RESUMO
Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.
Assuntos
Técnicas Citológicas/métodos , Genoma Humano , Células-Tronco Pluripotentes Induzidas/citologia , Antibacterianos/farmacologia , Composição de Bases/genética , Linhagem Celular , Clonagem Molecular , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , MutaçãoRESUMO
The 78-kDa glucose regulated protein (GRP78) commonly upregulated in a wide variety of tumors is an important prognostic marker and a promising target for suppressing tumorigenesis and treatment resistance. While GRP78 is well established as a major endoplasmic reticulum (ER) chaperone with anti-apoptotic properties and a master regulator of the unfolded protein response, its new role as a regulator of oncoprotein expression is just emerging. MYC is dysregulated in about 70 % of human cancers and is the most commonly activated oncoprotein. However, despite recent advances, therapeutic targeting of MYC remains challenging. Here we identify GRP78 as a new target for suppression of MYC expression. Using multiple MYC-dependent cancer models including head and neck squamous cell carcinoma and their cisplatin-resistant clones, breast and pancreatic adenocarcinoma, our studies revealed that GRP78 knockdown by siRNA or inhibition of its activity by small molecule inhibitors (YUM70 or HA15) reduced c-MYC expression, leading to onset of apoptosis and loss of cell viability. This was observed in 2D cell culture, 3D spheroid and in xenograft models. Mechanistically, we determined that the suppression of c-MYC is at the post-transcriptional level and that YUM70 and HA15 treatment potently upregulated the eukaryotic translation inhibitor 4E-BP1, which targets eIF4E critical for c-MYC translation initiation. Furthermore, knock-down of 4E-BP1 via siRNA rescued YUM70-mediated c-MYC suppression. As YUM70 is also capable of suppressing N-MYC expression, this study offers a new approach to suppress MYC protein expression through knockdown or inhibition of GRP78.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Chaperona BiP do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico , Proteínas Proto-Oncogênicas c-myc , Humanos , Chaperona BiP do Retículo Endoplasmático/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/antagonistas & inibidores , Camundongos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis, how ALK contributes to tumor formation remains unclear. Here, we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic, while ALK/MYCN tumors resembled mesenchymal, neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling, particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN, but not FN1, delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore, loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally, inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus, ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling.
Assuntos
Neuroblastoma , Receptores Proteína Tirosina Quinases , Humanos , Quinase do Linfoma Anaplásico/genética , Moléculas de Adesão Celular , Linhagem Celular Tumoral , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Via de Sinalização WntRESUMO
Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.
Assuntos
Diferenciação Celular , Variações do Número de Cópias de DNA , Proteína Proto-Oncogênica N-Myc , Crista Neural , Neuroblastoma , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Crista Neural/metabolismo , Crista Neural/patologia , Feminino , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Aberrações Cromossômicas , Células-Tronco Embrionárias Humanas/metabolismo , Transcriptoma , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
Deregulation of neuroblastoma-derived myc (N-myc) is a leading cause of malignant brain tumors in children. To target N-myc-driven medulloblastoma, most research has focused on identifying genomic alterations or on the analysis of the medulloblastoma transcriptome. Here, we have broadly characterized the translatome of medulloblastoma and shown that N-myc unexpectedly drives selective translation of transcripts that promote protein homeostasis. Cancer cells are constantly exposed to proteotoxic stress associated with alterations in protein production or folding. It remains poorly understood how cancers cope with proteotoxic stress to promote their growth. Here, our data revealed that N-myc regulates the expression of specific components (â¼5%) of the protein folding machinery at the translational level through the major cap binding protein, eukaryotic initiation factor eIF4E. Reducing eIF4E levels in mouse models of medulloblastoma blocked tumorigenesis. Importantly, targeting Hsp70, a protein folding chaperone translationally regulated by N-myc, suppressed tumor growth in mouse and human medulloblastoma xenograft models. These findings reveal a previously hidden molecular program that promotes medulloblastoma formation and identify new therapies that may have impact in the clinic. SIGNIFICANCE: Translatome analysis in medulloblastoma shows that N-myc drives selective translation of transcripts that promote protein homeostasis and that represent new therapeutic vulnerabilities.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Criança , Humanos , Camundongos , Animais , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Meduloblastoma/patologia , Fator de Iniciação 4E em Eucariotos/genética , Modelos Animais de Doenças , Neoplasias Cerebelares/patologiaRESUMO
