RESUMO
Approximately 30% of early-stage lung adenocarcinoma patients present with disease progression after successful surgical resection. Despite efforts of mapping the genetic landscape, there has been limited success in discovering predictive biomarkers of disease outcomes. Here we performed a systematic multi-omic assessment of 143 tumors and matched tumor-adjacent, histologically-normal lung tissue with long-term patient follow-up. Through histologic, mutational, and transcriptomic profiling of tumor and adjacent-normal tissue, we identified an inflammatory gene signature in tumor-adjacent tissue as the strongest clinical predictor of disease progression. Single-cell transcriptomic analysis demonstrated the progression-associated inflammatory signature was expressed in both immune and non-immune cells, and cell type-specific profiling in monocytes further improved outcome predictions. Additional analyses of tumor-adjacent transcriptomic data from The Cancer Genome Atlas validated the association of the inflammatory signature with worse outcomes across cancers. Collectively, our study suggests that molecular profiling of tumor-adjacent tissue can identify patients at high risk for disease progression.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Adenocarcinoma de Pulmão/genética , Inflamação/genética , Neoplasias Pulmonares/genética , Pulmão , Progressão da DoençaRESUMO
Tumor mutations can influence the surrounding microenvironment leading to suppression of anti-tumor immune responses and thereby contributing to tumor progression and failure of cancer therapies. Here we use genetically engineered lung cancer mouse models and patient samples to dissect how LKB1 mutations accelerate tumor growth by reshaping the immune microenvironment. Comprehensive immune profiling of LKB1 -mutant vs wildtype tumors revealed dramatic changes in myeloid cells, specifically enrichment of Arg1 + interstitial macrophages and SiglecF Hi neutrophils. We discovered a novel mechanism whereby autocrine LIF signaling in Lkb1 -mutant tumors drives tumorigenesis by reprogramming myeloid cells in the immune microenvironment. Inhibiting LIF signaling in Lkb1 -mutant tumors, via gene targeting or with a neutralizing antibody, resulted in a striking reduction in Arg1 + interstitial macrophages and SiglecF Hi neutrophils, expansion of antigen specific T cells, and inhibition of tumor progression. Thus, targeting LIF signaling provides a new therapeutic approach to reverse the immunosuppressive microenvironment of LKB1 -mutant tumors.
RESUMO
A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity.
Assuntos
Neoplasias Colorretais , Segunda Neoplasia Primária , Neoplasias Peritoneais , Neoplasias Colorretais/patologia , Humanos , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/secundário , Peritônio/metabolismo , Qualidade de VidaRESUMO
Pyrvinium is a quinoline-derived cyanine dye and an approved anti-helminthic drug reported to inhibit WNT signaling and have anti-proliferative effects in various cancer cell lines. To further understand the mechanism by which pyrvinium is cytotoxic, we conducted a pooled genome-wide CRISPR loss-of-function screen in the human HAP1 cell model. The top drug-gene sensitizer interactions implicated the malate-aspartate and glycerol-3-phosphate shuttles as mediators of cytotoxicity to mitochondrial complex I inhibition including pyrvinium. By contrast, perturbation of the poorly characterized gene C1orf115/RDD1 resulted in strong resistance to the cytotoxic effects of pyrvinium through dysregulation of the major drug efflux pump ABCB1/MDR1. Interestingly, C1orf115/RDD1 was found to physically associate with ABCB1/MDR1 through proximity-labeling experiments and perturbation of C1orf115 led to mis-localization of ABCB1/MDR1. Our results are consistent with a model whereby C1orf115 modulates drug efflux through regulation of the major drug exporter ABCB1/MDR1.
Assuntos
Antineoplásicos , Compostos de Pirvínio , Humanos , Compostos de Pirvínio/farmacologia , Via de Sinalização Wnt , Antineoplásicos/farmacologia , GenômicaRESUMO
The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1-3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.
