transplantation provides clear benefits in terms of increased insulin-independence and a reduced risk of hypoglycemia. However, a critical shortage of donor pancreata means that few can benefit from this approach. The ex vivo expansion of human ß-cells prior to transplantation could ameliorate this problem, however, attempts to grow large numbers of ß-cells that retain their native phenotype have thus far failed. Recent lineage tracing studies suggest that this problem is due to the inherent tendency of cultured human ß-cells to undergo a process reminiscent of epithelial-to-mesenchymal transition (EMT). EMT describes a highly complex process that culminates in a loss of epithelial cell polarity, severance of intercellular adhesive junctions and the acquisition of a highly motile mesenchymal phenotype. Interestingly, recent evidence suggests that a transient EMT-like process may also contribute to the delamination of endocrine progenitors and subsequent islet neogenesis. The inherent susceptibility of cultured human ß-cells to EMT, and the potential involvement of this process during islet neogenesis, raises important questions as to how this process is triggered and subsequently regulated. The primary purpose of this review is to describe those factors, pathways or processes that are complicit in inducing or regulating the mesenchymal transition of human ß-cells. This includes addressing the role of the extracellular matrix, the contribution of select signaling pathways, and the regulatory function of microRNAs. We propose that manipulation of these cues and pathways offers the greatest potential for restoring ß-cell function after ex vivo expansion.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Células Secretoras de Insulina/patologia , MicroRNAs/metabolismo , Caderinas/genética , Proliferação de Células , Diabetes Mellitus Tipo 1/genética , Humanos , Transdução de Sinais/genéticaRESUMO
Stepwise approaches for the derivation of ß-cells from human embryonic stem cells have been described. However, low levels of endocrine specification limit the final yield of insulin-producing ß-cells. In this study, we show that the pyrrolo-pyrimidine Src family kinase (SFK) inhibitor PP2 effectively promotes the endocrine specification of human embryonic stem cell derivatives based on its capacity to induce the expression of proendocrine transcription factors (NGN3, NEUROD1, NKX2.2, and PAX4) and to significantly increase the final yield of insulin-positive cells. We further demonstrate that PP2 inhibits the activation of focal adhesion kinase (FAK), and selective inhibition of this kinase is also sufficient to induce early endocrine commitment based on increased expression of NGN3, NEUROD1, and NKX2.2. Additional studies using dominant negative constructs and isolated human fetal pancreata suggest that c-Src is at least partially responsible for inhibiting early endocrine specification. Mechanistically, we propose that inhibition of SFK/FAK signaling can promote endocrine specification by limiting activation of the TGFßR/Smad2/3 pathway. Moreover, we show that inhibition of SFK/FAK signaling suppresses cell growth, increases the expression of the ß-cell-associated cyclin-dependent kinase inhibitor p57kip2, and simultaneously suppresses the expression of Id1 and Id2. This study has important implications for the derivation of ß-cells for the cell-based therapy of diabetes and sheds new light on the signaling events that regulate early endocrine specification.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Células Secretoras de Insulina/metabolismo , Células-Tronco Pluripotentes/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Antígenos de Diferenciação/biossíntese , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/terapia , Células-Tronco Embrionárias/citologia , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio , Humanos , Proteína 1 Inibidora de Diferenciação/biossíntese , Proteína 2 Inibidora de Diferenciação/biossíntese , Células Secretoras de Insulina/citologia , Proteínas Nucleares , Células-Tronco Pluripotentes/citologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Netrins have been extensively studied in the developing central nervous system as pathfinding guidance cues, and more recently in non-neural tissues where they mediate cell adhesion, migration and differentiation. Netrin-4, a distant relative of Netrins 1-3, has been proposed to affect cell fate determination in developing epithelia, though receptors mediating these functions have yet to be identified. METHODOLOGY/PRINCIPAL FINDINGS: Using human embryonic pancreatic cells as a model of developing epithelium, here we report that Netrin-4 is abundantly expressed in vascular endothelial cells and pancreatic ductal cells, and supports epithelial cell adhesion through integrins α2ß1 and α3ß1. Interestingly, we find that Netrin-4 recognition by embryonic pancreatic cells through integrins α2ß1 and α3ß1 promotes insulin and glucagon gene expression. In addition, full genome microarray analysis revealed that fetal pancreatic cell adhesion to Netrin-4 causes a prominent down-regulation of cyclins and up-regulation of negative regulators of the cell cycle. Consistent with these results, a number of other genes whose activities have been linked to developmental decisions and/or cellular differentiation are up-regulated. CONCLUSIONS/SIGNIFICANCE: Given the recognized function of blood vessels in epithelial tissue morphogenesis, our results provide a mechanism by which endothelial-derived Netrin-4 may function as a pro-differentiation cue for adjacent developing pancreatic cell populations expressing adhesion receptors α2ß1 and α3ß1 integrins.
