RESUMO
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal â¼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.
Assuntos
Processamento Alternativo/efeitos da radiação , DNA Helicases/genética , RNA não Traduzido/genética , Transcrição Gênica , Raios Ultravioleta , Linhagem Celular , Éxons , Humanos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética/efeitos da radiação , Iniciação da Transcrição Genética/efeitos da radiaçãoRESUMO
Many body surfaces harbor organ-specific γδ T cell compartments that contribute to tissue integrity. Thus, murine dendritic epidermal T cells (DETCs) uniquely expressing T cell receptor (TCR)-Vγ5 chains protect from cutaneous carcinogens. The DETC repertoire is shaped by Skint1, a butyrophilin-like (Btnl) gene expressed specifically by thymic epithelial cells and suprabasal keratinocytes. However, the generality of this mechanism has remained opaque, since neither Skint1 nor DETCs are evolutionarily conserved. Here, Btnl1 expressed by murine enterocytes is shown to shape the local TCR-Vγ7(+) γδ compartment. Uninfluenced by microbial or food antigens, this activity evokes the developmental selection of TCRαß(+) repertoires. Indeed, Btnl1 and Btnl6 jointly induce TCR-dependent responses specifically in intestinal Vγ7(+) cells. Likewise, human gut epithelial cells express BTNL3 and BTNL8 that jointly induce selective TCR-dependent responses of human colonic Vγ4(+) cells. Hence, a conserved mechanism emerges whereby epithelia use organ-specific BTNL/Btnl genes to shape local T cell compartments.
Assuntos
Butirofilinas/imunologia , Mucosa Intestinal/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Butirofilinas/genética , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Timo/imunologiaRESUMO
Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy1. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study2,3. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic-transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary-metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis.
Assuntos
Evolução Molecular , Genoma Humano , Neoplasias Pulmonares , Metástase Neoplásica , Transcriptoma , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Genômica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Metástase Neoplásica/genética , Transcriptoma/genética , Alelos , Aprendizado de Máquina , Genoma Humano/genéticaRESUMO
The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3' UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.
Assuntos
Antígeno B7-H1/imunologia , Neoplasias Colorretais/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/imunologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Tristetraprolina/imunologia , Evasão Tumoral , Animais , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Clivagem do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Tristetraprolina/genéticaRESUMO
Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.
Assuntos
Parasitos , Toxoplasma , Humanos , Animais , Camundongos , Toxoplasma/metabolismo , Parasitos/metabolismo , Interferon gama , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Virulência , Vacúolos/metabolismoRESUMO
Non-small-cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide. As the majority of patients present with invasive, metastatic disease, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma, in which it contributes to cancer progression and metastasis. Here we show that Hmga2 promotes lung cancer progression in mouse and human cells by operating as a competing endogenous RNA (ceRNA) for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are also observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-ß co-receptor Tgfbr3 (ref. 12) as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA through differential recruitment to Argonaute 2 (Ago2), and TGF-ß signalling driven by Tgfbr3 is important for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC-patient gene-expression data reveals that HMGA2 and TGFBR3 are coordinately regulated in NSCLC-patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis both as a protein-coding gene and as a non-coding RNA; such dual-function regulation of gene-expression networks reflects a novel means by which oncogenes promote disease progression.
Assuntos
Progressão da Doença , Proteína HMGA2/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Animais , Proteínas Argonautas/metabolismo , Ligação Competitiva/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Proteoglicanas/biossíntese , Proteoglicanas/deficiência , Proteoglicanas/genética , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/deficiência , Receptores de Fatores de Crescimento Transformadores beta/genética , Transcrição Gênica/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Alternative messenger RNA splicing is the main reason that vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription, and is greatly affected by the rate of transcript elongation. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle; this is the functional form of the nascent pre-mRNA and determines the fate of the mature transcript. However, factors that connect the transcribing polymerase with the mRNP particle and help to integrate transcript elongation with mRNA splicing remain unclear. Here we characterize the human interactome of chromatin-associated mRNP particles. This led us to identify deleted in breast cancer 1 (DBC1) and ZNF326 (which we call ZNF-protein interacting with nuclear mRNPs and DBC1 (ZIRD)) as subunits of a novel protein complex--named DBIRD--that binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in (A + T)-rich DNA, and is present at the affected exons. RNA-interference-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. Together, these data indicate that the DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with the regulation of alternative splicing.
