RESUMO
OBJECTIVE: Dedifferentiated endometrial cancer (DDEC) is an uncommon and clinically highly aggressive subtype of endometrial cancer characterized by genomic inactivation of SWItch/Sucrose Non-Fermentable (SWI/SNF) complex protein. It responds poorly to conventional systemic treatment and its rapidly progressive clinical course limits the therapeutic windows to trial additional lines of therapies. This underscores a pressing need for biologically accurate preclinical tumor models to accelerate therapeutic development. METHODS: DDEC tumor from surgical samples were implanted into immunocompromised mice for patient-derived xenograft (PDX) and cell line development. The histologic, immunophenotypic, genetic and epigenetic features of the patient tumors and the established PDX models were characterized. The SMARCA4-deficienct DDEC model was evaluated for its sensitivity toward a KDM6A/B inhibitor (GSK-J4) that was previously reported to be effective therapy for other SMARCA4-deficient cancer types. RESULTS: All three DDEC models exhibited rapid growth in vitro and in vivo, with two PDX models showing spontaneous development of metastases in vivo. The PDX tumors maintained the same undifferentiated histology and immunophenotype, and exhibited identical genomic and methylation profiles as seen in the respective parental tumors, including a mismatch repair (MMR)-deficient DDEC with genomic inactivation of SMARCA4, and two MMR-deficient DDECs with genomic inactivation of both ARID1A and ARID1B. Although the SMARCA4-deficient cell line showed low micromolecular sensitivity to GSK-J4, no significant tumor growth inhibition was observed in the corresponding PDX model. CONCLUSIONS: These established patient tumor-derived models accurately depict DDEC and represent valuable preclinical tools to gain therapeutic insights into this aggressive tumor type.
Assuntos
Neoplasias Encefálicas , Neoplasias Colorretais , Neoplasias do Endométrio , Feminino , Humanos , Animais , Camundongos , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Diferenciação Celular , Biomarcadores Tumorais/genética , DNA Helicases , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/genéticaRESUMO
OBJECTIVES: To investigate global changes in ureters at the transcriptional, translational and functional levels, both while stents are indwelling and after removal and recovery, and to study the effects of targeting pathways that play a potential role. METHODS: Pig ureters were stented for varying amounts of time (48 h, 72 h, 14 days) and the impact on peristalsis, dilatation and hydronephrosis were assessed. RNAseq, proteomic, histological and smooth muscle (SM) function analyses were performed on ureteric and kidney tissues to assess changes induced by stenting and recovery. Pathway analysis was performed using Ingenuity Pathway Analysis software. To study the impact of possible interventions, the effects of erythropoeitin (EPO) and a Gli1 inhibitor were assessed. RESULTS: Stenting triggers massive ureteric dilatation, aperistalsis and moderate hydronephrosis within 48 h. Pathways associated with obstruction, fibrosis and kidney injury were upregulated by stenting. Increased expression of GLI1, clusterin-α (a kidney injury marker) and collagen 4A2 (a fibrosis marker) was found in stented vs contralateral unstented ureters. EPO did not improve peristalsis or contraction force but did decrease non-purposeful spasming seen exclusively in stented ureters. Tamsulosin administration increased contractility but not rate of peristalsis in stented ureters. CONCLUSIONS: Ureters respond to stents similarly to how they respond to an obstruction, that is, with activation of pathways associated with hydronephrosis, fibrosis and kidney injury. This is driven by significant dilatation and associated ureteric SM dysfunction. EPO and tamsulosin induced mild favourable changes in SM physiology, suggesting that targeting specific pathways has potential to address stent-induced complications.
