RESUMO
Bladder cancer is the fifth most prevalent cancer in the U.S., yet is understudied, and few laboratory models exist that reflect the biology of the human disease. Here, we describe a biobank of patient-derived organoid lines that recapitulates the histopathological and molecular diversity of human bladder cancer. Organoid lines can be established efficiently from patient biopsies acquired before and after disease recurrence and are interconvertible with orthotopic xenografts. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Analyses of drug response using bladder tumor organoids show partial correlations with mutational profiles, as well as changes associated with treatment resistance, and specific responses can be validated using xenografts in vivo. Our studies indicate that patient-derived bladder tumor organoids represent a faithful model system for studying tumor evolution and treatment response in the context of precision cancer medicine.
Assuntos
Neoplasias da Bexiga Urinária/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Mutação , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Medicina de Precisão , Transplante Heterólogo , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismoRESUMO
Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Nav1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Nav1.7 channels, with a density of 0.1 to 0.15 channels per µm2 and 350 to 525 channels per cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrates that Nav1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Nav1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Nav channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Nav1.7 blockers regulate intracellular Ca2+ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.
Assuntos
Condrócitos , Canal de Sódio Disparado por Voltagem NAV1.7 , Osteoartrite , Bloqueadores do Canal de Sódio Disparado por Voltagem , Animais , Humanos , Camundongos , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Progressão da Doença , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/deficiência , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Neurônios/metabolismo , Osteoartrite/complicações , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , Osteoartrite/metabolismo , Dor/complicações , Dor/tratamento farmacológico , Dor/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
BACKGROUND: Considering the molecular heterogeneity of sarcomas and their immunologically quiet character, immunotherapy (e.g., immune checkpoint inhibitors) plays a viable role in only a subset of these tumors. This study aimed to determine the immune subtypes (IMSs) of sarcomas for selecting suitable patients from an extremely heterogeneous population. RESULTS: By performing consensus clustering analysis of the gene expression profiles of 538 patients with sarcomas in online databases, we stratified sarcomas into three IMSs characterized by different immune cell features, tumor mutational burdens (TMBs), gene mutations, and clinical outcomes. IMS1 showed an immune "hot" and immunosuppressive phenotype, the highest frequencies of CSMD3 mutation but the lowest frequencies of HMCN1 and LAMA2 mutations; these patients had the worst progression-free survival (PFS). IMS2 was defined by a high TMB and more gene mutations, but had the lowest frequency of MND1 mutations. IMS3 displayed the highest MDN1 expression level and an immune "cold" phenotype, these patients had the worst PFS. Each subtype was associated with different expression levels of immunogenic cell death modulators and immune checkpoints. Moreover, we applied graph learning-based dimensionality reduction to the immune landscape and identified significant intra-cluster heterogeneity within each IMS. Finally, we developed and validated an immune gene signature with good prognostic performance. CONCLUSIONS: Our results provide a conceptual framework for understanding the immunological heterogeneity of sarcomas. The identification of immune-related subtypes may facilitate optimal selection of sarcoma patients who will respond to appropriate therapeutic strategies.
Assuntos
Inibidores de Checkpoint Imunológico , Sarcoma , Biomarcadores Tumorais , Humanos , Imunoterapia/métodos , Prognóstico , Sarcoma/tratamento farmacológico , Sarcoma/terapiaRESUMO
OBJECTIVES: Osteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in the pathogenesis of OA. TNFR1 primarily mediates inflammation, whereas emerging evidences demonstrate that TNFR2 plays an anti-inflammatory and protective role in several diseases and conditions. This study aims to decipher TNFR2 signalling in chondrocytes and OA. METHODS: Biochemical copurification and proteomics screen were performed to isolate the intracellular cofactors of TNFR2 complex. Bulk and single cell RNA-seq were employed to determine 14-3-3 epsilon (14-3-3ε) expression in human normal and OA cartilage. Transcription factor activity screen was used to isolate the transcription factors downstream of TNFR2/14-3-3ε. Various cell-based assays and genetically modified mice with naturally occurring and surgically induced OA were performed to examine the importance of this pathway in chondrocytes and OA. RESULTS: Signalling molecule 14-3-3ε was identified as an intracellular component of TNFR2 complexes in chondrocytes in response to progranulin (PGRN), a growth factor known to protect against OA primarily through activating TNFR2. 14-3-3ε was downregulated in OA and its deficiency deteriorated OA. 14-3-3ε was required for PGRN regulation of chondrocyte metabolism. In addition, both global and chondrocyte-specific deletion of 14-3-3ε largely abolished PGRN's therapeutic effects against OA. Furthermore, PGRN/TNFR2/14-3-3ε signalled through activating extracellular signal-regulated kinase (ERK)-dependent Elk-1 while suppressing nuclear factor kappa B (NF-κB) in chondrocytes. CONCLUSIONS: This study identifies 14-3-3ε as an inducible component of TNFR2 receptor complex in response to PGRN in chondrocytes and presents a previously unrecognised TNFR2 pathway in the pathogenesis of OA.
