RESUMO
Patients with ER-negative breast cancer have the worst prognosis of all breast cancer subtypes, often experiencing rapid recurrence or progression to metastatic disease shortly after diagnosis. Given that metastasis is the primary cause of mortality in most solid tumors, understanding metastatic biology is crucial for effective intervention. Using a mouse systems genetics approach, we previously identified 12 genes associated with metastatic susceptibility. Here, we extend those studies to identify Resf1, a poorly characterized gene, as a novel metastasis susceptibility gene in ER- breast cancer. Resf1 is a large, unstructured protein with an evolutionarily conserved intron-exon structure, but with poor amino acid conservation. CRISPR or gene trap mouse models crossed to the Polyoma Middle-T antigen genetically engineered mouse model (MMTV-PyMT) demonstrated that reduction of Resf1 resulted in a significant increase in tumor growth, a shortened overall survival time, and increased incidence and number of lung metastases, consistent with patient data. Furthermore, an analysis of matched tail and primary tissues revealed loss of the wildtype copy in tumor tissue, consistent with Resf1 being a tumor suppressor. Mechanistic analysis revealed a potential role of Resf1 in transcriptional control through association with compound G4 quadruplexes in expressed sequences, particularly those associated with ribosomal biogenesis. These results suggest that loss of Resf1 enhances tumor progression in ER- breast cancer through multiple alterations in both transcriptional and translational control.
Assuntos
Proteínas Repressoras , Neoplasias de Mama Triplo Negativas , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Quadruplex G , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Breast cancer is the most frequently diagnosed cancer worldwide, constituting around 15% of all diagnosed cancers in 2023. The predominant cause of breast cancer-related mortality is metastasis to distant essential organs, and a lack of metastasis-targeted therapies perpetuates dismal outcomes for late-stage patients. However, through our use of meiotic genetics to study inherited transcriptional network regulation, we have identified a new class of "Goldilocks" genes that are promising candidates for the development of metastasis-targeted therapeutics. Building upon previous work that implicated the CCR4-NOT RNA deadenylase complex in metastasis, we now demonstrate that the RNA-binding proteins (RNA-BPs) NANOS1, PUM2, and CPSF4 also regulate metastatic potential. Using cell lines, 3D culture, mouse models, and clinical data, we pinpoint Smarcd1 mRNA as a key target of all three RNA-BPs. Strikingly, both high and low expression of Smarcd1 is associated with positive clinical outcomes, while intermediate expression significantly reduces the probability of survival. Applying the theory of "essential genes" from evolution, we identify an additional 50 genes that span several cellular processes and must be maintained within a discrete window of expression for metastasis to occur. In the case of Smarcd1, small perturbations in its expression level significantly reduce metastasis in laboratory mouse models and alter splicing programs relevant to the ER+/HER2-enriched breast cancer subtype. The identification of subtype-specific "Goldilocks" metastasis modifier genes introduces a new class of genes and potential catalogue of novel targets that, when therapeutically "nudged" in either direction, may significantly improve late-stage patient outcomes.
RESUMO
Acetylation of protein and RNA represent a critical event for development and cancer progression. NAT10 is the only known RNA acetylase that catalyzes the N4-actylcytidine (ac4C) modification of RNAs. Here, we show that the loss of NAT10 significantly decreases lung metastasis in allograft and genetically engineered mouse models of breast cancer. NAT10 interacts with a mechanosensitive, metastasis susceptibility protein complex at the nuclear pore. In addition to its canonical role in RNA acetylation, we find that NAT10 interacts with p300 at gene enhancers. NAT10 loss is associated with p300 mislocalization into heterochromatin regions. NAT10 depletion disrupts enhancer organization, leading to alteration of gene transcription necessary for metastatic progression, including reduced myeloid cell-recruiting chemokines that results in a less metastasis-prone tumor microenvironment. Our study uncovers a distinct role of NAT10 in enhancer organization of metastatic tumor cells and suggests its involvement in the tumor-immune crosstalk dictating metastatic outcomes.