Assuntos
Genoma/genética , Genômica , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Animais , Autofagia , Linhagem Celular Tumoral , Feminino , Genes Neoplásicos/genética , Humanos , Interferon gama/imunologia , Masculino , Camundongos , NF-kappa B/metabolismo , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
Small cell lung cancer (SCLC) is a particularly aggressive subset of lung cancer, and identification of new therapeutic options is of significant interest. We recently reported that SCLC cell lines display a specific vulnerability to inhibition of squalene epoxidase (SQLE), an enzyme in the cholesterol biosynthetic pathway that catalyzes the conversion of squalene to 2,3-oxidosqualene. Since it has been reported that SQLE inhibition can result in dermatitis in dogs, we conducted a series of experiments to determine if SQLE inhibitors would be tolerated at exposures predicted to drive maximal efficacy in SCLC tumors. Detailed profiling of the SQLE inhibitor NB-598 showed that dogs did not tolerate predicted efficacious exposures, with dose-limiting toxicity due to gastrointestinal clinical observations, although skin toxicities were also observed. To extend these studies, two SQLE inhibitors, NB-598 and Cmpd-4â³, and their structurally inactive analogs, NB-598.ia and Cmpd-4â³.ia, were profiled in monkeys. While both active SQLE inhibitors resulted in dose-limiting gastrointestinal toxicity, the structurally similar inactive analogs did not. Collectively, our data demonstrate that significant toxicities arise at exposures well below the predicted levels needed for anti-tumor activity. The on-target nature of the toxicities identified is likely to limit the potential therapeutic utility of SQLE inhibition for the treatment of SCLC.
Assuntos
Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/toxicidade , Esqualeno Mono-Oxigenase/antagonistas & inibidores , Esqualeno Mono-Oxigenase/sangue , Animais , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Macaca fascicularis , Masculino , Pele/efeitos dos fármacos , Pele/enzimologia , Pele/patologiaRESUMO
Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (â¼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.
Assuntos
Aciltransferases/genética , Neoplasias das Glândulas Suprarrenais/genética , Mutação em Linhagem Germinativa , Paraganglioma/genética , Feocromocitoma/genética , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinogênese , Domínio Catalítico , Ciclo do Ácido Cítrico , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-IdadeRESUMO
Squalene epoxidase (SQLE), also known as squalene monooxygenase, catalyzes the stereospecific conversion of squalene to 2,3(S)-oxidosqualene, a key step in cholesterol biosynthesis. SQLE inhibition is targeted for the treatment of hypercholesteremia, cancer, and fungal infections. However, lack of structure-function understanding has hindered further progression of its inhibitors. We have determined the first three-dimensional high-resolution crystal structures of human SQLE catalytic domain with small molecule inhibitors (2.3 Å and 2.5 Å). Comparison with its unliganded state (3.0 Å) reveals conformational rearrangements upon inhibitor binding, thus allowing deeper interpretation of known structure-activity relationships. We use the human SQLE structure to further understand the specificity of terbinafine, an approved agent targeting fungal SQLE, and to provide the structural insights into terbinafine-resistant mutants encountered in the clinic. Collectively, these findings elucidate the structural basis for the specificity of the epoxidation reaction catalyzed by SQLE and enable further rational development of next-generation inhibitors.
Assuntos
Esqualeno Mono-Oxigenase/química , Esqualeno Mono-Oxigenase/metabolismo , Animais , Domínio Catalítico , Linhagem Celular , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Insetos , Conformação Proteica , Domínios Proteicos , Esqualeno/metabolismo , Esqualeno Mono-Oxigenase/antagonistas & inibidoresRESUMO
Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers.
Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Esqualeno Mono-Oxigenase/antagonistas & inibidores , Esqualeno Mono-Oxigenase/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Colesterol/biossíntese , Deleção de Genes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , HumanosRESUMO
Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA) cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.
