RESUMEN
The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.
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Microbioma Gastrointestinal/genética , Regulación de la Expresión Génica/genética , Síndrome del Colon Irritable/metabolismo , Metaboloma , Purinas/metabolismo , Transcriptoma/genética , Animales , Ácidos y Sales Biliares/metabolismo , Biopsia , Butiratos/metabolismo , Cromatografía Liquida , Estudios Transversales , Epigenómica , Heces/microbiología , Femenino , Microbioma Gastrointestinal/fisiología , Regulación de la Expresión Génica/fisiología , Interacciones Microbiota-Huesped/genética , Humanos , Hipoxantina/metabolismo , Síndrome del Colon Irritable/genética , Síndrome del Colon Irritable/microbiología , Estudios Longitudinales , Masculino , Metaboloma/fisiología , Ratones , Estudios Observacionales como Asunto , Estudios Prospectivos , Programas Informáticos , Espectrometría de Masas en Tándem , Transcriptoma/fisiologíaRESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Patients with TNBC are primarily treated with neoadjuvant chemotherapy (NAC). The response to NAC is prognostic, with reductions in overall survival and disease-free survival rates in those patients who do not achieve a pathological complete response (pCR). Based on this premise, we hypothesized that paired analysis of primary and residual TNBC tumors following NAC could identify unique biomarkers associated with post-NAC recurrence. METHODS AND RESULTS: We investigated 24 samples from 12 non-LAR TNBC patients with paired pre- and post-NAC data, including four patients with recurrence shortly after surgery (< 24 months) and eight who remained recurrence-free (> 48 months). These tumors were collected from a prospective NAC breast cancer study (BEAUTY) conducted at the Mayo Clinic. Differential expression analysis of pre-NAC biopsies showed minimal gene expression differences between early recurrent and nonrecurrent TNBC tumors; however, post-NAC samples demonstrated significant alterations in expression patterns in response to intervention. Topological-level differences associated with early recurrence were implicated in 251 gene sets, and an independent assessment of microarray gene expression data from the 9 paired non-LAR samples available in the NAC I-SPY1 trial confirmed 56 gene sets. Within these 56 gene sets, 113 genes were observed to be differentially expressed in the I-SPY1 and BEAUTY post-NAC studies. An independent (n = 392) breast cancer dataset with relapse-free survival (RFS) data was used to refine our gene list to a 17-gene signature. A threefold cross-validation analysis of the gene signature with the combined BEAUTY and I-SPY1 data yielded an average AUC of 0.88 for six machine-learning models. Due to the limited number of studies with pre- and post-NAC TNBC tumor data, further validation of the signature is needed. CONCLUSION: Analysis of multiomics data from post-NAC TNBC chemoresistant tumors showed down regulation of mismatch repair and tubulin pathways. Additionally, we identified a 17-gene signature in TNBC associated with post-NAC recurrence enriched with down-regulated immune genes.