RESUMO
Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Saco Vitelino , Células-Tronco Hematopoéticas , Organoides , Atividades CotidianasRESUMO
A paradox of tumor immunology is that tumor-infiltrating lymphocytes are dysfunctional in situ, yet are capable of stem cell-like behavior including self-renewal, expansion, and multipotency, resulting in the eradication of large metastatic tumors. We find that the overabundance of potassium in the tumor microenvironment underlies this dichotomy, triggering suppression of T cell effector function while preserving stemness. High levels of extracellular potassium constrain T cell effector programs by limiting nutrient uptake, thereby inducing autophagy and reduction of histone acetylation at effector and exhaustion loci, which in turn produces CD8+ T cells with improved in vivo persistence, multipotency, and tumor clearance. This mechanistic knowledge advances our understanding of T cell dysfunction and may lead to novel approaches that enable the development of enhanced T cell strategies for cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Potássio/metabolismo , Células-Tronco/imunologia , Acetilcoenzima A/metabolismo , Acetilação , Animais , Autofagia/imunologia , Restrição Calórica , Diferenciação Celular/genética , Epigênese Genética , Histonas/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Microambiente TumoralRESUMO
Tumour heterogeneity poses a substantial problem for the clinical management of cancer. Somatic evolution of the cancer genome results in genetically distinct subclones in the primary tumour with different biological properties and therapeutic sensitivities. The problem of heterogeneity is compounded in metastatic disease owing to the complexity of the metastatic process and the multiple biological hurdles that the tumour cell must overcome to establish a clinically overt metastatic lesion. New advances in sequencing technology and clinical sample acquisition are providing insights into the phylogenetic relationship of metastases and primary tumours at the level of somatic tumour genetics while also illuminating fundamental mechanisms of the metastatic process. In addition to somatically acquired genetic heterogeneity in the tumour cells, inherited population-based genetic heterogeneity can profoundly modify metastatic biology and further complicate the development of effective, broadly applicable antimetastatic therapies. Here, we examine how genetic heterogeneity impacts metastatic disease and the implications of current knowledge for future research endeavours and therapeutic interventions.
Assuntos
Heterogeneidade Genética , Metástase Neoplásica/genética , Neoplasias/patologia , Animais , Predisposição Genética para Doença , Humanos , Camundongos , Metástase Neoplásica/terapia , Neoplasias/genética , Neoplasias Experimentais/genéticaRESUMO
Breast cancer mortality is primarily due to metastasis rather than primary tumors, yet relatively little is understood regarding the etiology of metastatic breast cancer. Previously, using a mouse genetics approach, we demonstrated that inherited germline polymorphisms contribute to metastatic disease, and that these single nucleotide polymorphisms (SNPs) could be used to predict outcome in breast cancer patients. In this study, a backcross between a highly metastatic (FVB/NJ) and low metastatic (MOLF/EiJ) mouse strain identified Arntl2, a gene encoding a circadian rhythm transcription factor, as a metastasis susceptibility gene associated with progression, specifically in estrogen receptor-negative breast cancer patients. Integrated whole genome sequence analysis with DNase hypersensitivity sites reveals SNPs in the predicted promoter of Arntl2. Using CRISPR/Cas9-mediated substitution of the MOLF promoter, we demonstrate that the SNPs regulate Arntl2 transcription and affect metastatic burden. Finally, analysis of SNPs associated with ARNTL2 expression in human breast cancer patients revealed reproducible associations of ARNTL2 expression quantitative trait loci (eQTL) SNPs with disease-free survival, consistent with the mouse studies.