Assuntos
Hidronefrose , Ureter , Obstrução Ureteral , Animais , Suínos , Proteína GLI1 em Dedos de Zinco , Proteômica , Tansulosina , Ureter/patologia , Hidronefrose/etiologia , Stents/efeitos adversosRESUMO
Prostate cancer (PCa) progression is driven by androgen receptor (AR) signaling. Unfortunately, androgen-deprivation therapy and the use of even more potent AR pathway inhibitors (ARPIs) cannot bring about a cure. ARPI resistance (ie, castration-resistant PCa, CRPC) will inevitably develop. Previously, we demonstrated that GRB10 is an AR transcriptionally repressed gene that functionally contributes to CRPC development and ARPI resistance. GRB10 expression is elevated prior to CRPC development in our patient-derived xenograft models and is significantly upregulated in clinical CRPC samples. Here, we analyzed transcriptomic data from GRB10 knockdown in PCa cells and found that AR signaling is downregulated. While the mRNA expression of AR target genes decreased upon GRB10 knockdown, AR expression was not affected at the mRNA or protein level. We further found that phosphorylation of AR serine 81 (S81), which is critical for AR transcriptional activity, is decreased by GRB10 knockdown and increased by its overexpression. Luciferase assay using GRB10-knockdown cells also indicate reduced AR activity. Immunoprecipitation coupled with mass spectrometry revealed an interaction between GRB10 and the PP2A complex, which is a known phosphatase of AR. Further validations and analyses showed that GRB10 binds to the PP2Ac catalytic subunit with its PH domain. Mechanistically, GRB10 knockdown increased PP2Ac protein stability, which in turn decreased AR S81 phosphorylation and reduced AR activity. Our findings indicate a reciprocal feedback between GRB10 and AR signaling, implying the importance of GRB10 in PCa progression.
Assuntos
Proteína Adaptadora GRB10/metabolismo , Neoplasias da Próstata/metabolismo , Proteína Fosfatase 2/metabolismo , Receptores Androgênicos/metabolismo , Animais , Linhagem Celular Tumoral , Proteína Adaptadora GRB10/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Xenoenxertos , Humanos , Masculino , Camundongos , Neoplasias da Próstata/genética , Proteína Fosfatase 2/antagonistas & inibidores , Transdução de SinaisRESUMO
MOTIVATION: Successful development and application of precision oncology approaches require robust elucidation of the genomic landscape of a patient's cancer and, ideally, the ability to monitor therapy-induced genomic changes in the tumour in an inexpensive and minimally invasive manner. Thanks to recent advances in sequencing technologies, 'liquid biopsy', the sampling of patient's bodily fluids such as blood and urine, is considered as one of the most promising approaches to achieve this goal. In many cancer patients, and especially those with advanced metastatic disease, deep sequencing of circulating cell free DNA (cfDNA) obtained from patient's blood yields a mixture of reads originating from the normal DNA and from multiple tumour subclones-called circulating tumour DNA or ctDNA. The ctDNA/cfDNA ratio as well as the proportion of ctDNA originating from specific tumour subclones depend on multiple factors, making comprehensive detection of mutations difficult, especially at early stages of cancer. Furthermore, sensitive and accurate detection of single nucleotide variants (SNVs) and indels from cfDNA is constrained by several factors such as the sequencing errors and PCR artifacts, and mapping errors related to repeat regions within the genome. In this article, we introduce SiNVICT, a computational method that increases the sensitivity and specificity of SNV and indel detection at very low variant allele frequencies. SiNVICT has the capability to handle multiple sequencing platforms with different error properties; it minimizes false positives resulting from mapping errors and other technology specific artifacts including strand bias and low base quality at read ends. SiNVICT also has the capability to perform time-series analysis, where samples from a patient sequenced at multiple time points are jointly examined to report locations of interest where there is a possibility that certain clones were wiped out by some treatment while some subclones gained selective advantage. RESULTS: We tested SiNVICT on simulated data as well as prostate cancer cell lines and cfDNA obtained from castration-resistant prostate cancer patients. On both simulated and biological data, SiNVICT was able to detect SNVs and indels with variant allele percentages as low as 0.5%. The lowest amounts of total DNA used for the biological data where SNVs and indels could be detected with very high sensitivity were 2.5 ng on the Ion Torrent platform and 10 ng on Illumina. With increased sequencing and mapping accuracy, SiNVICT might be utilized in clinical settings, making it possible to track the progress of point mutations and indels that are associated with resistance to cancer therapies and provide patients personalized treatment. We also compared SiNVICT with other popular SNV callers such as MuTect, VarScan2 and Freebayes. Our results show that SiNVICT performs better than these tools in most cases and allows further data exploration such as time-series analysis on cfDNA sequencing data. AVAILABILITY AND IMPLEMENTATION: SiNVICT is available at: https://sfu-compbio.github.io/sinvictSupplementary information: Supplementary data are available at Bioinformatics online. CONTACT: cenk@sfu.ca.