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Neoplasias de la Mama Triple Negativas , Humanos , Regulación hacia Abajo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Tubulina (Proteína) , Reparación de la Incompatibilidad de ADN , Multiómica , Estudios Prospectivos , Recurrencia Local de Neoplasia/genéticaRESUMEN
OBJECTIVE: Elevated serum chromogranin A (CGA) is associated with intrinsic or treatment-related neuroendocrine differentiation (NED) in men with metastatic castration-resistant prostate cancer (mCRPC). Fluctuations in serum CGA during treatment of mCRPC have had conflicting results. We analyzed the impact of (i) rising serum CGA and (ii) baseline CGA/PSA ratio during treatment to identify associations with abiraterone acetate (AA) therapy. METHODS: Between June 2013 and August 2015, 92 men with mCRPC were enrolled in a prospective trial with uniform serum CGA processing performed before initiating abiraterone acetate/prednisone (AA/P) and serially after 12 weeks of AA/P treatments. Serum CGA was measured using a homogenous automated immunofluorescent assay. Patients receiving proton pump inhibitors or with abnormal renal function were excluded due to possible false elevations of serum CGA (n = 21 excluded), therefore 71 patients were analyzed. All patients underwent a composite response assessment at 12-weeks. Kaplan-Meier estimates and Cox Regression models were used to calculate the association with time-to-treatment failure analyses and overall survival. RESULTS: An increase in chromogranin was associated with a lower risk of treatment failure (hazard ratio [HR]: 0.52, p = 0.0181). The median CGA/PSA ratio was 7.8 (2.6-16.0) and an elevated pretreatment CGA/PSA ratio above the median was associated with a lower risk of treatment failure (HR: 0.54 p value = 0.0185). An increase in CGA was not found to be associated with OS (HR: 0.71, 95% CI: 0.42-1.21, p = 0.207). An elevated baseline CGA/PSA ratio was not associated with OS (HR: 0.62, 95% CI: 0.37-1.03, p = 0.062). An increase in PSA after 12 weeks of treatment was associated with an increased risk of treatment failure (HR: 4.14, CI: 2.21-7.73, p = < 0.0001) and worse OS (HR: 2.93, CI: 1.57-4.45, p = < 0.0001). CONCLUSIONS: We show that an increasing chromogranin on AA/P and an elevated baseline CGA/PSA in patients with mCRPC were associated with a favorable response to AA/P with no changes in survival. There may be limited clinical utility in serum CGA testing to evaluate for lethal NED as AA/P did not induce lethal NED in this cohort. This highlights that not all patients with an increasing CGA have a worse OS.
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Acetato de Abiraterona , Neoplasias de la Próstata Resistentes a la Castración , Humanos , Masculino , Acetato de Abiraterona/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Cromogranina A , Cromograninas , Estudios Prospectivos , Antígeno Prostático Específico , Neoplasias de la Próstata Resistentes a la Castración/patología , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
We present Knowledge Engine for Genomics (KnowEnG), a free-to-use computational system for analysis of genomics data sets, designed to accelerate biomedical discovery. It includes tools for popular bioinformatics tasks such as gene prioritization, sample clustering, gene set analysis, and expression signature analysis. The system specializes in "knowledge-guided" data mining and machine learning algorithms, in which user-provided data are analyzed in light of prior information about genes, aggregated from numerous knowledge bases and encoded in a massive "Knowledge Network." KnowEnG adheres to "FAIR" principles (findable, accessible, interoperable, and reuseable): its tools are easily portable to diverse computing environments, run on the cloud for scalable and cost-effective execution, and are interoperable with other computing platforms. The analysis tools are made available through multiple access modes, including a web portal with specialized visualization modules. We demonstrate the KnowEnG system's potential value in democratization of advanced tools for the modern genomics era through several case studies that use its tools to recreate and expand upon the published analysis of cancer data sets.
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Algoritmos , Nube Computacional , Minería de Datos/métodos , Genómica/métodos , Programas Informáticos , Análisis por Conglomerados , Biología Computacional/métodos , Análisis de Datos , Conjuntos de Datos como Asunto , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Conocimiento , Aprendizaje Automático , Metabolómica/métodosRESUMEN
BACKGROUND & AIMS: The gut virome includes eukaryotic viruses and bacteriophages that can shape the gut bacterial community and elicit host responses. The virome can be implicated in diseases, such as irritable bowel syndrome (IBS), where gut bacteria play an important role in pathogenesis. We provide a comprehensive and longitudinal characterization of the virome, including DNA and RNA viruses and paired multi-omics data in a cohort of healthy subjects and patients with IBS. METHODS: We selected 2 consecutive stool samples per subject from a longitudinal study cohort and performed metagenomic sequencing on DNA and RNA viruses after enriching for viral-like particles. Viral sequence abundance was evaluated over time, as well as in the context of diet, bacterial composition and function, metabolite levels, colonic gene expression, host genetics, and IBS subsets. RESULTS: We found that the gut virome was temporally stable and correlated with the colonic transcriptome. We identified IBS-subset-specific changes in phage populations; Microviridae, Myoviridae, and Podoviridae species were elevated in diarrhea-predominant IBS, and other Microviridae and Myoviridae species were elevated in constipation-predominant IBS compared to healthy controls. We identified correlations between subsets of the virome and bacterial composition (unclassifiable "dark matter" and phages) and diet (eukaryotic viruses). CONCLUSIONS: We found that the gut virome is stable over time but varies among subsets of patients with IBS. It can be affected by diet and potentially influences host function via interactions with gut bacteria and/or altering host gene expression.
