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SUMMARY: Measuring cellular energetics is essential to understanding a matrix's (e.g. cell, tissue or biofluid) metabolic state. The Agilent Seahorse machine is a common method to measure real-time cellular energetics, but existing analysis tools are highly manual or lack functionality. The Cellular Energetics Analysis Software (ceas) R package fills this analytical gap by providing modular and automated Seahorse data analysis and visualization. AVAILABILITY AND IMPLEMENTATION: Ceas is available on CRAN (https://cran.r-project.org/package=ceas). Source code and installable tarballs are freely available for download at https://github.com/jamespeapen/ceas/releases/ under the MIT license. Package documentation may be found at https://jamespeapen.github.io/ceas/. ceas is implemented in R and is supported on macOS, Windows and Linux. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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PURPOSE OF REVIEW: Wnt signaling plays a central role in development and homeostasis, and its dysregulation is a common event in many types of human cancer. Here we explore in detail the contributions of Wnt signaling to the initiation and maintenance of three types of saroma: Ewing sarcoma, osteosarcoma, and malignant peripheral nerve sheath tumors. This review provides an overview of the Wnt signaling pathway and explores in detail the current knowledge about its role in the initiation or maintenance of three tumor types: Ewing sarcoma, osteosarcoma, and malignant peripheral nerve sheath tumors. RECENT FINDINGS: Recent work has assessed the role(s) of Wnt signaling within these cell types. This review provides an overview of the mechanistic insights that have been gained from a number of recent studies to set the foundation for potential therapeutic applications. Wnt signaling has emerged as a potentially critical pathway in maintaining the growth of these types of tumors. Given the fact that many new inhibitors of the pathway have recently or will soon enter Phase 1 clinical trials, it is likely that assessment of their activity in these tumor types will occur in human patients.
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Neoplasias Ósseas/metabolismo , Neurilemoma/metabolismo , Osteossarcoma/metabolismo , Sarcoma de Ewing/metabolismo , Via de Sinalização Wnt , Humanos , Neurofibromatose 1/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND: Osteosarcoma (OS) is the most common type of solid bone cancer, with latent metastasis being a typical mode of disease progression and a major contributor to poor prognosis. For this to occur, cells must resist anoikis and be able to recapitulate tumorigenesis in a foreign microenvironment. Finding novel approaches to treat osteosarcoma and target those cell subpopulations that possess the ability to resist anoikis and contribute to metastatic disease is imperative. Here we investigate anchorage-independent (AI) cell growth as a model to better characterize anoikis resistance in human osteosarcoma while using an expression profiling approach to identify and test targetable signaling pathways. METHODS: Established human OS cell lines and patient-derived human OS cell isolates were subjected to growth in either adherent or AI conditions using Ultra-Low Attachment plates in identical media conditions. Growth rate was assessed using cell doubling times and chemoresistance was assessed by determining cell viability in response to a serial dilution of either doxorubicin or cisplatin. Gene expression differences were examined using quantitative reverse-transcription PCR and microarray with principal component and pathway analysis. In-vivo OS xenografts were generated by either subcutaneous or intratibial injection of adherent or AI human OS cells into athymic nude mice. Statistical significance was determined using student's t-tests with significance set at α=0.05. RESULTS: We show that AI growth results in a global gene expression profile change accompanied by significant chemoresistance (up to 75 fold, p<0.05). AI cells demonstrate alteration of key mediators of mesenchymal differentiation (ß-catenin, Runx2), stemness (Sox2), proliferation (c-myc, Akt), and epigenetic regulation (HDAC class 1). AI cells were equally tumorigenic as their adherent counterparts, but showed a significantly decreased rate of growth in-vitro and in-vivo (p<0.05). Treatment with the pan-histone deacetylase inhibitor vorinostat and the DNA methyltransferase inhibitor 5-azacytidine mitigated AI growth, while 5-azacytidine sensitized anoikis-resistant cells to doxorubicin (p<0.05). CONCLUSIONS: These data demonstrate remarkable plasticity in anoikis-resistant human osteosarcoma subpopulations accompanied by a rapid development of chemoresistance and altered growth rates mirroring the early stages of latent metastasis. Targeting epigenetic regulation of this process may be a viable therapeutic strategy.