RESUMO
Metastasis remains the primary cause of patient morbidity and mortality in solid tumors and is due to the action of a large number of tumor-autonomous and non-autonomous factors. Here we report the results of a genome-wide integrated strategy to identify novel metastasis susceptibility candidate genes and molecular pathways in breast cancer metastasis. This analysis implicates a number of transcriptional regulators and suggests cell-mediated immunity is an important determinant. Moreover, the analysis identified novel or FDA-approved drugs as potentially useful for anti-metastatic therapy. Further explorations implementing this strategy may therefore provide a variety of information for clinical applications in the control and treatment of advanced neoplastic disease.
Assuntos
Predisposição Genética para Doença , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/genética , Animais , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Mamárias Animais/diagnóstico , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos NZB , Camundongos Transgênicos , Nectinas , Proteína com Dedos de Zinco da Leucemia Promielocítica , Interferência de RNA , RNA Interferente Pequeno/genética , Rosiglitazona , Tiazolidinedionas/farmacologiaRESUMO
Identification of conserved co-expression networks is a useful tool for clustering groups of genes enriched for common molecular or cellular functions [1]. The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2]. Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer. Here, we validate one of the highly connected genes as a metastasis associated gene. Tpx2, the most highly connected gene within a proliferation network specifically prognostic for estrogen receptor positive (ER+) breast cancers, enhances metastatic disease, but in a tumor autonomous, proliferation-independent manner. Histologic analysis suggests instead that variation of TPX2 levels within disseminated tumor cells may influence the transition between dormant to actively proliferating cells in the secondary site. These results support the co-expression network approach for identification of new metastasis-associated genes to provide new information regarding the etiology of breast cancer progression and metastatic disease.
Assuntos
Neoplasias da Mama/genética , Redes Reguladoras de Genes , Neoplasias Pulmonares/genética , Animais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Transplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prognóstico , Transcriptoma , Carga TumoralRESUMO
Metastasis remains the main cause for cancer-related deaths due to the lack of effective therapy. The clonal selection model has long been thought to be the primary mechanism of metastatic progression but many different mechanisms have been hypothesized for the progression from tumorigenesis to the successful dissemination and expansion of tumor cells at the secondary site. MicroRNAs, germline polymorphisms in combination with the tumor microenvironment are few of the different pathways to explain the metastatic cascade. Technological advances for high-throughput screening of cells such as expression profiling, next generation sequencing, as well as global network analyses have advanced the studies of these mechanisms. Combined with new insights into the various mechanisms of metastasis a systems biology approach has also been shown to be useful in identifying metastasis-specific gene signatures as well as predicting disease outcome. Furthermore, the results of these studies have been relevant for identifying biomarkers for metastatic disease.
Assuntos
Metástase Neoplásica , Biologia de Sistemas , Animais , Biomarcadores , Perfilação da Expressão Gênica , Humanos , Camundongos , MicroRNAsRESUMO
Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. BRD4, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Acetiltransferase N-Terminal E/metabolismo , Acetiltransferases N-Terminal , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Ligação Proteica , Isoformas de Proteínas , Transporte Proteico , Fatores de Transcrição/genética , Carga Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Warfarin is the most frequently prescribed anticoagulant in North America and Europe. It is administered as a racemate, but S-warfarin is principally responsible for its anticoagulant activity. Cytochrome P450 (CYP) 2C9 is the enzyme primarily responsible for the metabolism of S-warfarin. Numerous variant alleles of CYP2C9 have been identified. The CYP2C9*12 (rs9332239) allele harbors a P489S substitution in CYP2C9 which has been shown to result in a 40% decline in catalytic activity in vitro. CASES: Four Caucasian patients with a low mean weekly warfarin dose (MWWD) were genotyped for CYP2C9, VKORC1 and APOE variant alleles. None of the four patients carried the common CYP2C9 variant alleles (*2, *3, *5, *6, *7, *8, *9, *11, *13) despite a relatively low MWWD (23.4±7.94 mg) compared to 208 patients carrying the CYP29C9*1 genotype (32.2±12.65 mg). Given that CYP2C9*12 confers decreased in vitro activity to the enzyme, we investigated whether these patients carried this allele. All four patients were CYP2C9*12 CT heterozygotes. Individual comparisons with patients possessing the same VKORC1 and APOE genotypes also demonstrated lower dose requirements in the patients that possessed CYP2C9*12 allele. CONCLUSIONS: There are no reports of the clinical impact of rs9332239 on CYP2C9 substrates. This is the first report of patients with the rare CYP2C9*12 genotype and lower warfarin dose requirements.