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Dieta , Intestinos/virología , Síndrome del Colon Irritable/virología , Transcriptoma , Viroma , Virus/crecimiento & desarrollo , Adulto , Bacteriófagos/genética , Bacteriófagos/crecimiento & desarrollo , Estudios de Casos y Controles , Dieta/efectos adversos , Femenino , Microbioma Gastrointestinal , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Intestinos/microbiología , Síndrome del Colon Irritable/diagnóstico , Síndrome del Colon Irritable/genética , Síndrome del Colon Irritable/microbiología , Estudios Longitudinales , Masculino , Metagenoma , Metagenómica , Persona de Mediana Edad , Virología , Virus/genéticaRESUMEN
Nature and nurture have always been a prerogative of evolutionary biologists. The environment's role in shaping an organism's phenotype has always intrigued us. Since the inception of humankind, twinning has existed with an unsettled parley on the contribution of nature (i.e. genetics) versus nurture (i.e. environment), which can influence the phenotypes. The study of twins measures the genetic contribution and that of the environmental influence for a particular trait, acting as a catalyst, fine-tuning the phenotypic trajectories. This is further evident because a number of human diseases show a spectrum of clinical manifestations with the same underlying molecular aberration. As of now, there is no definite way to conclude just from the genomic data the severity of a disease or even to predict who will get affected. This greatly justifies initiating a twin registry for a country as diverse and populated as India. There is an unmet need to set up a nationwide database to carefully curate the information on twins, serving as a valuable biorepository to study their overall susceptibility to disease. Establishing a twin registry is of paramount importance to harness the wealth of human information related to the biomedical, anthropological, cultural, social and economic significance.
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Enfermedades en Gemelos , Gemelos , Enfermedades en Gemelos/epidemiología , Enfermedades en Gemelos/genética , Humanos , India/epidemiología , Sistema de Registros , Gemelos/genética , Recursos HumanosRESUMEN
OBJECTIVES: Based on our previous findings that postmenopausal women with estrone (E1) and estradiol (E2) concentrations at or above 1.3 pg/ml and 0.5 pg/ml, respectively, after 6 months of adjuvant anastrozole therapy had a three-fold risk of recurrence, we aimed to identify a single-nucleotide polymorphism (SNP)-based model that would predict elevated E1 and E2 and then validate it in an independent dataset. PATIENTS AND METHODS: The test set consisted of 322 women from the M3 study and the validation set consisted of 152 patients from MA.27. All patients were treated with adjuvant anastrozole, had on-anastrozole E1 and E2 concentrations and genome-wide genotyping. RESULTS: SNPs were identified from the M3 genome-wide association study. The best model to predict the E1-E2 phenotype with high balanced accuracy was a support vector machine model using clinical factors plus 46 SNPs. We did not have an independent cohort that is similar to the M3 study with clinical, E1-E2 phenotypes and genotype data to test our model. Hence, we chose a nested matched case-control cohort (MA.27 study) for testing. Our E1-E2 model was not validated but we found the MA.27 validation cohort was both clinically and genomically different. CONCLUSIONS: We identified a SNP-based model that had excellent performance characteristics for predicting the phenotype of elevated E1 and E2 in women treated with anastrozole. This model was not validated in an independent dataset but that dataset was clinically and genomically substantially different. The model will need validation in a prospective study.