Assuntos
Processamento Alternativo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromatina/genética , Cromatina/metabolismo , Éxons/genética , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/deficiência , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Camundongos , Complexos Multiproteicos/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
Ectopic expression of dual-specificity phosphatase 5 (DUSP5), an inducible mitogen-activated protein (MAP) kinase phosphatase, specifically inactivates and anchors extracellular signal-regulated kinase (ERK)1/2 in the nucleus. However, the role of endogenous DUSP5 in regulating the outcome of Ras/ERK kinase signaling under normal and pathological conditions is unknown. Here we report that mice lacking DUSP5 show a greatly increased sensitivity to mutant Harvey-Ras (HRas(Q61L))-driven papilloma formation in the 7,12-Dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) model of skin carcinogenesis. Furthermore, mouse embryo fibroblasts (MEFs) from DUSP5(-/-) mice show increased levels of nuclear phospho-ERK immediately after TPA stimulation and fail to accumulate total ERK in the nucleus compared with DUSP5(+/+) cells. Surprisingly, a microarray analysis reveals that only a small number of Ras/ERK-dependent TPA-responsive transcripts are up-regulated on deletion of DUSP5 in MEFs and mouse skin. The most up-regulated gene on DUSP5 loss encodes SerpinB2, an inhibitor of extracellular urokinase plasminogen activator and deletion of DUSP5 acts synergistically with mutant HRas(Q61L) and TPA to activate ERK-dependent SerpinB2 expression at the transcriptional level. SerpinB2 has previously been implicated as a mediator of DMBA/TPA-induced skin carcinogenesis. By analyzing DUSP5(-/-), SerpinB2(-/-) double knockout mice, we demonstrate that deletion of SerpinB2 abrogates the increased sensitivity to papilloma formation seen on DUSP5 deletion. We conclude that DUSP5 performs a key nonredundant role in regulating nuclear ERK activation, localization, and gene expression. Furthermore, our results suggest an in vivo role for DUSP5 as a tumor suppressor by modulating the oncogenic potential of activated Ras in the epidermis.
Assuntos
Núcleo Celular/enzimologia , Fosfatases de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes ras , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Neoplasias Cutâneas/prevenção & controle , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Fosfatases de Especificidade Dupla/genética , Camundongos , Camundongos Knockout , Transdução de Sinais , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
Adenomatous polyposis coli (APC ) mutations are found in most colorectal tumours. These mutations are almost always protein-truncating, deleting both central domains that regulate Wnt signalling and C-terminal domains that interact with the cytoskeleton. The importance of Wnt dysregulation for colorectal tumourigenesis is well characterized. It is, however, unclear whether loss of C-terminal functions contributes to tumourigenesis, although this protein region has been implicated in cellular processes--including polarity, migration, mitosis, and chromosomal instability (CIN)that have been postulated as critical for the development and progression of intestinal tumours. Since almost all APC mutations in human patients disrupt both central and C-terminal regions, we created a mouse model to test the role of the C-terminus of APC in intestinal tumourigenesis. This mouse (Apc(ΔSAMP)) carries an internal deletion within Apc that dysregulates Wnt by removing the beta-catenin-binding and SAMP repeats, but leaves the C-terminus intact. We compared Apc(ΔSAMP) mice with Apc(1322T) animals. The latter allele represented the most commonly found human APC mutation and was identical to Apc(ΔSAMP) except for absence of the entire C-terminus. Apc(ΔSAMP) mice developed numerous intestinal adenomas indistinguishable in number, location, and dysplasia from those seen in Apc(1322T) mice. No carcinomas were found in Apc(ΔSAMP) or Apc(1322T) animals. While similar disruption of the Wnt signalling pathway was observed in tumours from both mice, no evidence of differential C-terminus functions (such as cell migration, CIN, or localization of APC and EB1) was seen. We conclude that the C-terminus of APC does not influence intestinal adenoma development or progression.