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Anoikis , Neoplasias Ósseas/genética , Epigênese Genética , Perfilação da Expressão Gênica , Osteossarcoma/genética , Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Osteossarcoma/tratamento farmacológico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cutaneous neurofibromas (CNFs) are benign tumors that occur in the dermis of individuals with the inherited tumor predisposition disorder, neurofibromatosis type 1. CNFs cause disfigurement, pain, burning, and itching, resulting in substantially reduced QOL in patients with neurofibromatosis type 1. CNFs are benign tumors that exhibit cellular and molecular heterogeneity, making it difficult to develop tractable in vitro or in vivo models. As a result, CNF research and drug discovery efforts have been limited. To address this need, we developed a reproducible patient-derived explant (PDE) ex vivo culture model using CNF tumors from patients with neurofibromatosis type 1. CNF PDEs remain viable in culture for over 9 days and recapitulate the cellular composition and molecular signaling of CNFs. Using CNF PDEs as a model system, we found that proliferation was associated with increased T-cell infiltration. Furthermore, we identified a pattern of reciprocal inflammatory signaling in CNF PDEs in which tumors rely on prostaglandin or leukotriene-mediated signaling pathways. As proof of principle, we show that ex vivo glucocorticoid treatment reduced the expression of proinflammatory genes, confirming that CNF PDEs are a useful model for both mechanistic studies and preclinical drug testing.
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Neurofibroma , Neurofibromatose 1 , Neoplasias Cutâneas , Humanos , Neurofibromatose 1/patologia , Neurofibromatose 1/metabolismo , Neurofibromatose 1/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neurofibroma/patologia , Neurofibroma/genética , Neurofibroma/metabolismo , Proliferação de Células , Transdução de Sinais , Feminino , MasculinoRESUMO
OBJECTIVE: NF1 is a tumor suppressor gene and its protein product, neurofibromin, is a negative regulator of the RAS pathway. NF1 is one of the top driver mutations in sporadic breast cancer such that 27 % of breast cancers exhibit damaging NF1 alterations. NF1 loss-of-function is a frequent event in the genomic evolution of estrogen receptor (ER)+ breast cancer metastasis and endocrine resistance. Individuals with Neurofibromatosis type 1 (NF) - a disorder caused by germline NF1 mutations - have an increased risk of dying from breast cancer [1-4]. NF-related breast cancers are associated with decreased overall survival compared to sporadic breast cancer. Despite numerous studies interrogating the role of RAS mutations in tumor metabolism, no study has comprehensively profiled the NF1-deficient breast cancer metabolome to define patterns of energetic and metabolic reprogramming. The goals of this investigation were (1) to define the role of NF1 deficiency in estrogen receptor-positive (ER+) breast cancer metabolic reprogramming and (2) to identify potential targeted pathway and metabolic inhibitor combination therapies for NF1-deficient ER + breast cancer. METHODS: We employed two ER+ NF1-deficient breast cancer models: (1) an NF1-deficient MCF7 breast cancer cell line to model sporadic breast cancer, and (2) three distinct, Nf1-deficient rat models to model NF-related breast cancer [1]. IncuCyte proliferation analysis was used to measure the effect of NF1 deficiency on cell proliferation and drug response. Protein quantity was assessed by Western Blot analysis. We then used RNAseq to investigate the transcriptional effect of NF1 deficiency on global and metabolism-related transcription. We measured cellular energetics using Agilent Seahorse XF-96 Glyco Stress Test and Mito Stress Test assays. We performed stable isotope labeling and measured [U-13C]-glucose and [U-13C]-glutamine metabolite incorporation and measured total metabolite pools using mass spectrometry. Lastly, we used a Bliss synergy model to investigate NF1-driven changes in targeted and metabolic inhibitor synergy. RESULTS: Our results revealed that NF1 deficiency enhanced cell proliferation, altered neurofibromin expression, and increased RAS and PI3K/AKT pathway signaling while constraining oxidative ATP production and restricting energetic flexibility. Neurofibromin deficiency also increased glutamine influx into TCA intermediates and dramatically increased lipid pools, especially triglycerides (TG). Lastly, NF1 deficiency alters the synergy between metabolic inhibitors and traditional targeted inhibitors. This includes increased synergy with inhibitors targeting glycolysis, glutamine metabolism, mitochondrial fatty acid transport, and TG synthesis. CONCLUSIONS: NF1 deficiency drives metabolic reprogramming in ER+ breast cancer. This reprogramming is characterized by oxidative ATP constraints, glutamine TCA influx, and lipid pool expansion, and these metabolic changes introduce novel metabolic-to-targeted inhibitor synergies.