Advances in genome and tissue engineering have spurred significant progress and opportunity for innovation in cancer modeling. Human induced pluripotent stem cells (iPSCs) are an established and powerful tool to study cellular processes in the context of disease-specific genetic backgrounds; however, their application to cancer has been limited by the resistance of many transformed cells to undergo successful reprogramming. Here, we review the status of human iPSC modeling of solid tumors in the context of genetic engineering, including how base and prime editing can be incorporated into "bottom-up" cancer modeling, a term we coined for iPSC-based cancer models using genetic engineering to induce transformation. This approach circumvents the need to reprogram cancer cells while allowing for dissection of the genetic mechanisms underlying transformation, progression, and metastasis with a high degree of precision and control. We also discuss the strengths and limitations of respective engineering approaches and outline experimental considerations for establishing future models.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neoplasias , Sistemas CRISPR-Cas/genética , Edição de Genes , Humanos , Neoplasias/genética , Neoplasias/terapiaRESUMO
Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predisposed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma.
Assuntos
Síndrome do Nevo Basocelular/genética , Neoplasias Encefálicas/genética , Meduloblastoma/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Células-Tronco Neurais/fisiologia , Células Neuroepiteliais/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Síndrome do Nevo Basocelular/metabolismo , Síndrome do Nevo Basocelular/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/genética , RNA Helicases DEAD-box/genética , Modelos Animais de Doenças , Engenharia Genética , Predisposição Genética para Doença , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Camundongos SCID , Proteína Proto-Oncogênica N-Myc/genética , Proteínas de Neoplasias/genética , Receptor Patched-1/genética , Transplante de Células-Tronco , Transplante HeterólogoRESUMO
Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior "cranial" NCC form craniofacial bone, whereas solely posterior "trunk" NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages.
Assuntos
Diferenciação Celular , Crista Neural/citologia , Crista Neural/embriologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Biomarcadores , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes/efeitos dos fármacos , Transdução de Sinais , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
Glioma is the most common primary malignant brain tumor and arises throughout the central nervous system. Recent focus on stem-like glioma cells has implicated neural stem cells (NSCs), a minor precursor population restricted to germinal zones, as a potential source of gliomas. In this review, we focus on the relationship between oligodendrocyte progenitor cells (OPCs), the largest population of cycling glial progenitors in the postnatal brain, and gliomagenesis. OPCs can give rise to gliomas, with signaling pathways associated with NSCs also playing key roles during OPC lineage development. Gliomas can also undergo a switch from progenitor- to stem-like phenotype after therapy, consistent with an OPC-origin even for stem-like gliomas. Future in-depth studies of OPC biology may shed light on the etiology of OPC-derived gliomas and reveal new therapeutic avenues.
Assuntos
Transformação Celular Neoplásica , Glioma/patologia , Células-Tronco Neurais/fisiologia , Neuroglia/fisiologia , Animais , Diferenciação Celular/genética , Linhagem da Célula/fisiologia , Transformação Celular Neoplásica/patologia , Glioma/genética , Glioma/terapia , Humanos , Terapia de Alvo Molecular , Células-Tronco Neurais/patologia , Neuroglia/patologiaRESUMO
Recurrent mutations in H3F3A at K27 and G34 are frequent in pediatric glioblastoma, but it is unclear how these mutations promote tumorigenesis. In this issue of Cancer Discovery, Bjerke and colleagues identify mutations at G34 in H3F3A that result in elevated expression of MYCN as a potential mechanism in gliomagenesis.
Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Histonas/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Criança , Humanos , Mutação , Proteína Proto-Oncogênica N-Myc , Isoformas de Proteínas/genéticaRESUMO
Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to originate from undifferentiated neural crest cells. Amplification of the MYC family member, MYCN, is found in â¼25% of cases and correlates with high-risk disease and poor prognosis. Currently, amplification of MYCN remains the best-characterized genetic marker of risk in neuroblastoma. This article reviews roles for MYCN in neuroblastoma and highlights recent identification of other driver mutations. Strategies to target MYCN at the level of protein stability and transcription are also reviewed.
Assuntos
Mutação/genética , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Apoptose/genética , Ciclo Celular/genética , Proliferação de Células , Amplificação de Genes/genética , Expressão Gênica , Genes myc/genética , Marcadores Genéticos/genética , Humanos , Proteína Proto-Oncogênica N-Myc , Metástase Neoplásica , Neovascularização Patológica/genética , Neuroblastoma/patologia , Estabilidade Proteica , Fatores de Risco , Transcrição Gênica/genéticaRESUMO
Neural crest (NC) cells contribute to the development of many complex tissues of all three germ layers during embryogenesis, and its abnormal development accounts for several congenital birth defects. Generating NC cells-including specific subpopulations such as cranial, cardiac, and trunk NC cells-from human pluripotent stem cells will provide a valuable model system to study human development and disease. Here, we describe a rapid and robust NC differentiation method called "LSB-short" that is based on dual SMAD pathway inhibition. This protocol yields high percentages of NC cell populations from multiple human induced pluripotent stem and human embryonic stem cell lines in 8 days. The resulting cells can be propagated easily, retain NC marker expression over multiple passages, and can spontaneously differentiate into several NC-derived cell lineages, including smooth muscle cells, peripheral neurons, and Schwann cells. NC cells generated by this method represent cranial, cardiac and trunk NC subpopulations based on global gene expression analyses, are similar to in vivo analogues, and express a common set of NC alternative isoforms. Functionally, they are also able to migrate appropriately in response to chemoattractants such as SDF-1, FGF8b, and Wnt3a. By yielding NC cells that likely represent all NC subpopulations in a shorter time frame than other published methods, our LSB-short method provides an ideal model system for further studies of human NC development and disease.
RESUMO
Mammalian pluripotent stem cells (PSCs) represent an important venue for understanding basic principles regulating tissue-specific differentiation and discovering new tools that may facilitate clinical applications. Mechanisms that direct neural differentiation of PSCs involve growth factor signaling and transcription regulation. However, it is unknown whether and how electrical activity influences this process. Here we report a high throughput imaging-based screen, which uncovers that selamectin, an anti-helminthic therapeutic compound with reported activity on invertebrate glutamate-gated chloride channels, promotes neural differentiation of PSCs. We show that selamectin's pro-neurogenic activity is mediated by γ2-containing GABAA receptors in subsets of neural rosette progenitors, accompanied by increased proneural and lineage-specific transcription factor expression and cell cycle exit. In vivo, selamectin promotes neurogenesis in developing zebrafish. Our results establish a chemical screening platform that reveals activity-dependent neural differentiation from PSCs. Compounds identified in this and future screening might prove therapeutically beneficial for treating neurodevelopmental or neurodegenerative disorders. DOI:http://dx.doi.org/10.7554/eLife.00508.001.