Assuntos
Ácido Aspártico/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , RNA Interferente Pequeno/metabolismoRESUMO
UNLABELLED: Epithelial-to-mesenchymal transition (EMT) has been implicated in models of tumor cell migration, invasion, and metastasis. In a search for candidate therapeutic targets to reverse this process, nontumorigenic MCF10A breast epithelial cells were infected with an arrayed lentiviral kinome shRNA library and screened for either suppression or enhancement of a 26-gene EMT RNA signature. No individual kinase gene knockdown was sufficient to induce EMT. In contrast, grouped epithelial markers were induced by knockdown of multiple kinases, including mitogen activated protein kinase 7 (MAPK7). In breast cancer cells, suppression of MAPK7 increased E-cadherin (CDH1) expression and inhibited cell migration. In an orthotopic mouse model, MAPK7 suppression reduced the generation of circulating tumor cells and the appearance of lung metastases. Together, these observations raise the possibility that targeting kinases that maintain mesenchymal cell properties in cancer cells, such as MAPK7, may lessen tumor invasiveness. IMPLICATIONS: Suppression of MAPK7 induces epithelial markers, reduces generation of circulating tumor cells and appearance of lung metastases.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Animais , Antígenos CD , Neoplasias da Mama/sangue , Caderinas/biossíntese , Caderinas/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteína Quinase 7 Ativada por Mitógeno/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , TranscriptomaRESUMO
Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial markers were repressed within 6 hr, and mesenchymal genes were induced at 24 hr. Snail-1 binding to its target promoters was transient (6-48 hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Transição Epitelial-Mesenquimal , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Acetilação , Carcinogênese/metabolismo , Epigênese Genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Células MCF-7 , Metilação , Regiões Promotoras Genéticas , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genéticaRESUMO
Accelerated glucose metabolism is a common feature of cancer cells. Hexokinases catalyze the first committed step of glucose metabolism. Hexokinase 2 (HK2) is expressed at high level in cancer cells, but only in a limited number of normal adult tissues. Using Hk2 conditional knockout mice, we showed that HK2 is required for tumor initiation and maintenance in mouse models of KRas-driven lung cancer, and ErbB2-driven breast cancer, despite continued HK1 expression. Similarly, HK2 ablation inhibits the neoplastic phenotype of human lung and breast cancer cells in vitro and in vivo. Systemic Hk2 deletion is therapeutic in mice bearing lung tumors without adverse physiological consequences. Hk2 deletion in lung cancer cells suppressed glucose-derived ribonucleotides and impaired glutamine-derived carbon utilization in anaplerosis.
Assuntos
Neoplasias da Mama/enzimologia , Hexoquinase/metabolismo , Neoplasias Pulmonares/enzimologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Glicólise , Hexoquinase/biossíntese , Hexoquinase/genética , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Knockout , Transplante HeterólogoRESUMO
Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell-cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.
Assuntos
Ativação Enzimática/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas/química , Animais , Biologia Computacional/métodos , Células HeLa , Humanos , Ligantes , Termodinâmica , Peixe-Zebra , Quinases da Família src/metabolismoRESUMO
To define the functional pathways regulating epithelial cell migration, we performed a genome-wide RNAi screen using 55,000 pooled lentiviral shRNAs targeting â¼11,000 genes, selecting for transduced cells with increased motility. A stringent validation protocol generated a set of 31 genes representing diverse pathways whose knockdown dramatically enhances cellular migration. Some of these pathways share features of epithelial-to-mesenchymal transition (EMT), and together they implicate key regulators of transcription, cellular signaling, and metabolism, as well as novel modulators of cellular trafficking, such as DLG5. In delineating downstream pathways mediating these migration phenotypes, we observed universal activation of ERKs and a profound dependence on their RSK effectors. Pharmacological inhibition of RSK dramatically suppresses epithelial cell migration induced by knockdown of all 31 genes, suggesting that convergence of diverse migratory pathways on this kinase may provide a therapeutic opportunity in disorders of cell migration, including cancer metastasis.
Assuntos
Movimento Celular/genética , Estudo de Associação Genômica Ampla , Interferência de RNA , Proteínas Quinases S6 Ribossômicas/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Humanos , Proteínas de Membrana/metabolismo , Mesoderma/citologia , Reprodutibilidade dos Testes , Proteínas Supressoras de Tumor/metabolismoRESUMO
The Hippo signalling pathway regulates cellular proliferation and survival, thus has profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP (yes-associated protein), a transcriptional co-activator amplified in mouse and human cancers, where it promotes epithelial to mesenchymal transition (EMT) and malignant transformation. So far, studies of YAP target genes have focused on cell-autonomous mediators; here we show that YAP-expressing MCF10A breast epithelial cells enhance the proliferation of neighbouring untransfected cells, implicating a non-cell-autonomous mechanism. We identify the gene for the epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) as a transcriptional target of YAP, whose induction contributes to YAP-mediated cell proliferation and migration, but not EMT. Knockdown of AREG or addition of an EGFR kinase inhibitor abrogates the proliferative effects of YAP expression. Suppression of the negative YAP regulators LATS1 and 2 (large tumour suppressor 1 and 2) is sufficient to induce AREG expression, consistent with physiological regulation of AREG by the Hippo pathway. Genetic interaction between the Drosophila YAP orthologue Yorkie and Egfr signalling components supports the link between these two highly conserved signalling pathways. Thus, YAP-dependent secretion of AREG indicates that activation of EGFR signalling is an important non-cell-autonomous effector of the Hippo pathway, which has implications for the regulation of both physiological and malignant cell proliferation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Anfirregulina , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Família de Proteínas EGF , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
Epithelial-to-mesenchymal transition (EMT) plays an important role during normal embryogenesis, and it has been implicated in cancer invasion and metastasis. Here, we report that Ladybird homeobox 1 (LBX1), a developmentally regulated homeobox gene, directs expression of the known EMT inducers ZEB1, ZEB2, Snail1, and transforming growth factor beta2 (TGFB2). In mammary epithelial cells, overexpression of LBX1 leads to morphological transformation, expression of mesenchymal markers, enhanced cell migration, increased CD44(high)/CD24(low) progenitor cell population, and tumorigenic cooperation with known oncogenes. In human breast cancer, LBX1 is up-regulated in the unfavorable estrogen receptor (ER)/progesterone (PR)/HER2 triple-negative basal-like subtype. Thus, aberrant expression of LBX1 may lead to the activation of a developmentally regulated EMT pathway in human breast cancer.