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Neurofibromatose 1 , Neurofibromina 1 , Animais , Ratos , Trifosfato de Adenosina/metabolismo , Glutamina/metabolismo , Lipídeos , Reprogramação Metabólica , Neurofibromatose 1/genética , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismoRESUMO
Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.
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Resistencia a Medicamentos Antineoplásicos , Neurofibromatose 1 , Inibidores de Proteínas Quinases , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Camundongos , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/patologia , Neoplasias de Bainha Neural/tratamento farmacológico , Linhagem Celular Tumoral , Transdução de Sinais , Linhagem da Célula/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Neurofibrossarcoma/genética , Neurofibrossarcoma/patologia , Neurofibrossarcoma/tratamento farmacológico , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/genéticaRESUMO
Background: Neurofibromin, coded by the NF1 tumor suppressor gene, is the main negative regulator of the RAS pathway and is frequently mutated in various cancers. Women with Neurofibromatosis Type I (NF1)-a tumor predisposition syndrome caused by a germline NF1 mutation-have an increased risk of developing aggressive breast cancer with poorer prognosis. The mechanism by which NF1 mutations lead to breast cancer tumorigenesis is not well understood. Therefore, the objective of this work was to identify stromal alterations before tumor formation that result in the increased risk and poorer outcome seen among NF1 patients with breast cancer. Approach: To accurately model the germline monoallelic NF1 mutations in NF1 patients, we utilized an Nf1-deficient rat model with accelerated mammary development before presenting with highly penetrant breast cancer. Results: We identified increased collagen content in Nf1-deficient rat mammary glands before tumor formation that correlated with age of tumor onset. Additionally, gene expression analysis revealed that Nf1-deficient mature adipocytes in the rat mammary gland have increased collagen expression and shifted to a fibroblast and preadipocyte expression profile. This alteration in lineage commitment was also observed with in vitro differentiation, however, flow cytometry analysis did not show a change in mammary adipose-derived mesenchymal stem cell abundance. Conclusion: Collectively, this study uncovered the previously undescribed role of Nf1 in mammary collagen deposition and regulating adipocyte differentiation. In addition to unraveling the mechanism of tumor formation, further investigation of adipocytes and collagen modifications in preneoplastic mammary glands will create a foundation for developing early detection strategies of breast cancer among NF1 patients.
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Skeletal metastases result in significant morbidity and mortality. This is particularly true of cancers with a strong predilection for the bone, such as breast, prostate, and lung cancers. There is currently no reliable cure for skeletal metastasis, and palliative therapy options are limited. The Wnt signaling pathway has been found to play an integral role in the process of skeletal metastasis and may be an important clinical target. Several experimental models of skeletal metastasis have been used to find new biomarkers and test new treatments. In this review, we discuss pathologic process of bone metastasis, the roles of the Wnt signaling, and the available experimental models and treatments.