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Anastrozol/efectos adversos , Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Recurrencia Local de Neoplasia/genética , Adulto , Anastrozol/administración & dosificación , Inhibidores de la Aromatasa/administración & dosificación , Inhibidores de la Aromatasa/efectos adversos , Neoplasias de la Mama/sangre , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/patología , Estradiol/sangre , Estrona/sangre , Femenino , Genoma Humano/genética , Estudio de Asociación del Genoma Completo , Humanos , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/patología , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
MOTIVATION: Estimation of isoform-level gene expression from RNA-seq data depends on simplifying assumptions, such as uniform read distribution, that are easily violated in real data. Such violations typically lead to biased estimates. Most existing methods provide bias correction step(s), which is based on biological considerations-such as GC content-and applied in single samples separately. The main problem is that not all biases are known. RESULTS: We have developed a novel method called XAEM based on a more flexible and robust statistical model. Existing methods are essentially based on a linear model Xß, where the design matrix X is known and is computed based on the simplifying assumptions. In contrast XAEM considers Xß as a bilinear model with both X and ß unknown. Joint estimation of X and ß is made possible by a simultaneous analysis of multi-sample RNA-seq data. Compared to existing methods, XAEM automatically performs empirical correction of potentially unknown biases. We use an alternating expectation-maximization (AEM) algorithm, alternating between estimation of X and ß. For speed XAEM utilizes quasi-mapping for read alignment, thus leading to a fast algorithm. Overall XAEM performs favorably compared to recent advanced methods. For simulated datasets, XAEM obtains higher accuracy for multiple-isoform genes. In a differential-expression analysis of a real single-cell RNA-seq dataset, XAEM achieves substantially better rediscovery rates in independent validation sets. AVAILABILITY AND IMPLEMENTATION: The method and pipeline are implemented as a tool and freely available for use at http://fafner.meb.ki.se/biostatwiki/xaem/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Perfilación de la Expresión Génica , RNA-Seq , Algoritmos , Isoformas de Proteínas/genética , Análisis de Secuencia de ARN , Programas InformáticosRESUMEN
SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1 gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using "one-at-a-time" functional systems. Deep mutational scanning (DMS) using a "landing pad cell-based system" is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the SLCO1B1 ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into "landing pad" cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and SLCO1B1 variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for SLCO1B1-dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1. SIGNIFICANCE STATEMENT: The functional implications of a large numbers of open reading frame (ORF) "variants of unknown significance" (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of SLCO1B1(s olute carrier organic anion transporter family member 1B1). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by SLCO1B1.
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Variación Genética/genética , Genómica/métodos , Transportador 1 de Anión Orgánico Específico del Hígado/genética , Mutación Missense/genética , Células HEK293 , HumanosRESUMEN
At the stroke of the New Year 2020, COVID-19, a zoonotic disease that would turn into a global pandemic, was identified in the Chinese city of Wuhan. Although unique in its transmission and virulence, COVID-19 is similar to zoonotic diseases, including other SARS variants (e.g., SARS-CoV) and MERS, in exhibiting severe flu-like symptoms and acute respiratory distress. Even at the molecular level, many parallels have been identified between SARS and COVID-19 so much so that the COVID-19 virus has been named SARS-CoV-2. These similarities have provided several opportunities to treat COVID-19 patients using clinical approaches that were proven to be effective against SARS. Importantly, the identification of similarities in how SARS-CoV and SARS-CoV-2 access the host, replicate, and trigger life-threatening pathological conditions have revealed opportunities to repurpose drugs that were proven to be effective against SARS. In this article, we first provided an overview of COVID-19 etiology vis-à-vis other zoonotic diseases, particularly SARS and MERS. Then, we summarized the characteristics of droplets/aerosols emitted by COVID-19 patients and how they aid in the transmission of the virus among people. Moreover, we discussed the molecular mechanisms that enable SARS-CoV-2 to access the host and become more contagious than other betacoronaviruses such as SARS-CoV. Further, we outlined various approaches that are currently being employed to diagnose and symptomatically treat COVID-19 in the clinic. Finally, we reviewed various approaches and technologies employed to develop vaccines against COVID-19 and summarized the attempts to repurpose various classes of drugs and novel therapeutic approaches.