Assuntos
Análise Mutacional de DNA/métodos , DNA de Neoplasias/sangue , Mutação INDEL , Neoplasias/genética , Mutação Puntual , Software , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Neoplasias/sangue , Sensibilidade e EspecificidadeRESUMO
Type 2 innate lymphoid cells (ILC2s) perform vital functions in orchestrating humoral immune responses, facilitating tissue remodelling, and ensuring tissue homeostasis. Additionally, in a role that has garnered considerably less attention, ILC2s can also enhance Th1-related cytolytic T lymphocyte immune responses against tumours. Studies have thus far generally failed to address the mystery of how one ILC2 cell-type can participate in a multiplicity of functions. Here we utilized single cell RNA sequencing analysis to create the first comprehensive atlas of naïve and tumour-associated lung ILC2s and discover multiple unique subtypes of ILC2s equipped with developmental gene programs that become skewed during tumour expansion favouring inflammation, antigen processing, immunological memory and Th1-related anti-tumour CTL responses. The discovery of these new subtypes of ILC2s challenges current paradigms of ILC2 biology and provides an explanation for their diversity of function.
Assuntos
Imunidade Inata , Neoplasias , Humanos , Linfócitos , Pulmão/patologia , Inflamação/patologia , Neoplasias/genética , Neoplasias/patologiaRESUMO
Next-generation sequencing is making sequence-based molecular pathology and personalized oncology viable. We selected an individual initially diagnosed with conventional but aggressive prostate adenocarcinoma and sequenced the genome and transcriptome from primary and metastatic tissues collected prior to hormone therapy. The histology-pathology and copy number profiles were remarkably homogeneous, yet it was possible to propose the quadrant of the prostate tumour that likely seeded the metastatic diaspora. Despite a homogeneous cell type, our transcriptome analysis revealed signatures of both luminal and neuroendocrine cell types. Remarkably, the repertoire of expressed but apparently private gene fusions, including C15orf21:MYC, recapitulated this biology. We hypothesize that the amplification and over-expression of the stem cell gene MSI2 may have contributed to the stable hybrid cellular identity. This hybrid luminal-neuroendocrine tumour appears to represent a novel and highly aggressive case of prostate cancer with unique biological features and, conceivably, a propensity for rapid progression to castrate-resistance. Overall, this work highlights the importance of integrated analyses of genome, exome and transcriptome sequences for basic tumour biology, sequence-based molecular pathology and personalized oncology.
Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Genômica , Neoplasias da Próstata/genética , Adenocarcinoma/secundário , Adenocarcinoma/terapia , Terapia Combinada , DNA de Neoplasias/análise , Amplificação de Genes , Dosagem de Genes , Perfilação da Expressão Gênica , Fusão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Células Neuroendócrinas/metabolismo , Células Neuroendócrinas/patologia , Prognóstico , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
The current paradigm of cancer care relies on predictive nomograms which integrate detailed histopathology with clinical data. However, when predictions fail, the consequences for patients are often catastrophic, especially in prostate cancer where nomograms influence the decision to therapeutically intervene. We hypothesized that the high dimensional data afforded by massively parallel sequencing (MPS) is not only capable of providing biological insights, but may aid molecular pathology of prostate tumours. We assembled a cohort of six patients with high-risk disease, and performed deep RNA and shallow DNA sequencing in primary tumours and matched metastases where available. Our analysis identified copy number abnormalities, accurately profiled gene expression levels, and detected both differential splicing and expressed fusion genes. We revealed occult and potentially dormant metastases, unambiguously supporting the patients' clinical history, and implicated the REST transcriptional complex in the development of neuroendocrine prostate cancer, validating this finding in a large independent cohort. We massively expand on the number of novel fusion genes described in prostate cancer; provide fresh evidence for the growing link between fusion gene aetiology and gene expression profiles; and show the utility of fusion genes for molecular pathology. Finally, we identified chromothripsis in a patient with chronic prostatitis. Our results provide a strong foundation for further development of MPS-based molecular pathology.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias Hormônio-Dependentes/genética , Células Neuroendócrinas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Adenocarcinoma/terapia , Idoso , Processamento Alternativo , Biomarcadores Tumorais/sangue , Colúmbia Britânica , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Análise por Conglomerados , Técnicas de Apoio para a Decisão , Dosagem de Genes , Fusão Gênica , Predisposição Genética para Doença , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Neoplasias Hormônio-Dependentes/terapia , Células Neuroendócrinas/patologia , Nomogramas , Seleção de Pacientes , Fenótipo , Medicina de Precisão , Prognóstico , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Interferência de RNA , TransfecçãoRESUMO
Infertility affects 10-15% of couples, with half attributed to male factors. An improved understanding of the cell-type-specific dysfunction contributing to male infertility is needed to improve available therapies; however, human testicular tissues are difficult to obtain for research purposes. To overcome this, researchers have begun to use human induced pluripotent stem cells (hiPSCs) to generate various testis-specific cell types in vitro. Peritubular myoid cells (PTMs) are one such testicular cell type that serves a critical role in the human testis niche but, to date, have not been derived from hiPSCs. This study set forth to generate a molecular-based differentiation method for deriving PTMs from hiPSCs, mirroring in vivo patterning factors. Whole transcriptome profiling and quantitative polymerase chain reaction (qPCR) show that this differentiation method is sufficient to derive cells with PTM-like transcriptomes, including upregulation of hallmark PTM functional genes, secreted growth and matrix factors, smooth muscle, integrins, receptors, and antioxidants. Hierarchical clustering shows that they acquire transcriptomes similar to primary isolated PTMs, and immunostaining shows the acquisition of a smooth muscle phenotype. Overall, these hiPSC-PTMs will allow in vitro study of patient-specific PTM development and function in spermatogenesis and infertility.
Assuntos
Células-Tronco Pluripotentes Induzidas , Infertilidade Masculina , Humanos , Masculino , Testículo/metabolismo , Espermatogênese/genética , Diferenciação Celular/genética , Infertilidade Masculina/metabolismoRESUMO
Heart disease is the leading cause of global morbidity and mortality. This is in part because, despite an abundance of animal and in vitro models, it has been a challenge to date to study human heart tissue with sufficient depth and resolution to develop disease-modifying therapies for common cardiac conditions. Single-nucleus RNA sequencing (snRNA-seq) has emerged as a powerful tool capable of analyzing cellular function and signaling in health and disease, and has already contributed to significant advances in areas such as oncology and hematology. Employing snRNA-seq technology on flash-frozen human tissue has the potential to unlock novel disease mechanisms and pathways in any organ. Studying the human heart using snRNA-seq is a key priority for the field of cardiovascular sciences; however, progress to date has been slowed by numerous barriers. One key challenge is the fact that the human heart is very resistant to shearing and stress, making tissue dissociation and nuclear isolation difficult. Here, we describe a tissue dissociation method allowing the efficient and cost-effective isolation of high-quality nuclei from flash-frozen human heart tissue collected in surgical operating rooms. Our protocol addresses the challenge of nuclear isolation from human hearts, enables snRNA-seq of the human heart, and paves the way for an improved understanding of the human heart in health and disease. Ultimately, this will be key to uncovering signaling pathways and networks amenable to therapeutic intervention and the development of novel biomarkers and disease-modifying therapies. © 2022 Wiley Periodicals LLC. Basic Protocol: Human heart tissue dissociation and nuclear isolation for snRNA-seq.