Assuntos
Proteínas 14-3-3/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteoartrite/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Animais , Cartilagem Articular/citologia , Humanos , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Progranulinas/metabolismo , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in â¼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/enzimologia , Neoplasias/genética , Receptores Proteína Tirosina Quinases/genética , Iniciação da Transcrição Genética , Alelos , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Feminino , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Íntrons/genética , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/genética , Lisina/metabolismo , Metilação , Camundongos , Dados de Sequência Molecular , Peso Molecular , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Oncogenes/genética , Estrutura Terciária de Proteína/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/química , Transdução de SinaisRESUMO
Inverted urothelial papilloma (IUP) and urothelial papilloma (UP) are rare urothelial neoplasms that typically follow a benign clinical course. Oncogenic mutations in FGFR3, HRAS, and the TERT promoter have been reported in these entities but no comprehensive molecular analysis has been performed. We sought to characterize the genomic landscape of IUP and UP using whole-exome and targeted next-generation sequencing. In IUP, 10 of 11 tumors harbored oncogenic hotspot mutations in HRAS and the remaining tumor had an oncogenic KRAS mutation. None of the IUP tumors harbored TERT promoter or FGFR3 mutations. In UP, 8 of 11 tumors had oncogenic KRAS mutations and two had oncogenic HRAS mutations. One UP tumor had oncogenic mutations in FGFR3, PIK3CA, and the TERT promoter, and arose in a patient with recurrent non-invasive papillary urothelial carcinomas. In contrast to urothelial carcinoma, the APOBEC mutational signature was not present in any IUP and UP tumors, and oncogenic alterations in chromatin remodeling genes were uncommon in both IUP and UP. The current study suggests that IUP and UP are driven primarily by RAS pathway activation and lack the more common genomic features of urothelial cancers. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Papiloma Invertido/patologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Bexiga Urinária/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/patologia , Bases de Dados Genéticas , Feminino , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Papiloma Invertido/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Progranulin (PGRN) restrains inflammation and is therapeutic against inflammatory arthritis; however, the underlying immunological mechanism remains unknown. In this study, we demonstrated that anti-inflammatory cytokine IL-10 was a critical mediator for PGRN-mediated anti-inflammation in collagen-induced arthritis by using PGRN and IL-10 genetically modified mouse models. IL-10 green fluorescent protein reporter mice revealed that regulatory T (Treg) cells were the predominant source of IL-10 in response to PGRN. In addition, PGRN-mediated expansion and activation of Treg cells, as well as IL-10 production, depends on JNK signaling, but not on known PGRN-activated ERK and PI3K pathways. Furthermore, microarray and chromatin immunoprecipitation sequencing screens led to the discovery of forkhead box protein O4 and signal transducer and activator of transcription 3 as the transcription factors required for PGRN induction of IL-10 in Treg cells. These findings define a previously unrecognized signaling pathway that underlies IL-10 production by PGRN in Treg cells and present new insights into the mechanisms by which PGRN resolves inflammation in inflammatory conditions and autoimmune diseases, particularly inflammatory arthritis.-Fu, W., Hu, W., Shi, L., Mundra, J. J. Xiao, G., Dustin, M. L., Liu, C. Foxo4- and Stat3-dependent IL-10 production by progranulin in regulatory T cells restrains inflammatory arthritis.