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COVID-19/transmisión , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/prevención & control , COVID-19/terapia , Vacunas contra la COVID-19/inmunología , HumanosRESUMEN
BACKGROUND: The tamoxifen metabolite, Z-endoxifen, demonstrated promising antitumor activity in endocrine-resistant estrogen receptor-positive (ER+) breast cancer. We compared the antitumor activity of Z-endoxifen with tamoxifen and letrozole in the letrozole-sensitive MCF7 aromatase expressing model (MCF7AC1), as well as with tamoxifen, fulvestrant, exemestane, and exemestane plus everolimus in a letrozole-resistant MCF7 model (MCF7LR). METHODS: MCF7AC1 tumor-bearing mice were randomized to control (no drug), letrozole (10 µg/day), tamoxifen (500 µg/day), or Z-endoxifen (25 and 75 mg/kg). Treatment in the letrozole arm was continued until resistance developed. MCF7LR tumor-bearing mice were then randomized to Z-endoxifen (50 mg/kg) or tamoxifen for 4 weeks and tumors harvested for microarray and immunohistochemistry analysis. The antitumor activity of Z-endoxifen in the MCF7LR tumors was further compared in a second in vivo study with exemestane, exemestane plus everolimus, and fulvestrant. RESULTS: In the MCF7AC1 tumors, both Z-endoxifen doses were significantly superior to control and tamoxifen in reducing tumor volumes at 4 weeks. Additionally, the 75 mg/kg Z-endoxifen dose was additionally superior to letrozole. Prolonged letrozole exposure resulted in resistance at 25 weeks. In MCF7LR tumor-bearing mice, Z-endoxifen significantly reduced tumor volumes compared to tamoxifen, letrozole, and exemestane, with no significant differences compared to exemestane plus everolimus and fulvestrant. Additionally, compared to tamoxifen, Z-endoxifen markedly inhibited ERα target genes, Ki67 and Akt expression in vivo. CONCLUSION: In endocrine-sensitive and letrozole-resistant breast tumors, Z-endoxifen results in robust antitumor and antiestrogenic activity compared to tamoxifen and aromatase inhibitor monotherapy. These data support the ongoing development of Z-endoxifen.
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Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Receptores de Estrógenos/metabolismo , Tamoxifeno/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Humanos , Letrozol/farmacología , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Tamoxifeno/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MOTIVATION: Both single-cell RNA sequencing (scRNA-seq) and DNA sequencing (scDNA-seq) have been applied for cell-level genomic profiling. For mutation profiling, the latter seems more natural. However, the task is highly challenging due to the limited input materials from only two copies of DNA molecules, while whole-genome amplification generates biases and other technical noises. ScRNA-seq starts with a higher input amount, so generally has better data quality. There exists various methods for mutation detection from DNA sequencing, it is not clear whether these methods work for scRNA-seq data. RESULTS: Mutation detection methods developed for either bulk-cell sequencing data or scDNA-seq data do not work well for the scRNA-seq data, as they produce substantial numbers of false positives. We develop a novel and robust statistical method-called SCmut-to identify specific cells that harbor mutations discovered in bulk-cell data. Statistically SCmut controls the false positives using the 2D local false discovery rate method. We apply SCmut to several scRNA-seq datasets. In scRNA-seq breast cancer datasets SCmut identifies a number of highly confident cell-level mutations that are recurrent in many cells and consistent in different samples. In a scRNA-seq glioblastoma dataset, we discover a recurrent cell-level mutation in the PDGFRA gene that is highly correlated with a well-known in-frame deletion in the gene. To conclude, this study contributes a novel method to discover cell-level mutation information from scRNA-seq that can facilitate investigation of cell-to-cell heterogeneity. AVAILABILITY AND IMPLEMENTATION: The source codes and bioinformatics pipeline of SCmut are available at https://github.com/nghiavtr/SCmut. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Mutación , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Programas InformáticosRESUMEN
The rapid advancement of high-throughput technologies and sharp decrease in cost have opened up the possibility to generate large amount of multi-omics data on an individual basis. The development of high-throughput -omics, including genomics, epigenomics, transcriptomics, proteomics, metabolomics and microbiomics, enables the application of multi-omics technologies in the clinical settings. Combination therapy, defined as disease treatment with two or more drugs to achieve efficacy with lower doses or lower drug toxicity, is the basis for the care of diseases like cancer. Patient-specific multi-omics data integration can help the identification and development of combination therapies. In this review, we provide an overview of different -omics platforms, and discuss the methods for multi-omics, high-throughput, data integration, personalized combination therapy.