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Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/radioterapia , Neoplasias Ósseas/cirurgia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , beta Catenina/metabolismoRESUMO
Cutaneous neurofibromas (cNFs) are benign tumors of the skin that affect >95% of adults with neurofibromatosis type 1. Despite their benign histology, cNFs can significantly impact QOL due to disfigurement, pain, and pruritus. There are no approved therapies for cNFs. Existing treatments are limited to surgery or laser-based treatments that have had mixed success and cannot be readily applied to a large number of tumors. We review cNF treatment options that are currently available and under investigation, discuss the regulatory considerations specific to cNFs, and propose strategies to improve cNF clinical trial design and standardize clinical trial endpoints.
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Neurofibroma , Neurofibromatose 1 , Neoplasias Cutâneas , Adulto , Humanos , Qualidade de Vida , Neurofibroma/patologia , Neurofibroma/terapia , Neurofibromatose 1/terapia , Neoplasias Cutâneas/patologia , PruridoRESUMO
Neurofibromatosis type 1 is one of the most common genetic disorders of the nervous system and predisposes patients to develop benign and malignant tumors. Cutaneous neurofibromas (cNFs) are NF1-associated benign tumors that affect nearly 100% of patients with NF1. cNFs dramatically reduce patients' QOL owing to their unaesthetic appearance, physical discomfort, and corresponding psychological burden. There is currently no effective drug therapy option, and treatment is restricted to surgical removal. One of the greatest hurdles for cNF management is the variability of clinical expressivity in NF1, resulting in intrapatient and interpatient cNF tumor burden heterogeneity, that is, the variability in the presentation and evolution of these tumors. There is growing evidence that a wide array of factors are involved in the regulation of cNF heterogeneity. Understanding the mechanisms underlying this heterogeneity of cNF at the molecular, cellular, and environmental levels can facilitate the development of innovative and personalized treatment regimens.
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Neurofibroma , Neurofibromatose 1 , Neoplasias Cutâneas , Humanos , Neurofibromatose 1/genética , Neurofibromatose 1/terapia , Qualidade de Vida , Carga Tumoral , Neurofibroma/genética , Neoplasias Cutâneas/genéticaRESUMO
Neurofibromatosis type 1 (NF1) is caused by a nonfunctional copy of the NF1 tumor suppressor gene that predisposes patients to the development of cutaneous neurofibromas (cNFs), the skin tumor that is the hallmark of this condition. Innumerable benign cNFs, each appearing by an independent somatic inactivation of the remaining functional NF1 allele, form in nearly all patients with NF1. One of the limitations in developing a treatment for cNFs is an incomplete understanding of the underlying pathophysiology and limitations in experimental modeling. Recent advances in preclinical in vitro and in vivo modeling have substantially enhanced our understanding of cNF biology and created unprecedented opportunities for therapeutic discovery. We discuss the current state of cNF preclinical in vitro and in vivo model systems, including two- and three-dimensional cell cultures, organoids, genetically engineered mice, patient-derived xenografts, and porcine models. We highlight the models' relationship to human cNFs and how they can be used to gain insight into cNF development and therapeutic discovery.
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Neurofibroma , Neurofibromatose 1 , Neoplasias Cutâneas , Camundongos , Humanos , Animais , Suínos , Neurofibromatose 1/genética , Neurofibromatose 1/terapia , Mutação , Neurofibroma/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , AlelosRESUMO
Cutaneous neurofibromas (cNFs) are the most common tumor in people with the rasopathy neurofibromatosis type 1. They number in hundreds or even thousands throughout the body, and currently, there are no effective interventions to prevent or treat these skin tumors. To facilitate the identification of novel and effective therapies, essential studies including a more refined understanding of cNF biology and the role of RAS signaling and downstream effector pathways responsible for cNF initiation, growth, and maintenance are needed. This review highlights the current state of knowledge of RAS signaling in cNF pathogenesis and therapeutic development for cNF treatment.