Assuntos
Adenoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias Intestinais/genética , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/química , Animais , Western Blotting , Movimento Celular/genética , Progressão da Doença , Imunofluorescência , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Intestinais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Análise de Sequência com Séries de Oligonucleotídeos , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genética , Via de Sinalização WntRESUMO
Chromosomal instability (CIN) has been implicated in multidrug resistance and the silencing of the ceramide transporter, CERT, promotes sensitization to diverse cytotoxics. An improved understanding of mechanisms governing multidrug sensitization might provide insight into pathways contributing to the death of CIN cancer cells. Using an integrative functional genomics approach, we find that CERT-specific multidrug sensitization is associated with enhanced autophagosome-lysosome flux, resulting from the expression of LAMP2 following CERT silencing in colorectal and HER2(+) breast cancer cell lines. Live cell microscopy analysis revealed that CERT depletion induces LAMP2-dependent death of polyploid cells following exit from mitosis in the presence of paclitaxel. We find that CERT is relatively over-expressed in HER2(+) breast cancer and CERT protein expression acts as an independent prognostic variable and predictor of outcome in adjuvant chemotherapy-treated patients with primary breast cancer. These data suggest that the induction of LAMP2-dependent autophagic flux through CERT targeting may provide a rational approach to enhance multidrug sensitization and potentiate the death of polyploid cells following paclitaxel exposure to limit the acquisition of CIN and intra-tumour heterogeneity.
Assuntos
Autofagia/fisiologia , Neoplasias da Mama/tratamento farmacológico , Instabilidade Cromossômica/fisiologia , Proteínas Serina-Treonina Quinases/deficiência , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/genética , Ceramidas/metabolismo , Ceramidas/farmacologia , Cisplatino/farmacologia , Resistência a Múltiplos Medicamentos/genética , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Expressão Gênica , Inativação Gênica/fisiologia , Humanos , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/fisiologia , Pessoa de Meia-Idade , Moduladores de Mitose/farmacologia , Poliploidia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Receptor ErbB-2 , Células Tumorais CultivadasRESUMO
The evolution of established cancers is driven by selection of cells with enhanced fitness. Subclonal mutations in numerous epigenetic regulator genes are common across cancer types, yet their functional impact has been unclear. Here, we show that disruption of the epigenetic regulatory network increases the tolerance of cancer cells to unfavorable environments experienced within growing tumors by promoting the emergence of stress-resistant subpopulations. Disruption of epigenetic control does not promote selection of genetically defined subclones or favor a phenotypic switch in response to environmental changes. Instead, it prevents cells from mounting an efficient stress response via modulation of global transcriptional activity. This "transcriptional numbness" lowers the probability of cell death at early stages, increasing the chance of long-term adaptation at the population level. Our findings provide a mechanistic explanation for the widespread selection of subclonal epigenetic-related mutations in cancer and uncover phenotypic inertia as a cellular trait that drives subclone expansion.
Assuntos
Neoplasias , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , FenótipoRESUMO
Murine intraepithelial γδ T cells include distinct tissue-protective cells selected by epithelial butyrophilin-like (BTNL) heteromers. To determine whether this biology is conserved in humans, we characterized the colonic γδ T cell compartment, identifying a diverse repertoire that includes a phenotypically distinct subset coexpressing T cell receptor Vγ4 and the epithelium-binding integrin CD103. This subset was disproportionately diminished and dysregulated in inflammatory bowel disease, whereas on-treatment CD103+γδ T cell restoration was associated with sustained inflammatory bowel disease remission. Moreover, CD103+Vγ4+cell dysregulation and loss were also displayed by humans with germline BTNL3/BTNL8 hypomorphism, which we identified as a risk factor for penetrating Crohn's disease (CD). Thus, BTNL-dependent selection and/or maintenance of distinct tissue-intrinsic γδ T cells appears to be an evolutionarily conserved axis limiting the progression of a complex, multifactorial, tissue-damaging disease of increasing global incidence.
Assuntos
Butirofilinas , Doenças Inflamatórias Intestinais , Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T , Animais , Humanos , Camundongos , Butirofilinas/genética , Colo/imunologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Subpopulações de Linfócitos T/imunologia , Mucosa Intestinal/imunologiaRESUMO
Microtubule-stabilizing (MTS) agents, such as taxanes, are important chemotherapeutics with a poorly understood mechanism of action. We identified a set of genes repressed in multiple cell lines in response to MTS agents and observed that these genes are overexpressed in tumors exhibiting chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these "CIN-survival" genes is associated with poor outcome in estrogen receptor-positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane resistance but carboplatin sensitivity, indicating that CIN may determine MTS response in vivo. Thus, pretherapeutic assessment of CIN may optimize treatment stratification and clinical trial design using these agents.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Instabilidade Cromossômica/efeitos dos fármacos , Instabilidade Cromossômica/genética , Taxoides/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Microtúbulos/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Paclitaxel/toxicidade , Reação em Cadeia da Polimerase , PrognósticoRESUMO
Specification of the germ layers by Nodal signaling has long been regarded as an archetype of how graded morphogens induce different cell fates. However, this deterministic model cannot explain why only a subset of cells at the early zebrafish embryo margin adopt the endodermal fate, whereas their immediate neighbours, experiencing a similar signaling environment, become mesoderm. Combining pharmacology, quantitative imaging and single cell transcriptomics, we demonstrate that sustained Nodal signaling establishes a bipotential progenitor state from which cells can switch to an endodermal fate or differentiate into mesoderm. Switching is a random event, the likelihood of which is modulated by Fgf signaling. This inherently imprecise mechanism nevertheless leads to robust endoderm formation because of buffering at later stages. Thus, in contrast to previous deterministic models of morphogen action, Nodal signaling establishes a temporal window when cells are competent to undergo a stochastic cell fate switch, rather than determining fate itself.