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Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biología Computacional/métodos , Modelos Estadísticos , Neoplasias/terapia , Medicina de Precisión , Ensayos Clínicos como Asunto , Terapia Combinada , Epigenómica/métodos , Genómica/métodos , Humanos , Metabolómica/métodos , Neoplasias/genética , Neoplasias/metabolismo , Pronóstico , Proteómica/métodosRESUMEN
BACKGROUND: Our previous genome-wide association study using the MA.27 aromatase inhibitors adjuvant trial identified SNPs in the long noncoding RNA MIR2052HG associated with breast cancer-free interval. MIR2052HG maintained ERα both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated ERα degradation. Our goal was to further elucidate MIR2052HG's mechanism of action. METHODS: RNA-binding protein immunoprecipitation assays were performed to demonstrate that the transcription factor, early growth response protein 1 (EGR1), worked together with MIR2052HG to regulate that lemur tyrosine kinase-3 (LMTK3) transcription in MCF7/AC1 and CAMA-1 cells. The location of EGR1 on the LMTK3 gene locus was mapped using chromatin immunoprecipitation assays. The co-localization of MIR2052HG RNA and the LMTK3 gene locus was determined using RNA-DNA dual fluorescent in situ hybridization. Single-nucleotide polymorphisms (SNP) effects were evaluated using a panel of human lymphoblastoid cell lines. RESULTS: MIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing protein kinase C (PKC) activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor-dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels. CONCLUSIONS: Our findings support a model in which the MIR2052HG regulates LMTK3 via EGR1, and LMTK3 regulates ERα stability via the PKC/MEK/ERK/RSK1 axis. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibilities of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer.
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Inhibidores de la Aromatasa/farmacología , Resistencia a Antineoplásicos/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Receptor alfa de Estrógeno/genética , Proteínas de la Membrana/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Largo no Codificante/genética , Biomarcadores , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Polimorfismo de Nucleótido Simple , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Transducción de Señal , Transcripción GenéticaRESUMEN
OBJECTIVE: To identify additional genetic variants beyond those observed in a previous genome-wide association study (GWAS) in women treated on the MA.27 clinical trial in which women were randomized to 5 years of adjuvant therapy with anastrozole or exemestane. PATIENTS AND METHODS: We performed a matched case-control study in 234 women who had a recurrence of breast cancer (cases) and 649 women who had not (controls). The analysis was restricted to White women with an estrogen receptor-positive breast cancer. Multiplex PCR-based targeted deep sequencing was performed of the MIR2052HG region on chromosome 8 between positions 75.4 and 75.7, a span of 300 kb, in an attempt to identify additional functional single nucleotide polymorphisms (SNPs). RESULTS: A total of 4677 unique variants were identified that had not been identified in the previous GWAS. Clinical Annotation of Variants analysis revealed 10 variants, including eight SNPs and two insertion-deletion mutations with moderate or high impact. However, none of the common and variant regions was significant after adjustment for the most significant SNP (rs13260300) identified in our previous GWAS. We performed haplotype analysis that revealed two regions in which the haplotypes lost significance when adjusted for this prior GWAS SNP and one region with two significant haplotypes (P = 0.046 and 0.031) after adjusting for the GWAS SNP. CONCLUSION: We were unable to identify common or rare variant regions that added value to the findings from our previous GWAS. We did find two haplotypes that were significant after adjusting for our top GWAS SNP but these were considered to be of marginal value.