Assuntos
Diferenciação Celular , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Animais , Linhagem da Célula , Células Cultivadas , Ensaios de Triagem em Larga Escala , Ivermectina/análogos & derivados , Ivermectina/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacosRESUMO
Receptor tyrosine kinases and integrins play an essential role in tumor cell invasion and metastasis. We previously showed that EGF and other growth factors induce human carcinoma cell invasion and metastasis mediated by integrin αvß5 that is prevented by Src blockade. MUC1, a transmembrane glycoprotein, is expressed in most epithelial tumors as a heterodimer consisting of an extracellular and a transmembrane subunit. The MUC1 cytoplasmic domain of the transmembrane subunit (MUC1.CD) translocates to the nucleus where it promotes the transcription of a metastatic gene signature associated with epithelial to mesenchymal transition. Here, we demonstrate a requirement for MUC1 in carcinoma cell metastasis dependent on EGFR and Src without affecting primary tumor growth. EGF stimulates Src-dependent MUC1 cleavage and nuclear localization leading to the expression of genes linked to metastasis. Moreover, expression of MUC1.CD results in its nuclear localization and is sufficient for transcription of the metastatic gene signature and tumor cell metastasis. These results demonstrate that EGFR and Src activity contribute to carcinoma cell invasion and metastasis mediated by integrin αvß5 in part by promoting proteolytic cleavage of MUC1 and highlight the ability of MUC1.CD to promote metastasis in a context-dependent manner. Our findings may have implications for the use and future design of targeted therapies in cancers known to express EGFR, Src, or MUC1.
Assuntos
Fator de Crescimento Epidérmico , Receptores ErbB , Mucina-1 , Invasividade Neoplásica/genética , Proteínas Tirosina Quinases , Receptores de Vitronectina , Animais , Proteína Tirosina Quinase CSK , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Embrião de Galinha , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Transição Epitelial-Mesenquimal , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Mucina-1/genética , Mucina-1/metabolismo , Metástase Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Transdução de Sinais , Quinases da Família srcRESUMO
RAF kinases regulate cell proliferation and survival and can be dysregulated in tumors. The role of RAF in cell proliferation has been linked to its ability to activate mitogen-activated protein kinase kinase 1 (MEK) and mitogen-activated protein kinase 1 (ERK). Here we identify a MEK-independent role for RAF in tumor growth. Specifically, in mitotic cells, CRAF becomes phosphorylated on Ser338 and localizes to the mitotic spindle of proliferating tumor cells in vitro as well as in murine tumor models and in biopsies from individuals with cancer. Treatment of tumors with allosteric inhibitors, but not ATP-competitive RAF inhibitors, prevents CRAF phosphorylation on Ser338 and localization to the mitotic spindle and causes cell-cycle arrest at prometaphase. Furthermore, we identify phospho-Ser338 CRAF as a potential biomarker for tumor progression and a surrogate marker for allosteric RAF blockade. Mechanistically, CRAF, but not BRAF, associates with Aurora kinase A (Aurora-A) and Polo-like kinase 1 (Plk1) at the centrosomes and spindle poles during G2/M. Indeed, allosteric or genetic inhibition of phospho-Ser338 CRAF impairs Plk1 activation and accumulation at the kinetochores, causing prometaphase arrest, whereas a phospho-mimetic Ser338D CRAF mutant potentiates Plk1 activation, mitosis and tumor progression in mice. These findings show a previously undefined role for RAF in tumor progression beyond the RAF-MEK-ERK paradigm, opening new avenues for targeting RAF in cancer.
Assuntos
MAP Quinase Quinase 1/metabolismo , Mitose , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Aurora Quinase A , Aurora Quinases , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Centrossomo/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Cinetocoros/metabolismo , MAP Quinase Quinase 1/genética , Camundongos , Camundongos Nus , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Transdução de Sinais , Fuso Acromático/metabolismo , Quinase 1 Polo-LikeRESUMO
Although it is well established that tumors initiate an angiogenic switch, the molecular basis of this process remains incompletely understood. Here we show that the miRNA miR-132 acts as an angiogenic switch by targeting p120RasGAP in the endothelium and thereby inducing neovascularization. We identified miR-132 as a highly upregulated miRNA in a human embryonic stem cell model of vasculogenesis and found that miR-132 was highly expressed in the endothelium of human tumors and hemangiomas but was undetectable in normal endothelium. Ectopic expression of miR-132 in endothelial cells in vitro increased their proliferation and tube-forming capacity, whereas intraocular injection of an antagomir targeting miR-132, anti-miR-132, reduced postnatal retinal vascular development in mice. Among the top-ranking predicted targets of miR-132 was p120RasGAP, which we found to be expressed in normal but not tumor endothelium. Endothelial expression of miR-132 suppressed p120RasGAP expression and increased Ras activity, whereas a miRNA-resistant version of p120RasGAP reversed the vascular response induced by miR-132. Notably, administration of anti-miR-132 inhibited angiogenesis in wild-type mice but not in mice with an inducible deletion of Rasa1 (encoding p120RasGAP). Finally, vessel-targeted nanoparticle delivery of anti-miR-132 restored p120RasGAP expression in the tumor endothelium, suppressed angiogenesis and decreased tumor burden in an orthotopic xenograft mouse model of human breast carcinoma. We conclude that miR-132 acts as an angiogenic switch by suppressing endothelial p120RasGAP expression, leading to Ras activation and the induction of neovascularization, whereas the application of anti-miR-132 inhibits neovascularization by maintaining vessels in the resting state.