Assuntos
Adesão Celular , Diferenciação Celular , Endotélio Vascular/metabolismo , Células Epiteliais/metabolismo , Glucagon/metabolismo , Insulina/metabolismo , Integrina alfa2beta1/metabolismo , Integrina alfa3beta1/metabolismo , Fatores de Crescimento Neural/metabolismo , Ductos Pancreáticos/citologia , Biomarcadores/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Endotélio Vascular/citologia , Feto/citologia , Feto/metabolismo , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Glucagon/genética , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Insulina/genética , Fatores de Crescimento Neural/genética , Netrinas , Análise de Sequência com Séries de Oligonucleotídeos , Ductos Pancreáticos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A critical shortage of donor pancreata currently prevents the development of a universal cell-based therapy for type I diabetes. The ex vivo expansion of insulin-producing beta-cells offers a potential solution but is problematic due to the inherent tendency of these cells to transition into mesenchymal-like cells that are devoid of function. Here, we demonstrate for the first time that exposure to elements of the extracellular matrix (ECM) directly potentiates the mesenchymal transition of cultured fetal beta-cells and causes associated declines in insulin gene expression. Individual ECM constituents varied in their ability to induce such responses, with collagen-IV (C-IV) and fibronectin inducing strong responses, whereas laminin-1 had no significant effect. Mesenchymal transition and concomitant losses in insulin gene expression observed on C-IV were found to be dependent on beta(1)-integrin ligation and were augmented in the presence of hepatocyte growth factor. Importantly, selective inhibition of c-Src, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) prior to exposure to C-IV prevented mesenchymal transition and effectively preserved insulin expression. Fetal beta-cells undergoing mesenchymal transition were found to acquire alpha(1)beta(1) expression, and ligation of this integrin then promotes declines in insulin gene expression and a marked increase in beta-cell motility. Inhibition of Src-, ERK-, or JNK-dependent signaling combined with the selective regulation of matrix exposure may ultimately facilitate the development of more effective beta-cell expansion protocols.
Assuntos
Transdiferenciação Celular , Proteínas da Matriz Extracelular/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/genética , Integrina alfa1beta1/metabolismo , Mesoderma/metabolismo , Transdução de Sinais , Idoso , Proteína Tirosina Quinase CSK , Adesão Celular , Movimento Celular , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Regulação para Baixo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibronectinas/metabolismo , Idade Gestacional , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Laminina/metabolismo , Pessoa de Meia-Idade , Pâncreas/embriologia , Pâncreas/metabolismo , Fenótipo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção , Vimentina/metabolismo , Quinases da Família srcRESUMO
Using human embryonic stem cells (hESCs), we describe a novel method for the rapid derivation and enrichment of cells that are comparable to primordial germ cells (PGCs) and Sertoli cells. The methodology described is based on modest changes to the growth conditions commonly used to expand hESCs and does not require genetic manipulation or complex three-dimensional culture. Remarkably, we have determined that simply reducing the size of cultured ESC colonies and manipulating the number of feeding cycles, results in the rapid emergence of cells that are comparable to migratory PGCs. Importantly, these cells can be monitored and purified on the basis of the expression of the chemokine receptor CXCR4. Under more stringent differentiating conditions these cells mature and upregulate the expression of specific germ cell markers. Importantly, this process is accompanied by the development of Sertoli-like support cells. Such cells normally provide trophic support and immunoprotection to developing germ cells and may have significant clinical utility in the prevention of graft rejection. The putative Sertoli-germ cell cocultures generated in this study may ultimately be developed to study and manipulate interactions and processes involved in human gametogenesis.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Células Germinativas/citologia , Células de Sertoli/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Movimento Celular , Forma Celular , Sobrevivência Celular , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Células Germinativas/ultraestrutura , Humanos , Masculino , Camundongos , Fenótipo , Receptores CXCR4/metabolismo , Células de Sertoli/metabolismo , Células de Sertoli/ultraestruturaRESUMO
Named after the Sanskrit word netr, which means 'one who guides', the netrin family of secreted proteins provides migrational cues in the developing central nervous system. Recently, netrins have also been shown to regulate diverse processes (such as cell adhesion, motility, proliferation, differentiation and, ultimately, cell survival) in a number of non-neuronal tissues. In some cases, netrins affect these functions through non-classic netrin receptors, prompting a renewed interest in these factors in and beyond the nervous system.