Assuntos
Núcleo Celular , Perfilação da Expressão Gênica , Animais , Núcleo Celular/genética , Perfilação da Expressão Gênica/métodos , Coração , Humanos , RNA Nuclear Pequeno/genética , Análise de Sequência de RNA/métodosRESUMO
Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors. IMPLICATIONS: We have established highly clinically relevant PDXs of ADT-induced dormant prostate cancer and identified two dormancy subtypes, leading to the development of a novel predicative gene signature that allows robust risk stratification of patients with prostate cancer to ADT or androgen-receptor pathway inhibitors.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Antagonistas de Androgênios/farmacologia , Antagonistas de Receptores de Andrógenos , Androgênios/uso terapêutico , Animais , Humanos , Masculino , Camundongos , Recidiva Local de Neoplasia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologiaRESUMO
Germline CDH1 point or small frameshift mutations can be identified in 30-50% of hereditary diffuse gastric cancer (HDGC) families. We hypothesized that CDH1 genomic rearrangements would be found in HDGC and identified 160 families with either two gastric cancers in first-degree relatives and with at least one diffuse gastric cancer (DGC) diagnosed before age 50, or three or more DGC in close relatives diagnosed at any age. Sixty-seven carried germline CDH1 point or small frameshift mutations. We screened germline DNA from the 93 mutation negative probands for large genomic rearrangements by Multiplex Ligation-Dependent Probe Amplification. Potential deletions were validated by RT-PCR and breakpoints cloned using a combination of oligo-CGH-arrays and long-range-PCR. In-silico analysis of the CDH1 locus was used to determine a potential mechanism for these rearrangements. Six of 93 (6.5%) previously described mutation negative HDGC probands, from low GC incidence populations (UK and North America), carried genomic deletions (UK and North America). Two families carried an identical deletion spanning 193 593 bp, encompassing the full CDH3 sequence and CDH1 exons 1 and 2. Other deletions affecting exons 1, 2, 15 and/or 16 were identified. The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions. When all mutations and deletions are considered, the overall frequency of CDH1 alterations in HDGC is approximately 46% (73/160). CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences. As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families.
Assuntos
Caderinas/genética , Mutação em Linhagem Germinativa , Deleção de Sequência , Neoplasias Gástricas/genética , Adulto , Antígenos CD , Sequência de Bases , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Mutação PuntualRESUMO
A better understanding of the secondary injury mechanisms that occur after traumatic spinal cord injury (SCI) is essential for the development of novel neuroprotective strategies linked to the restoration of metabolic deficits. We and others have shown that Ketogenic diet (KD), a high fat, moderate in proteins and low in carbohydrates is neuroprotective and improves behavioural outcomes in rats with acute SCI. Ketones are alternative fuels for mitochondrial ATP generation, and can modulate signaling pathways via targeting specific receptors. Here, we demonstrate that ad libitum administration of KD for 7 days after SCI rescued mitochondrial respiratory capacity, increased parameters of mitochondrial biogenesis, affected the regulation of mitochondrial-related genes, and activated the NRF2-dependent antioxidant pathway. This study demonstrates that KD improves post-SCI metabolism by rescuing mitochondrial function and supports the potential of KD for treatment of acute SCI in humans.