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Neurofibroma , Neurofibromatose 1 , Neoplasias Cutâneas , Humanos , Neurofibroma/metabolismo , Neurofibroma/patologia , Neurofibromatose 1/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Transdução de SinaisRESUMO
The patient was a 45-year-old male who initially presented with a left hydrocele. During radiographic work-up, a 26 cm right retroperitoneal lipoma was incidentally discovered. Despite a recommendation for preoperative radiation therapy followed by surgery from the sarcoma multispecialty team, the patient opted for surgery alone, in the hopes of avoiding damage or loss of his right kidney. Following surgical excision of the 39 cm well-differentiated liposarcoma, with removal of the perinephric fat adjacent to the tumor thereby preserving the kidney, he was discharged home after two nights in the hospital. Follow-up imaging eight months later showed no recurrence.
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Benign peripheral nerve sheath tumors are the clinical hallmark of Neurofibromatosis Type 1. They account for substantial morbidity and mortality in NF1. Cutaneous (CNF) and plexiform neurofibromas (PNF) share nearly identical histology, but maintain different growth rates and risk of malignant conversion. The reasons for this disparate clinical behavior are not well explained by recent genome or transcriptome profiling studies. We hypothesized that CNFs and PNFs are epigenetically distinct tumor types that exhibit differential signaling due to genome-wide and site-specific methylation events. We interrogated the methylation profiles of 45 CNFs and 17 PNFs from NF1 subjects with the Illumina EPIC 850K methylation array. Based on these profiles, we confirm that CNFs and PNFs are epigenetically distinct tumors with broad differences in higher-order chromatin states and specific methylation events altering genes involved in key biological and cellular processes, such as inflammation, RAS/MAPK signaling, actin cytoskeleton rearrangement, and oxytocin signaling. Based on our identification of two separate DMRs associated with alternative leading exons in MAP2K3, we demonstrate differential RAS/MKK3/p38 signaling between CNFs and PNFs. Epigenetic reinforcement of RAS/MKK/p38 was a defining characteristic of CNFs leading to pro-inflammatory signaling and chromatin conformational changes, whereas PNFs signaled predominantly through RAS/MEK. Tumor size also correlated with specific CpG methylation events. Taken together, these findings confirm that NF1 deficiency influences the epigenetic regulation of RAS signaling fates, accounting for observed differences in CNF and PNF clinical behavior. The extension of these findings is that CNFs may respond differently than PNFs to RAS-targeted therapeutics raising the possibility of targeting p38-mediated inflammation for CNF treatment.
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Neurofibroma Plexiforme , Neurofibromatose 1 , Epigênese Genética , Epigenômica , Humanos , Neurofibroma Plexiforme/genética , Neurofibromatose 1/genética , Transdução de SinaisRESUMO
PURPOSE: Hand amputations cause marked functional loss for patients. In patients with large soft tissue sarcomas of the hand, partial hand preservation is extremely challenging for surgeons attempting a complete resection of the tumor with negative resection margins. We conducted this review to examine the oncologic outcome, including local recurrence rate and patient overall survival, and functional outcome after resections for large soft tissue sarcomas with partial hand preservation. METHODS: We performed a retrospective review of all patients with soft tissue sarcomas of the hand treated at our institution from 1995 to 2007. We identified 8 patients who had tumors at least 5 cm in maximum dimension and had tumor resection with partial hand preservation. The mean age at the time of surgery was 49 years (range, 10-80 years). Two patients had myxofibrosarcoma, 2 patients had synovial sarcoma, 2 patients had malignant fibrous histiocytoma, 1 patient had a malignant peripheral nerve sheath tumor, and 1 patient had a liposarcoma. Two patients had low-grade tumors, and 6 patients had high-grade tumors. Two patients had American Joint Committee on Cancer stage 1b tumors, and 6 patients had American Joint Committee on Cancer stage 3 tumors. No patients had distant metastases at the time of surgery. Hand function was evaluated using Musculoskeletal Tumor Society criteria. RESULTS: Of the 8 patients, 1 died of distant metastatic disease, 1 developed local tumor recurrence and is alive with locally recurrent disease, and the other 6 patients are completely disease-free. The mean Musculoskeletal Tumor Society score was 26 (range, 19-29), with the 2 patients who had received double-ray amputations having the lower scores (19 and 24). CONCLUSIONS: Partial hand preservation is possible in selected patients with large soft tissue sarcomas of the hand, obtaining low local recurrence rates, good overall survival, and good functional outcome. However, all effort should be made to achieve negative resection margins. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.