Assuntos
Sistema Nervoso Central/metabolismo , Fatores de Crescimento Neural/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Humanos , Modelos Biológicos , Neovascularização Fisiológica , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Receptores de Netrina , Receptores de Superfície Celular/metabolismoRESUMO
The impact of extracellular matrix on insulin production needs to be understood both to optimize the derivation of functional beta-cells for transplantation and to understand mechanisms controlling islet neogenesis and glucose homeostasis. In this study, we present evidence that adhesion to some common matrix constituents has a profound impact on the transcription, secretion, and storage of insulin by human beta-cells. The integrin-dependent adhesion of fetal beta-cells to both collagen IV and vitronectin induces significant glucose-independent insulin secretion and a substantial reciprocal decline in insulin content. Collagen IV, but not vitronectin, induces comparable responses in adult beta-cells. Inhibition of extracellular signal-regulated kinase activation abrogates matrix-induced insulin secretion and effectively preserves the insulin content of adherent beta-cells. Using real-time PCR, we demonstrate that adhesion of both fetal and adult beta-cells to collagen IV and vitronectin also results in the marked suppression of insulin gene transcription. Based on these findings, we contend that integrin-dependent adhesion and signaling in response to certain matrices can have a significant negative impact on insulin production by primary human beta-cells. Such responses were not found to be associated with cell death but may precede beta-cell dedifferentiation.
Assuntos
Matriz Extracelular/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Integrinas/fisiologia , Butadienos/farmacologia , Adesão Celular/fisiologia , Células Cultivadas , Colágeno Tipo IV/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feto/citologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Nitrilas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/fisiologia , Vitronectina/fisiologiaRESUMO
Collagens have been shown to influence the survival and function of cultured beta-cells; however, the utilization and function of individual collagen receptors in beta-cells is largely unknown. The integrin superfamily contains up to five collagen receptors, but we have determined that alpha(1)beta(1) is the primary receptor utilized by both fetal and adult beta-cells. Cultured beta-cells adhered to and migrated on collagen type IV (Col-IV), and these responses were mediated almost exclusively by alpha(1)beta(1). The migration of cultured beta-cells to Col-IV significantly exceeded that to other matrix components suggesting that this substrate is of unique importance for beta-cell motility. The interaction of alpha(1)beta(1) with Col-IV also resulted in significant insulin secretion at basal glucose concentrations. A subset of beta-cells in developing islets was confirmed to express alpha(1)beta(1), and this expression co-localized with Col-IV in the basal membranes of juxtaposed endothelial cells. Our findings indicate that alpha(1)beta(1) and Col-IV contribute to beta-cell functions known to be important for islet morphogenesis and glucose homeostasis.
Assuntos
Colágeno Tipo IV/fisiologia , Insulina/metabolismo , Integrina alfa1beta1/metabolismo , Ilhotas Pancreáticas/metabolismo , Idoso , Adesão Celular , Movimento Celular , Separação Celular , Sobrevivência Celular , Células Cultivadas , Colágeno/metabolismo , Citometria de Fluxo , Glucose/metabolismo , Humanos , Imuno-Histoquímica , Secreção de Insulina , Integrinas/metabolismo , Microscopia de Fluorescência , Pessoa de Meia-Idade , Pâncreas/embriologia , Fatores de TempoRESUMO
The cell adhesion molecule L1 has been implicated in a variety of motile processes, including neurite extension, cerebellar cell migration, extravasation, and metastasis. Homophilic or heterophilic L1 binding and concomitant signaling have been shown to promote cell motility in the short term. In this report, L1 is also shown to induce and maintain a motile and invasive phenotype by promoting gene transcription. In the presence of serum or platelet-derived growth factor, L1 promotes heightened and sustained activation of the extracellular signal-regulated kinase pathway. Activation of this pathway then induces the expression of motility- and invasion-associated gene products, including the beta(3)-integrin subunit, small GTPases, and the cysteine proteases cathepsin-L and -B. Induction of integrin alpha(v)beta(3) and rac-1 is shown to contribute directly to L1-dependent haptotaxis, whereas induction of cathepsins-L and -B promotes matrix invasion. This study provides a novel translational mechanism to account for the association between L1 expression and motile processes involved in metastasis and development.