Assuntos
Medula Cervical/patologia , Metabolismo Energético/genética , Expressão Gênica/genética , Genes Mitocondriais/genética , Mitocôndrias/genética , Traumatismos da Medula Espinal/genética , Animais , Dieta Cetogênica/métodos , Modelos Animais de Doenças , Corpos Cetônicos/genética , Masculino , Biogênese de Organelas , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/genética , Transdução de Sinais/genética , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologiaRESUMO
The emergence of bacteria resistant to antibiotics and the resulting infections are increasingly becoming a public health issue. Multidrug-resistant (MDR) bacteria are responsible for infections leading to increased morbidity and mortality in hospitals, prolonged time of hospitalization, and additional burden to financial costs. Therefore, there is an urgent need for novel antibacterial agents that will both treat MDR infections and outsmart the bacterial evolutionary mechanisms, preventing further resistance development. In this study, a green synthesis employing nontoxic lignin as both reducing and capping agents was adopted to formulate stable and biocompatible silver-lignin nanoparticles (NPs) exhibiting antibacterial activity. The resulting silver-lignin NPs were approximately 20 nm in diameter and did not agglomerate after one year of storage at 4 °C. They were able to inhibit the growth of a panel of MDR clinical isolates, including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, at concentrations that did not affect the viability of a monocyte-derived THP-1 human cell line. Furthermore, the exposure of silver-lignin NPs to the THP-1 cells led to a significant increase in the secretion of the anti-inflammatory cytokine IL-10, demonstrating the potential of these particles to act as an antimicrobial and anti-inflammatory agent simultaneously. P. aeruginosa genes linked with efflux, heavy metal resistance, capsular biosynthesis, and quorum sensing were investigated for changes in gene expression upon sublethal exposure to the silver-lignin NPs. Genes encoding for membrane proteins with an efflux function were upregulated. However, all other genes were membrane proteins that did not efflux metals and were downregulated.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Lignina/química , Nanopartículas Metálicas , Prata/química , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Humanos , Inflamação/prevenção & controle , Testes de Sensibilidade Microbiana , Células THP-1RESUMO
Hip implants are a successful solution for osteoarthritis; however, some individuals with metal-on-metal (MoM) and metal-on-polyethylene (MoP) prosthetics develop adverse local tissue reactions (ALTRs). While MoM and MoP ALTRs are presumed to be delayed hypersensitivity reactions to corrosion products, MoM- and MoP-associated ALTRs present with different histological characteristics. We compared MoM- and MoP-associated ALTRs histopathology with cobalt and chromium levels in serum and synovial fluid. We analyzed the gene expression levels of leukocyte aggregates and synovial fluid chemokines/cytokines to resolve potential pathophysiologic differences. In addition, we classified ALTRs from 79 patients according to their leukocyte infiltrates as macrophage-dominant, mixed, and lymphocyte-dominant. Immune-related transcript profiles from lymphocyte-dominant MoM- and MoP-associated ALTR patients with perivascular lymphocytic aggregates were similar. Cell signatures indicated predominantly macrophage, Th1 and Th2 lymphocytic infiltrate, with strong exhausted CD8+ signature, and low Th17 and B cell, relative to healthy lymph nodes. Lymphocyte-dominant ALTR-associated synovial fluid contained higher levels of induced protein 10 (IP-10), interleukin-1 receptor antagonist (IL-1RN), IL-8, IL-6, IL-16, macrophage inflammatory protein 1 (MIP-1α), IL-18, MCP-2, and lower cell-attracting chemokine levels, when compared with prosthetic revisions lacking ALTRs. In addition, the higher levels of IP-10, IL-8, IL-6, MIP-1α, and MCP-2 were observed within the synovial fluid of the lymphocyte-dominant ALTRs relative to the macrophage-dominant ALTRs. Not all cytokines/chemokines were detected in the perivascular aggregate transcripts, suggesting the existence of other sources in the affected synovia. Our results support the hypothesis of common hypersensitivity pathogenesis in lymphocyte-dominant MoM and MoP ALTRs. The exhausted lymphocyte signature indicates chronic processes and an impaired immune response, although the cause of the persistent T-cell activation remains unclear. The cytokine/chemokine signature of lymphocyte-dominant-associated ATLRs may be of utility for diagnosing this more aggressive pathogenesis.