Assuntos
Endotélio Vascular/patologia , MicroRNAs/fisiologia , Neovascularização Patológica/genética , Proteína p120 Ativadora de GTPase/genética , Animais , Anticorpos Monoclonais/farmacologia , Proliferação de Células , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Neovascularização Patológica/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Artéria Retiniana/efeitos dos fármacos , Artéria Retiniana/metabolismo , Artéria Retiniana/patologia , Regulação para Cima/genética , Regulação para Cima/fisiologia , Proteína p120 Ativadora de GTPase/metabolismoRESUMO
Tyrosine kinase receptors and integrins play essential roles in tumor cell invasion and metastasis. Previously, we showed that epidermal growth factor (EGF) stimulation of pancreatic carcinoma cells led to invasion and metastasis that was blocked by antagonists of integrin alpha(v)beta(5). Here, we show that EGF stimulates metastasis of carcinoma cells via a Src-dependent phosphorylation of p130 CAS leading to activation of Rap1, a small GTPase involved in integrin activation. Specifically, EGF receptor (EGFR)-induced Src activity leads to phosphorylation of a region within the CAS substrate domain, which is essential for Rap1 and alpha(v)beta(5) activation. This pathway induces alpha(v)beta(5)-mediated invasion and metastasis in vivo yet does not influence primary tumor growth or activation of other integrins on these cells. These findings show cross-talk between a tyrosine kinase receptor and an integrin involved in carcinoma cell invasion and metastasis and may explain in part how inhibitors of EGFR affect malignant disease.
Assuntos
Carcinoma/patologia , Receptores ErbB/fisiologia , Neoplasias Pancreáticas/patologia , Receptor Cross-Talk/fisiologia , Receptores de Vitronectina/fisiologia , Animais , Carcinoma/genética , Movimento Celular , Embrião de Galinha , Primers do DNA , Fator de Crescimento Epidérmico/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Sequências Repetidas Invertidas/genética , Pulmão/embriologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Pancreáticas/genética , Reação em Cadeia da Polimerase , RNA Neoplásico/genética , Receptores de Vitronectina/genética , Células Tumorais CultivadasRESUMO
Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin alpha(v)beta(3) is associated with progression of a variety of human tumors. Here we reveal a previously undescribed adhesion-independent role for integrin alpha(v)beta(3) in pancreatic cancer and other carcinomas. Specifically, alpha(v)beta(3) expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. These effects required recruitment of c-Src to the beta(3) integrin cytoplasmic tail, leading to c-Src activation, Crk-associated substrate (CAS) phosphorylation and tumor cell survival that, unexpectedly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Pharmacological blockade of c-Src kinase activity or decreased expression of endogenous alpha(v)beta(3) integrin or c-Src not only inhibited anchorage-independent growth but also suppressed metastasis in vivo, yet these manipulations did not affect tumor cell migration or invasion. These data define an unexpected role for an integrin as a mediator of anchorage independence, suggesting that an alpha(v)beta(3)-c-Src signaling module may account for the aggressive behavior of integrin alpha(v)beta(3)-expressing tumors in humans.