Assuntos
Artrite Experimental/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-10/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Proteínas de Ciclo Celular , Células Cultivadas , Granulinas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Interleucina-10/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos DBA , Fosfatidilinositol 3-Quinases/metabolismo , Progranulinas , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Hematopoietic stem cells (HSCs) possess the ability to generate all hematopoietic cell types and to self-renew over long periods, but the mechanisms that regulate their unique properties are incompletely understood. Herein, we show that homozygous deletion of the miR-29a/b-1 bicistron results in decreased numbers of hematopoietic stem and progenitor cells (HSPCs), decreased HSC self-renewal, and increased HSC cell cycling and apoptosis. The HSPC phenotype is specifically due to loss of miR-29a, because miR-29b expression is unaltered in miR-29a/b-1-null HSCs, and only ectopic expression of miR-29a restores HSPC function both in vitro and in vivo. HSCs lacking miR-29a/b-1 exhibit widespread transcriptional dysregulation and adopt gene expression patterns similar to normal committed progenitors. A number of predicted miR-29 target genes, including Dnmt3a, are significantly upregulated in miR-29a/b-1-null HSCs. The loss of negative regulation of Dnmt3a by miR-29a is a major contributor to the miR-29a/b-1-null HSPC phenotype, as both in vitro Dnmt3a short hairpin RNA knockdown assays and a genetic haploinsufficiency model of Dnmt3a restored the frequency and long-term reconstitution capacity of HSCs from miR-29a/b-1-deficient mice. Overall, these data demonstrate that miR-29a is critical for maintaining HSC function through its negative regulation of Dnmt3a.
Assuntos
Diferenciação Celular , DNA (Citosina-5-)-Metiltransferases/fisiologia , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/fisiologia , Animais , Apoptose , Ciclo Celular , Proliferação de Células , Células Cultivadas , DNA Metiltransferase 3A , Citometria de Fluxo , Perfilação da Expressão Gênica , Hematopoese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Precision oncology relies on the accurate molecular characterization of individual patients with cancer at the time of treatment initiation. However, tumor molecular profiles are not static, and cancers continually evolve because of ongoing mutagenesis and clonal selection. Here, we performed genomic analyses of primary tumors, metastases, and plasma collected from individual patients to define the concordance of actionable genomic alterations and to identify drivers of metastatic disease progression. We observed a high degree of discordance of actionable genomic alterations, with 23% discordant between primary and metastatic disease sites. Among chromatin-modifying genes, ARID1A mutations, when discordant, were exclusive to the metastatic tumor samples. Our findings indicate that the high degree of lesion-to-lesion genomic heterogeneity may be a barrier to precision oncology approaches for bladder cancer and that circulating tumor DNA profiling may be preferred to tumor sequencing for a subset of patients.
Assuntos
DNA Tumoral Circulante , Neoplasias da Bexiga Urinária , Humanos , Medicina de Precisão , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Genômica , Mutação/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors.
Assuntos
Carcinoma de Células Escamosas , Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Humanos , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células de Transição/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Filogenia , Neoplasias da Bexiga Urinária/patologia , Linhagem da CélulaRESUMO
Cancers develop from the accumulation of somatic mutations, yet it remains unclear how oncogenic lesions cooperate to drive cancer progression. Using a mouse model harboring NRasG12D and EZH2 mutations that recapitulates leukemic progression, we employ single-cell transcriptomic profiling to map cellular composition and gene expression alterations in healthy or diseased bone marrows during leukemogenesis. At cellular level, NRasG12D induces myeloid lineage-biased differentiation and EZH2-deficiency impairs myeloid cell maturation, whereas they cooperate to promote myeloid neoplasms with dysregulated transcriptional programs. At gene level, NRasG12D and EZH2-deficiency independently and synergistically deregulate gene expression. We integrate results from histopathology, leukemia repopulation, and leukemia-initiating cell assays to validate transcriptome-based cellular profiles. We use this resource to relate developmental hierarchies to leukemia phenotypes, evaluate oncogenic cooperation at single-cell and single-gene levels, and identify GEM as a regulator of leukemia-initiating cells. Our studies establish an integrative approach to deconvolute cancer evolution at single-cell resolution in vivo.
Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Leucemia/genética , Leucemia/metabolismo , Análise de Célula Única , Animais , Apoptose , Ciclo Celular , Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigenômica , GTP Fosfo-Hidrolases , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Leucemia Mieloide Aguda , Proteínas de Membrana , Camundongos Knockout , Mutação , Células Mieloides , Oncogenes , Fenótipo , TranscriptomaRESUMO
TNFR1 and TNFR2 have received prominent attention because of their dominance in the pathogenesis of inflammation and autoimmunity. TNFR1 has been extensively studied and primarily mediates inflammation. TNFR2 remains far less studied, although emerging evidence demonstrates that TNFR2 plays an antiinflammatory and immunoregulatory role in various conditions and diseases. Herein, we report that TNFR2 regulates macrophage polarization, a highly dynamic process controlled by largely unidentified intracellular regulators. Using biochemical copurification and mass spectrometry approaches, we isolated the signaling molecule 14-3-3ε as a component of TNFR2 complexes in response to progranulin stimulation in macrophages. In addition, 14-3-3ε was essential for TNFR2 signaling-mediated regulation of macrophage polarization and switch. Both global and myeloid-specific deletion of 14-3-3ε resulted in exacerbated inflammatory arthritis and counteracted the protective effects of progranulin-mediated TNFR2 activation against inflammation and autoimmunity. TNFR2/14-3-3ε signaled through PI3K/Akt/mTOR to restrict NF-κB activation while simultaneously stimulating C/EBPß activation, thereby instructing macrophage plasticity. Collectively, this study identifies 14-3-3ε as a previously unrecognized vital component of the TNFR2 receptor complex and provides new insights into the TNFR2 signaling, particularly its role in macrophage polarization with therapeutic implications for various inflammatory and autoimmune diseases with activation of the TNFR2/14-3-3ε antiinflammatory pathway.
Assuntos
Proteínas 14-3-3/imunologia , Macrófagos/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Proteínas 14-3-3/química , Proteínas 14-3-3/deficiência , Proteínas 14-3-3/metabolismo , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Autoimunidade , Humanos , Inflamação/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Complexos Multiproteicos/química , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Progranulinas/imunologia , Progranulinas/metabolismo , Células RAW 264.7 , Receptores Tipo II do Fator de Necrose Tumoral/química , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/imunologiaRESUMO
Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury, although the repair mechanisms are unclear. Here, we found that Bach2 deficiency promotes intestinal epithelial cell proliferation during homeostasis. Moreover, genetic inactivation of Bach2 in mouse intestinal epithelium facilitated crypt regeneration after irradiation, resulting in a reduction in mortality. RNA-sequencing analysis of isolated crypts revealed that Bach2 deficiency altered the expression of numerous genes, including those regulating double-strand break repair. Mechanistic characterizations indicated that Bach2 deletion facilitated DNA repair in intestinal crypt cells, as evidenced by faster resolution of γ-H2AX and 53BP1 foci in Bach2-/- crypt cells, compared with Bach2+/+ control. Together, our studies highlight that Bach2 deficiency promotes intestinal regeneration by accelerating DNA repair in intestinal stem cells after radiation damage.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Reparo do DNA , Intestinos/fisiologia , Regeneração/fisiologia , Células-Tronco/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Histonas/genética , Mucosa Intestinal/citologia , Intestinos/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Radiação Ionizante , Células-Tronco/citologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
Mutational activation of the PI3K/AKT pathway is among the most common pro-oncogenic events in human cancers. The clinical utility of PI3K and AKT inhibitors has, however, been modest to date. Here, we used CRISPR-mediated gene editing to study the biological consequences of AKT1 E17K mutation by developing an AKT1 E17K-mutant isogenic system in a TP53-null background. AKT1 E17K expression under the control of its endogenous promoter enhanced cell growth and colony formation, but had a paradoxical inhibitory effect on cell migration and invasion. The mechanistic basis by which activated AKT1 inhibited cell migration and invasion was increased E-cadherin expression mediated by suppression of ZEB1 transcription via altered