Assuntos
Artroplastia de Quadril , Prótese de Quadril , Próteses Articulares Metal-Metal , Artroplastia de Quadril/efeitos adversos , Linfócitos T CD8-Positivos , Quimiocina CCL3 , Quimiocina CXCL10 , Prótese de Quadril/efeitos adversos , Humanos , Interleucina-6 , Interleucina-8 , Linfócitos , Próteses Articulares Metal-Metal/efeitos adversos , Metais , Polietileno , Desenho de Prótese , Falha de Prótese , ReoperaçãoRESUMO
Adverse local tissue reactions (ALTRs) are a prominent cause of hip implant failure. ALTRs are characterized by aseptic necrosis and leukocyte infiltration of synovial tissue. The prevalence of ALTRs in hips with failing metal implants, with highest rates occurring in patients with metal-on-metal articulations, suggests a role for CoCrMo corrosion in ALTR formation. Although hypersensitivity reactions are the most accepted etiology, the precise cellular mechanism driving ALTR pathogenesis remains enigmatic. Here we show that cobalt ions released by failing hip implants induce mitochondrial stress and cytokine secretion by synovial fibroblasts: the presumptive initiators of ALTR pathogenesis. We found that in-vitro treatment of synovial fibroblasts with cobalt, but not chromium, generated gene expression changes indicative of hypoxia and mitophagy responses also observed in ALTRs biopsies. Inflammatory factors secreted by cobalt-exposed synovial fibroblasts were among those most concentrated in ALTR synovial fluid. Furthermore, both conditioned media from cobalt-exposed synovial fibroblasts, and synovial fluid from ALTRs patients, elicit endothelial activation and monocyte migration. Finally, we identify the IL16/CTACK ratio in synovial fluid as a possible diagnostic marker of ALTRs. Our results provide evidence suggesting that metal ions induce cell stress in synovial fibroblasts that promote an inflammatory response consistent with initiating ALTR formation. STATEMENT OF SIGNIFICANCE: We demonstrate that the cytotoxic effects of cobalt ions on the synovial cells (fibroblast) is sufficient to trigger inflammation on hip joints with metal implants. Cobalt ions affect mitochondrial function, leading to the auto phagocytosis of mitochondria and trigger a hypoxic response. The cell's hypoxic response includes secretion of cytokines that are capable of trigger inflammation by activating blood vessels and enhancing leukocyte migration. Among the secreted cytokines is IL-16, which is highly concentrated in the synovial fluid of the patients with adverse local tissue reactions and could be use as diagnostic marker. In conclusion we define the cells of the hip joint as key players in triggering the adverse reactions to hip implants and providing biomarkers for early diagnosis of adverse reactions to hip implants.
Assuntos
Artroplastia de Quadril , Prótese de Quadril , Próteses Articulares Metal-Metal , Quimiocinas , Cromo , Cobalto/toxicidade , Citocinas , Fibroblastos , Prótese de Quadril/efeitos adversos , Humanos , Íons , Desenho de Prótese , Falha de Prótese , Estresse FisiológicoRESUMO
Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. SIGNIFICANCE: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.
Assuntos
Biomarcadores Farmacológicos/análise , Cistadenocarcinoma Seroso/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Genômica/métodos , Humanos , Metabolômica/métodos , Gradação de Tumores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteômica/métodos , Integração de SistemasRESUMO
Well-differentiated papillary mesothelioma (WDPM) is an uncommon mesothelial proliferation that is most commonly encountered as an incidental finding in the peritoneal cavity. There is controversy in the literature about whether WDPM is a neoplasm or a reactive process and, if neoplastic, whether it is a variant or precursor of epithelial malignant mesothelioma or is a different entity. Using whole exome sequencing of five WDPMs of the peritoneum, we have identified distinct mutations in EHD1, ATM, FBXO10, SH2D2A, CDH5, MAGED1, and TP73 shared by WDPM cases but not reported in malignant mesotheliomas. Furthermore, we show that WDPM is strongly enriched with C > A transversion substitution mutations, a pattern that is also not found in malignant mesotheliomas. The WDPMs lacked the alterations involving BAP1, SETD2, NF2, CDKN2A/B, LASTS1/2, PBRM1, and SMARCC1 that are frequently found in malignant mesotheliomas. We conclude that WDPMs are neoplasms that are genetically distinct from malignant mesotheliomas and, based on observed mutations, do not appear to be precursors of malignant mesotheliomas.