Assuntos
Peixe-Zebra , AnimaisRESUMO
Activating mutations in KRAS occur in 32% of lung adenocarcinomas (LUAD). Despite leading to aggressive disease and resistance to therapy in preclinical studies, the KRAS mutation does not predict patient outcome or response to treatment, presumably due to additional events modulating RAS pathways. To obtain a broader measure of RAS pathway activation, we developed RAS84, a transcriptional signature optimised to capture RAS oncogenic activity in LUAD. We report evidence of RAS pathway oncogenic activation in 84% of LUAD, including 65% KRAS wild-type tumours, falling into four groups characterised by coincident alteration of STK11/LKB1, TP53 or CDKN2A, suggesting that the classifications developed when considering only KRAS mutant tumours have significance in a broader cohort of patients. Critically, high RAS activity patient groups show adverse clinical outcome and reduced response to chemotherapy. Patient stratification using oncogenic RAS transcriptional activity instead of genetic alterations could ultimately assist in clinical decision-making.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Genes ras/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas rasRESUMO
Recently developed KRASG12C inhibitory drugs are beneficial to lung cancer patients harboring KRASG12C mutations, but drug resistance frequently develops. Because of the immunosuppressive nature of the signaling network controlled by oncogenic KRAS, these drugs can indirectly affect antitumor immunity, providing a rationale for their combination with immune checkpoint blockade. In this study, we have characterized how KRASG12C inhibition reverses immunosuppression driven by oncogenic KRAS in a number of preclinical lung cancer models with varying levels of immunogenicity. Mechanistically, KRASG12C inhibition up-regulates interferon signaling via Myc inhibition, leading to reduced tumor infiltration by immunosuppressive cells, enhanced infiltration and activation of cytotoxic T cells, and increased antigen presentation. However, the combination of KRASG12C inhibitors with immune checkpoint blockade only provides synergistic benefit in the most immunogenic tumor model. KRASG12C inhibition fails to sensitize cold tumors to immunotherapy, with implications for the design of clinical trials combining KRASG12C inhibitors with anti-PD1 drugs.
Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Inibidores de Checkpoint Imunológico , Interferons , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Genetic investigations of Upper Palaeolithic Europe have revealed a complex and transformative history of human population movements and ancestries, with evidence of several instances of genetic change across the European continent in the period following the Last Glacial Maximum (LGM). Concurrent with these genetic shifts, the post-LGM period is characterized by a series of significant climatic changes, population expansions and cultural diversification. Britain lies at the extreme northwest corner of post-LGM expansion and its earliest Late Glacial human occupation remains unclear. Here we present genetic data from Palaeolithic human individuals in the United Kingdom and the oldest human DNA thus far obtained from Britain or Ireland. We determine that a Late Upper Palaeolithic individual from Gough's Cave probably traced all its ancestry to Magdalenian-associated individuals closely related to those from sites such as El Mirón Cave, Spain, and Troisième Caverne in Goyet, Belgium. However, an individual from Kendrick's Cave shows no evidence of having ancestry related to the Gough's Cave individual. Instead, the Kendrick's Cave individual traces its ancestry to groups who expanded across Europe during the Late Glacial and are represented at sites such as Villabruna, Italy. Furthermore, the individuals differ not only in their genetic ancestry profiles but also in their mortuary practices and their diets and ecologies, as evidenced through stable isotope analyses. This finding mirrors patterns of dual genetic ancestry and admixture previously detected in Iberia but may suggest a more drastic genetic turnover in northwestern Europe than in the southwest.