ß-catenin subcellular localization. This phenotypic effect was AKT1-specific, as AKT2 activation had the opposite effect, a reduction in E-cadherin expression. Consistent with the opposing effects of AKT1 and AKT2 activation on E-cadherin expression, a pro-migratory effect of AKT1 activation was not observed in breast cancer cells with PTEN loss or expression of an activating PIK3CA mutation, alterations which induce the activation of both AKT isoforms. The results suggest that the use of AKT inhibitors in patients with breast cancer could paradoxically accelerate metastatic progression in some genetic contexts and may explain the frequent coselection for CDH1 mutations in AKT1-mutated breast tumors. IMPLICATIONS: AKT1 E17K mutation in breast cancer impairs migration/invasiveness via sequestration of ß-catenin to the cell membrane leading to decreased ZEB1 transcription, resulting in increased E-cadherin expression and a reversal of epithelial-mesenchymal transition.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Humanos , Mutação , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de SinaisRESUMO
Genomic rearrangements leading to the aberrant expression of ERG are the most common early events in prostate cancer and are significantly enriched for the concomitant loss of PTEN. Genetically engineered mouse models reveal that ERG overexpression alone is not sufficient to induce tumorigenesis, but combined loss of PTEN results in an aggressive invasive phenotype. Here, we show that oncogenic ERG repressed PI3K signaling through direct transcriptional suppression of IRS2, leading to reduced RTK levels and activity. In accordance with this finding, ERG-positive human prostate cancers had a repressed AKT gene signature and transcriptional downregulation of IRS2. Although overexpression of IRS2 activated PI3K signaling, promoting cell migration in a PI3K-dependent manner, this did not fully recapitulate the phenotype seen with loss of PTEN as PI3K signaling is not as robust as observed in the setting of loss of PTEN. Importantly, deletions of the PTEN locus, which promotes active PI3K signaling, were among the most significant copy-number alterations that co-occurred with ERG genomic rearrangements. This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote oncogenic signaling during tumor evolution. SIGNIFICANCE: This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote tumorigenesis.
Assuntos
Proteínas Substratos do Receptor de Insulina/genética , Proteínas Oncogênicas/metabolismo , Neoplasias da Próstata/genética , Regulador Transcricional ERG/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Rearranjo Gênico , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Camundongos , Proteínas Oncogênicas/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , Próstata/patologia , Neoplasias da Próstata/patologia , RNA-Seq , Transdução de Sinais/genética , Regulador Transcricional ERG/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on MAP2K1 and MAP2K2 mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part in silico methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. In silico prediction accurately distinguished functional from benign MAP2K1 and MAP2K2 mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted in silico modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response. SIGNIFICANCE: Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation.See related commentary by Whitehead and Sebolt-Leopold, p. 4042.
Assuntos
Benchmarking , Neoplasias , Simulação por Computador , Humanos , Mutação , Neoplasias/genética , Estudos ProspectivosRESUMO
Treatment paradigms for patients with upper tract urothelial carcinoma (UTUC) are typically extrapolated from studies of bladder cancer despite their distinct clinical and molecular characteristics. The advancement of UTUC research is hampered by the lack of disease-specific models. Here, we report the establishment of patient derived xenograft (PDX) and cell line models that reflect the genomic and biological heterogeneity of the human disease. Models demonstrate high genomic concordance with the corresponding patient tumors, with invasive tumors more likely to successfully engraft. Treatment of PDX models with chemotherapy recapitulates responses observed in patients. Analysis of a HER2 S310F-mutant PDX suggests that an antibody drug conjugate targeting HER2 would have superior efficacy versus selective HER2 kinase inhibitors. In sum, the biological and phenotypic concordance between patient and PDXs suggest that these models could facilitate studies of intrinsic and acquired resistance and the development of personalized medicine strategies for UTUC patients.