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Mãos/cirurgia , Salvamento de Membro/métodos , Recidiva Local de Neoplasia/patologia , Sarcoma/diagnóstico , Sarcoma/cirurgia , Neoplasias de Tecidos Moles/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Amputação Cirúrgica/métodos , Braço , Membros Artificiais , Criança , Estudos de Coortes , Feminino , Seguimentos , Mãos/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/radioterapia , Estadiamento de Neoplasias , Ajuste de Prótese , Estudos Retrospectivos , Medição de Risco , Sarcoma/mortalidade , Neoplasias de Tecidos Moles/mortalidade , Neoplasias de Tecidos Moles/patologia , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary epithelial cell isolation; 2) rat mammary fibroblast isolation; 3) culturing rat mammary epithelial cells; and characterization of rat mammary cells by 4) flow cytometric analysis; and 5) immunofluorescence. Cells derived from this protocol can be used for many purposes, including RNAseq, drug studies, functional assays, gene/protein expression analyses, and image analysis.
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Neurofibromatosis Type 1 (NF1)-related Malignant Peripheral Nerve Sheath Tumors (MPNST) are highly resistant sarcomas that account for significant mortality. The mechanisms of therapy resistance are not well-understood in MPNSTs, particularly with respect to kinase inhibition strategies. In this study, we aimed to quantify the impact of both the genomic context and targeted therapy on MPNST resistance using reverse phase phosphoproteome array (RPPA) analysis. We treated tumorgrafts from three genetically engineered mouse models using MET (capmatinib) and MEK (trametinib) inhibitors and doxorubicin, and assessed phosphosignaling at 4 h, 2 days, and 21 days. Baseline kinase signaling in our mouse models recapitulated an MET-addicted state (NF1-MET), P53 mutation (NF1-P53), and HGF overexpression (NF1). Following perturbation with the drug, we observed broad and redundant kinome adaptations that extended well beyond canonical RAS/ERK or PI3K/AKT/mTOR signaling. MET and MEK inhibition were both associated with an initial inflammatory response mediated by kinases in the JAK/STAT pathway and NFkB. Growth signaling predominated at the 2-day and 21-day time points as a result of broad RTK and intracellular kinase activation. Interestingly, AXL and NFkB were strongly activated at the 2-day and 21-day time points, and tightly correlated, regardless of the treatment type or genomic context. The degree of kinome adaptation observed in innately resistant tumors was significantly less than the surviving fractions of responsive tumors that exhibited a latency period before reinitiating growth. Lastly, doxorubicin resistance was associated with kinome adaptations that strongly favored growth and survival signaling. These observations confirm that MPNSTs are capable of profound signaling plasticity in the face of kinase inhibition or DNA damaging agent administration. It is possible that by targeting AXL or NFkB, therapy resistance can be mitigated.