Assuntos
Neoplasias da Mama/fisiopatologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proteínas de Homeodomínio/genética , Humanos , Receptores de Hialuronatos/metabolismo , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Oncogenes/fisiologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição da Família Snail , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta2/metabolismo , Regulação para Cima , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
RNAi is widely applied to inhibit expression of specific genes, but it is limited by variable efficiency and specificity of empirically designed siRNA or shRNA constructs. This complicates studies targeting individual genes and significantly impairs large-scale screens using genome-wide knockdown libraries. Here, we show that ectopic expression of the RISC slicer Argonaute-2 (Ago2, eIF2C2) dramatically enhances RNAi specifically for mRNA targets with perfectly matched binding sites. This effect depends on its endonuclease activity and is uncoupled from its regulation of microRNA expression. To model the application of Ago2 coexpression with shRNA knockdown, we targeted the EGF receptor (EGFR) in lung cancer cells exhibiting oncogene addiction to EGFR. Whereas multiple empirically designed shRNA constructs exhibited highly divergent efficiencies in mediating EGFR knockdown and cell killing, coexpression of Ago2 resulted in uniform and highly specific target gene suppression and apoptosis in EGFR-dependent cells. Codelivery of Ago2 with shRNA constructs or siRNA duplexes thus provides a strategy to enhance the efficacy and the specificity of RNAi in experimental and potentially therapeutic settings.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Interferência de RNA , Proteínas Argonautas , Sítios de Ligação , Morte Celular , Linhagem Celular , Receptores ErbB/metabolismo , Humanos , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: Desmoplastic Small Round Cell Tumor (DSRCT) is a highly aggressive malignancy that affects mainly adolescents and young adults. A defining characteristic of DSRCT is a specific chromosomal translocation, t(11;22)(p13;q12), that fuses EWS with WT1, leading to a production of two isoforms of chimeric transcription factor, EWS/WT1(-KTS) and EWS/WT1(+KTS). The chimeric proteins are thought to play critical roles in various stages of oncogenesis through aberrant transcription of different genes, but only a few of these genes have been identified. METHODOLOGY/PRINCIPAL FINDINGS: We report the identification of a new target of EWS/WT1, ENT4 (equilibrative nucleoside transporter 4) which encodes a pH-dependent adenosine transporter. ENT4 is transcriptionally activated by both isoforms of EWS/WT1 as evidenced by promoter-reporter and chromatin immunoprecipitation (ChIP) analyses. Furthermore, ENT4 is highly and specifically expressed in primary tumors of DSRCT as well as in a DSRCT cell line, JN-DSRCT-1. Treatment of JN-DSRCT-1 cells with adenosine analogs, such as 2-chloro-2'-deoxyadenosine (2-CdA), resulted in an increased cytotoxic response in dose- and pH-dependent manner. CONCLUSIONS/SIGNIFICANCE: Our detailed analyses of a novel target of EWS/WT1 in DSRCT reveal an insight into the oncogenic mechanism of EWS-fusion chromosomal translocation gene products and provide a new marker for DSRCT. Furthermore, identification of ENT4 as a highly expressed transcript in DSRCT may represent an attractive pathway for targeting chemotherapeutic drugs into DSRCT.