Assuntos
Anticoagulantes/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Mutação , Tromboembolia/genética , Varfarina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Alelos , Substituição de Aminoácidos , Anticoagulantes/uso terapêutico , Apolipoproteínas E/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sequência de Bases , Biotransformação , Citocromo P-450 CYP2C9 , Cálculos da Dosagem de Medicamento , Feminino , Genótipo , Técnicas de Genotipagem , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Tromboembolia/enzimologia , Tromboembolia/patologia , Tromboembolia/prevenção & controle , Vitamina K Epóxido Redutases/genética , Varfarina/uso terapêuticoRESUMO
Even though the hyaluronan-mediated motility receptor (HMMR), a cell surface oncogenic protein, is widely up-regulated in human cancers and correlates well with cell motility and invasion, the underlying molecular and nature of its putative upstream regulation remain unknown. Here, we found for the first time that MTA1 (metastatic tumor antigen 1), a master chromatin modifier, regulates the expression of HMMR and, consequently, its function in breast cancer cell motility and invasiveness. We recognized a positive correlation between the levels of MTA1 and HMMR in human cancer. Furthermore, MTA1 is required for optimal expression of HMMR. The underlying mechanism includes interaction of the MTA1·RNA polymerase II·c-Jun coactivator complex with the HMMR promoter to stimulates its transcription. Accordingly, selective siRNA-mediated knockdown of HMMR in breast cancer cells substantially reduces the invasion and migration of cells. These findings reveal a regulatory role for MTA1 as an upstream coactivator of HMMR expression and resulting biological phenotypes.
Assuntos
Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/genética , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Proteínas Repressoras/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Invasividade Neoplásica , RNA Polimerase II/metabolismo , Transativadores , Transcrição Gênica/genéticaRESUMO
Triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). However, pathways responsible for downregulation of therapeutic receptors, as well as subsequent aggressiveness, remain unknown. In this study, we discovered that lactoferrin (Lf) efficiently downregulates levels of ER-α, PR, and HER-2 in a proteasome-dependent manner in breast cancer cells, and it accounts for the loss of responsiveness to ER- or HER-2-targeted therapies. Furthermore, we found that lactoferrin increases migration and invasiveness of both non-TNBC and TNBC cell lines. We discovered that lactoferrin directly stimulates the transcription of endothelin-1 (ET-1), a secreted proinvasive polypeptide that acts through a specific receptor, ET(A)R, leading to secretion of the bioactive ET-1 peptide. Interestingly, a therapeutic ET-1 receptor-antagonist blocked lactoferrin-dependent motility and invasiveness of breast cancer cells. The physiologic significance of this newly discovered Lf-ET-1 axis in the manifestation of TNBC phenotypes is revealed by elevated plasma and tissue lactoferrin and ET-1 levels in patients with TNBC compared with those in ER(+) cases. These findings describe the first physiologically relevant polypeptide as a functional determinant in downregulating all three therapeutic receptors in breast cancer, which uses another secreted ET-1 system to confer invasiveness. Results presented in this article provide proof-of-principle evidence in support of the therapeutic effectiveness of ET-1 receptor antagonist to completely block the lactoferrin-induced motility and invasiveness of the TNBC as well as non-TNBC cells, and thus, open a remarkable opportunity to treat TNBC by targeting the Lf-ET-1 axis using an approved developmental drug.