RESUMO
Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It develops mainly via NE transdifferentiation of prostate adenocarcinoma in response to androgen receptor (AR)-inhibition therapy. The study of NEPC development has been hampered by a lack of clinically relevant models. We previously established a unique and first-in-field patient-derived xenograft (PDX) model of adenocarcinoma (LTL331)-to-NEPC (LTL331R) transdifferentiation. In this study, we applied conditional reprogramming (CR) culture to establish a LTL331 PDX-derived cancer cell line named LTL331_CR_Cell. These cells retain the same genomic mutations as the LTL331 parental tumor. They can be continuously propagated in vitro and can be genetically manipulated. Androgen deprivation treatment on LTL331_CR_Cells had no effect on cell proliferation. Transcriptomic analyses comparing the LTL331_CR_Cell to its parental tumor revealed a profound downregulation of the androgen response pathway and an upregulation of stem and basal cell marker genes. The transcriptome of LTL331_CR_Cells partially resembles that of post-castrated LTL331 xenografts in mice. Notably, when grafted under the renal capsules of male NOD/SCID mice, LTL331_CR_Cells spontaneously gave rise to NEPC tumors. This is evidenced by the histological expression of the NE marker CD56 and the loss of adenocarcinoma markers such as PSA. Transcriptomic analyses of the newly developed NEPC tumors further demonstrate marked enrichment of NEPC signature genes and loss of AR signaling genes. This study provides a novel research tool derived from a unique PDX model. It allows for the investigation of mechanisms underlying NEPC development by enabling gene manipulations ex vivo and subsequent functional evaluations in vivo.
Assuntos
Carcinogênese/patologia , Reprogramação Celular , Tumores Neuroendócrinos/patologia , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Androgênios/farmacologia , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Reprogramação Celular/efeitos dos fármacos , Humanos , Masculino , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
BACKGROUND: Malignant peritoneal mesothelioma (PeM) is a rare and fatal cancer that originates from the peritoneal lining of the abdomen. Standard treatment of PeM is limited to cytoreductive surgery and/or chemotherapy, and no effective targeted therapies for PeM exist. Some immune checkpoint inhibitor studies of mesothelioma have found positivity to be associated with a worse prognosis. METHODS: To search for novel therapeutic targets for PeM, we performed a comprehensive integrative multi-omics analysis of the genome, transcriptome, and proteome of 19 treatment-naïve PeM, and in particular, we examined BAP1 mutation and copy number status and its relationship to immune checkpoint inhibitor activation. RESULTS: We found that PeM could be divided into tumors with an inflammatory tumor microenvironment and those without and that this distinction correlated with haploinsufficiency of BAP1. To further investigate the role of BAP1, we used our recently developed cancer driver gene prioritization algorithm, HIT'nDRIVE, and observed that PeM with BAP1 haploinsufficiency form a distinct molecular subtype characterized by distinct gene expression patterns of chromatin remodeling, DNA repair pathways, and immune checkpoint receptor activation. We demonstrate that this subtype is correlated with an inflammatory tumor microenvironment and thus is a candidate for immune checkpoint blockade therapies. CONCLUSIONS: Our findings reveal BAP1 to be a potential, easily trackable prognostic and predictive biomarker for PeM immunotherapy that refines PeM disease classification. BAP1 stratification may improve drug response rates in ongoing phases I and II clinical trials exploring the use of immune checkpoint blockade therapies in PeM in which BAP1 status is not considered. This integrated molecular characterization provides a comprehensive foundation for improved management of a subset of PeM patients.
Assuntos
Biomarcadores Tumorais/genética , Haploinsuficiência , Mesotelioma/genética , Neoplasias Peritoneais/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Biomarcadores Tumorais/metabolismo , Humanos , Imunoterapia , Mesotelioma/classificação , Mesotelioma/terapia , Mutação , Neoplasias Peritoneais/classificação , Neoplasias Peritoneais/terapia , Microambiente Tumoral , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. HP1α knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management.Significance: Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(10); 2691-704. ©2018 AACR.