Assuntos
Cavernas , Ecologia , Humanos , Reino Unido , Europa (Continente) , CefotaximaRESUMO
Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumor regression but ultimately fail to elicit cures. As a result, there is an intense interest in how to best combine targeted therapies with other treatments, such as immunotherapies. However, preclinical systems for studying the interaction of lung tumors with the host immune system are inadequate, in part due to the low tumor mutational burden in genetically engineered mouse models. Here we set out to develop mouse models of mutant KRAS-driven lung cancer with an elevated tumor mutational burden by expressing the human DNA cytosine deaminase, APOBEC3B, to mimic the mutational signature seen in human lung cancer. This failed to substantially increase clonal tumor mutational burden and autochthonous tumors remained refractory to immunotherapy. However, establishing clonal cell lines from these tumors enabled the generation of an immunogenic syngeneic transplantation model of KRAS-mutant lung adenocarcinoma that was sensitive to immunotherapy. Unexpectedly, antitumor immune responses were not directed against neoantigens but instead targeted derepressed endogenous retroviral antigens. The ability of KRASG12C inhibitors to cause regression of KRASG12C -expressing tumors was markedly potentiated by the adaptive immune system, highlighting the importance of using immunocompetent models for evaluating targeted therapies. Overall, this model provides a unique opportunity for the study of combinations of targeted and immunotherapies in immune-hot lung cancer. SIGNIFICANCE: This study develops a mouse model of immunogenic KRAS-mutant lung cancer to facilitate the investigation of optimal combinations of targeted therapies with immunotherapies.
Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Animais , Citidina Desaminase/genética , Citosina Desaminase/genética , Citosina Desaminase/uso terapêutico , Modelos Animais de Doenças , Receptores ErbB/genética , Humanos , Imunoterapia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/terapia , Camundongos , Antígenos de Histocompatibilidade Menor , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
The transcriptional effector SMAD4 is a core component of the TGF-ß family signaling pathways. However, its role in vertebrate embryo development remains unresolved. To address this, we deleted Smad4 in zebrafish and investigated the consequences of this on signaling by the TGF-ß family morphogens, BMPs and Nodal. We demonstrate that in the absence of Smad4, dorsal/ventral embryo patterning is disrupted due to the loss of BMP signaling. However, unexpectedly, Nodal signaling is maintained, but lacks robustness. This Smad4-independent Nodal signaling is sufficient for mesoderm specification, but not for optimal endoderm specification. Furthermore, using Optical Projection Tomography in combination with 3D embryo morphometry, we have generated a BMP morphospace and demonstrate that Smad4 mutants are morphologically indistinguishable from embryos in which BMP signaling has been genetically/pharmacologically perturbed. Smad4 is thus differentially required for signaling by different TGF-ß family ligands, which has implications for diseases where Smad4 is mutated or deleted.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteína Nodal/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , Desenvolvimento Embrionário , Endoderma/metabolismo , Técnicas de Inativação de Genes , Mesoderma/metabolismo , Morfogênese , Transdução de Sinais , Proteína Smad4/deficiência , Proteína Smad4/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND & AIMS: We previously demonstrated that the 2 APC mutations in human colorectal tumors are coselected, because tumorigenesis requires an optimal level of Wnt signaling. We and others subsequently showed that the truncated APC proteins in colorectal tumors usually retain a total of 1-2 beta-catenin binding/degradation repeats (20AARs); very few intestinal tumors have proteins with no 20AARs. The coselection of the "2 hits" at APC makes it difficult to undertake further mechanistic studies in this area in humans. In mice, however, second hits appear to vary with the strain or genetic background used. This suggested the possibility of creating suboptimal Apc genotypes in the mouse. METHODS: We have constructed a mouse, Apc(1322T), with a mutant protein retaining one 20AAR. After repeated backcrossing to the C57BL/6J background, we compared the 1322T animals with the widely used Min mouse in which the mutant Apc protein has zero 20AARs. RESULTS: In both mice, intestinal adenomas showed copy-neutral loss of heterozygosity, making them homozygous for the mutant Apc allele. 1322T animals had markedly more severe polyposis, with earlier-onset, larger, more numerous, and more severely dysplastic adenomas. 1322T tumors also had more marked Paneth cell differentiation and higher frequencies of crypt fission. Somewhat surprisingly, nuclear beta-catenin expression was lower in 1322T than Min tumors. CONCLUSIONS: We propose that the Apc(1322T) mutation produces submaximal beta-catenin levels that promote early tumor growth more effectively than the Apc(Min) mutation.