Assuntos
Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Urotélio/patologia , Idoso , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Biópsia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Feminino , Perfilação da Expressão Gênica , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoconjugados/farmacologia , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Transplante de Neoplasias , Fenótipo , Medicina de Precisão , Estudos Prospectivos , Quinolinas/farmacologia , Estudos Retrospectivos , Análise de Sequência de RNA , TrastuzumabRESUMO
Heat shock proteins (Hsps) are molecular chaperons, which function in protein folding and assembly, protein intracellular localization and secretion, and degradation of misfolded and truncated proteins. Heat shock factors (Hsfs) are the transcriptional activators of Hsps. It has been reported that Hsps and Hsfs are widely involved in response to various abiotic stresses such as heat, drought, salinity and cold. To elucidate the function and regulation of rice Hsp and Hsf genes, we examined a global expression profiling with heat stressed rice seedling, and then compared our results with the previous rice data under cold, drought and salt stresses. The comparison revealed that, while most Hsfs and Hsps had highly similar and overlapped response and regulation patterns under different stresses, some of those genes showed significantly specific response to distinct stress. We also found that heat-responsive gene profiling differed largely from those under cold/drought/salt stresses, and that drought treatment was more effective to up-regulate Hsf expression in rice than in Arabidopsis. Overall, our data suggests that Hsps and Hsfs might be important elements in cross-talk of different stress signal transduction networks.
RESUMO
BACKGROUND & AIMS: Identification and validation of new functionally relevant and pharmacologically actionable targets for pancreatic ductal adenocarcinoma (PDAC) remains a great challenge. Premalignant acinar cell reprogramming (acinar-to-ductal metaplasia [ADM]) is a precursor of pancreatic intraepithelial neoplasia (PanIN) lesions that can progress to PDAC. This study investigated the role of proline-rich tyrosine kinase 2 (PYK2) in mutant Kras-induced and pancreatitis-associated ADM and PanIN formation, as well as in PDAC maintenance. METHODS: Genetically engineered mouse models of mutant Kras (glycine 12 to aspartic acid) and Pyk2 deletion were used for investigating the role of PYK2 in PDAC genesis in mice. In vitro ADM assays were conducted using primary pancreatic acinar cells isolated from mice. Immunohistochemistry, immunofluorescence, and a series of biochemical experiments were used to investigate upstream regulators/downstream targets of PYK2 in pancreatic carcinogenesis. PDAC cell line xenograft experiments were performed to study the role of PYK2 and its downstream target in PDAC maintenance. RESULTS: PYK2 was increased substantially in ADM lesions induced by mutant Kras or inflammatory injury. Pyk2 deletion remarkably suppressed ADM and PanIN formation in a mutant Kras-driven and pancreatitis-associated PDAC model, whereas PYK2 knockdown substantially inhibited PDAC cell growth in vitro and in nude mice. This study uncovered a novel yes-associated protein 1/transcriptional co-activator with PDZ binding motif/signal transducer and activator of transcription 3/PYK2/ß-catenin regulation axis in PDAC. Our results suggest that PYK2 contributes to PDAC genesis and maintenance by activating the Wnt/ß-catenin pathway through directly phosphorylating ß-cateninY654. CONCLUSIONS: The current study uncovers PYK2 as a novel downstream effector of mutant KRAS signaling, a previously unrecognized mediator of pancreatitis-induced ADM and a novel intervention target for PDAC.
Assuntos
Carcinoma de Células Acinares/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Neoplasias Pancreáticas/metabolismo , Lesões Pré-Cancerosas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma de Células Acinares/genética , Carcinoma de Células Acinares/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proteínas de Ciclo Celular/metabolismo , Reprogramação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Quinase 2 de Adesão Focal/genética , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Via de Sinalização Wnt , Proteínas de Sinalização YAPRESUMO
On the basis of our previous work defining the molecular rationale for combined targeting of the PI3K and AR pathways in PTEN loss prostate cancer, the first clinical trial was recently reported demonstrating a significant benefit for combination therapy in patients with metastatic prostate cancer. In this phase II trial, loss of PTEN was a biomarker predictive of response to combined AKT and AR inhibition. Given that PTEN loss prostate cancers are significantly enriched for ERG genomic rearrangements, we evaluated how the aberrant expression of ERG may impact response to PI3K/AR-targeted therapy. Here, we show that overexpression of ERG in the setting of Pten loss promotes resistance to combined PI3K and AR pathway inhibition with associated maintenance of AR target gene expression. Importantly, following AR knockout in the setting of ERG overexpression, there is maintenance of a subset of AR lineage-specific target genes, making AR dispensable in this context. This has important clinical implications as even in the setting of the androgen-regulated TMPRSS2:ERG genomic rearrangement, ERG expression is never abolished following AR inhibition and may allow for cell survival following AR (lineage)-targeted therapies.