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Antineoplásicos/uso terapêutico , Sistema de Sinalização das MAP Quinases , Neoplasias de Bainha Neural/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteoma/metabolismo , Animais , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica , Benzamidas , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imidazóis/administração & dosagem , Imidazóis/uso terapêutico , Camundongos , Camundongos SCID , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias de Bainha Neural/genética , Neurofibromina 1/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Proteoma/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridonas/administração & dosagem , Piridonas/uso terapêutico , Pirimidinonas/administração & dosagem , Pirimidinonas/uso terapêutico , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Inibidores da Topoisomerase II/administração & dosagem , Inibidores da Topoisomerase II/uso terapêutico , Triazinas/administração & dosagem , Triazinas/uso terapêutico , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Malignant peripheral nerve sheath tumors (MPNST) are highly resistant sarcomas that occur in up to 13% of individuals with neurofibromatosis type I (NF1). Genomic analysis of longitudinally collected tumor samples in a case of MPNST disease progression revealed early hemizygous microdeletions in NF1 and TP53, with progressive amplifications of MET, HGF, and EGFR To examine the role of MET in MPNST progression, we developed mice with enhanced MET expression and Nf1 ablation (Nf1fl/ko;lox-stop-loxMETtg/+;Plp-creERTtg/+ ; referred to as NF1-MET). NF1-MET mice express a robust MPNST phenotype in the absence of additional mutations. A comparison of NF1-MET MPNSTs with MPNSTs derived from Nf1ko/+;p53R172H;Plp-creERTtg/+ (NF1-P53) and Nf1ko/+;Plp-creERTtg/+ (NF1) mice revealed unique Met, Ras, and PI3K signaling patterns. NF1-MET MPNSTs were uniformly sensitive to the highly selective MET inhibitor, capmatinib, whereas a heterogeneous response to MET inhibition was observed in NF1-P53 and NF1 MPNSTs. Combination therapy of capmatinib and the MEK inhibitor trametinib resulted in reduced response variability, enhanced suppression of tumor growth, and suppressed RAS/ERK and PI3K/AKT signaling. These results highlight the influence of concurrent genomic alterations on RAS effector signaling and therapy response to tyrosine kinase inhibitors. Moreover, these findings expand our current understanding of the role of MET signaling in MPNST progression and identify a potential therapeutic niche for NF1-related MPNSTs.Significance: Longitudinal genomic analysis reveals a positive selection for MET and HGF copy number gain early in malignant peripheral nerve sheath tumor progression. Cancer Res; 78(13); 3672-87. ©2018 AACR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/genética , Neurofibromatose 1/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Adolescente , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzamidas , Biomarcadores Tumorais/antagonistas & inibidores , Modelos Animais de Doenças , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Amplificação de Genes , Dosagem de Genes , Fator de Crescimento de Hepatócito/genética , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Estudos Longitudinais , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Triazinas/farmacologia , Triazinas/uso terapêuticoRESUMO
The key negative regulatory gene of the RAS pathway, NF1, is mutated or deleted in numerous cancer types and is associated with increased cancer risk and drug resistance. Even though women with neurofibromatosis (germline NF1 mutations) have a substantially increased breast cancer risk at a young age and NF1 is commonly mutated in sporadic breast cancers, we have a limited understanding of the role of NF1 in breast cancer. We utilized CRISPR-Cas9 gene editing to create Nf1 rat models to evaluate the effect of Nf1 deficiency on tumorigenesis. The resulting Nf1 indels induced highly penetrant, aggressive mammary adenocarcinomas that express estrogen receptor (ER) and progesterone receptor (PR). We identified distinct Nf1 mRNA and protein isoforms that were altered during tumorigenesis. To evaluate NF1 in human breast cancer, we analyzed genomic changes in a data set of 2000 clinically annotated breast cancers. We found NF1 shallow deletions in 25% of sporadic breast cancers, which correlated with poor clinical outcome. To identify biological networks impacted by NF1 deficiency, we constructed gene co-expression networks using weighted gene correlation network analysis (WGCNA) and identified a network connected to ESR1 (estrogen receptor). Moreover, NF1-deficient cancers correlated with established RAS activation signatures. Estrogen-dependence was verified by estrogen-ablation in Nf1 rats where rapid tumor regression was observed. Additionally, Nf1 deficiency correlated with increased estrogen receptor phosphorylation in mammary adenocarcinomas. These results demonstrate a significant role for NF1 in both NF1-related breast cancer and sporadic breast cancer, and highlight a potential functional link between neurofibromin and the estrogen receptor.