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Inhibidores de la Aromatasa/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación INDEL , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/etnología , Neoplasias de la Mama/genética , Estudios de Casos y Controles , Quimioterapia Adyuvante , Cromosomas Humanos Par 8/genética , Femenino , Estudio de Asociación del Genoma Completo , Haplotipos , Humanos , Persona de Mediana Edad , Análisis de Secuencia de ADNRESUMEN
Motivation: RNA sequencing of single cells enables characterization of transcriptional heterogeneity in seemingly homogeneous cell populations. Single-cell sequencing has been applied in a wide range of researches fields. However, few studies have focus on characterization of isoform-level expression patterns at the single-cell level. In this study, we propose and apply a novel method, ISOform-Patterns (ISOP), based on mixture modeling, to characterize the expression patterns of isoform pairs from the same gene in single-cell isoform-level expression data. Results: We define six principal patterns of isoform expression relationships and describe a method for differential-pattern analysis. We demonstrate ISOP through analysis of single-cell RNA-sequencing data from a breast cancer cell line, with replication in three independent datasets. We assigned the pattern types to each of 16 562 isoform-pairs from 4929 genes. Among those, 26% of the discovered patterns were significant (P<0.05), while remaining patterns are possibly effects of transcriptional bursting, drop-out and stochastic biological heterogeneity. Furthermore, 32% of genes discovered through differential-pattern analysis were not detected by differential-expression analysis. Finally, the effects of drop-out events and expression levels of isoforms on ISOP's performances were investigated through simulated datasets. To conclude, ISOP provides a novel approach for characterization of isoform-level preference, commitment and heterogeneity in single-cell RNA-sequencing data. Availability and implementation: The ISOP method has been implemented as a R package and is available at https://github.com/nghiavtr/ISOP under a GPL-3 license. Supplementary information: Supplementary data are available at Bioinformatics online.
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Perfilación de la Expresión Génica/métodos , Expresión Génica , Isoformas de ARN/genética , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , HumanosRESUMEN
BACKGROUND: Transfer of genetic material from microbes or viruses into the host genome is known as horizontal gene transfer (HGT). The integration of viruses into the human genome is associated with multiple cancers, and these can now be detected using next-generation sequencing methods such as whole genome sequencing and RNA-sequencing. RESULTS: We designed a novel computational workflow, HGT-ID, to identify the integration of viruses into the human genome using the sequencing data. The HGT-ID workflow primarily follows a four-step procedure: i) pre-processing of unaligned reads, ii) virus detection using subtraction approach, iii) identification of virus integration site using discordant and soft-clipped reads and iv) HGT candidates prioritization through a scoring function. Annotation and visualization of the events, as well as primer design for experimental validation, are also provided in the final report. We evaluated the tool performance with the well-understood cervical cancer samples. The HGT-ID workflow accurately detected known human papillomavirus (HPV) integration sites with high sensitivity and specificity compared to previous HGT methods. We applied HGT-ID to The Cancer Genome Atlas (TCGA) whole-genome sequencing data (WGS) from liver tumor-normal pairs. Multiple hepatitis B virus (HBV) integration sites were identified in TCGA liver samples and confirmed by HGT-ID using the RNA-Seq data from the matched liver pairs. This shows the applicability of the method in both the data types and cross-validation of the HGT events in liver samples. We also processed 220 breast tumor WGS data through the workflow; however, there were no HGT events detected in those samples. CONCLUSIONS: HGT-ID is a novel computational workflow to detect the integration of viruses in the human genome using the sequencing data. It is fast and accurate with functions such as prioritization, annotation, visualization and primer design for future validation of HGTs. The HGT-ID workflow is released under the MIT License and available at http://kalarikrlab.org/Software/HGT-ID.html .
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Transferencia de Gen Horizontal/genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Integración Viral/genética , Algoritmos , Secuencia de Bases , Neoplasias de la Mama/virología , Línea Celular Tumoral , Simulación por Computador , Femenino , Humanos , Curva ROC , Programas Informáticos , Secuenciación Completa del Genoma , Flujo de TrabajoRESUMEN
Metformin is currently considered as a promising anticancer agent in addition to its anti-diabetic effect. To better individualize metformin therapy and explore novel molecular mechanisms in cancer treatment, we conducted a pharmacogenomic study using 266 lymphoblastoid cell lines (LCLs). Metformin cytotoxicity assay was performed using the MTS assay. Genome-wide association (GWA) analyses were performed in LCLs using 1.3 million SNPs, 485k DNA methylation probes, 54k mRNA expression probe sets, and metformin cytotoxicity (IC50s). Top candidate genes were functionally validated using siRNA screening, followed by MTS assay in breast cancer cell lines. Further study of one top candidate, STUB1, was performed to elucidate the mechanisms by which STUB1 might contribute to metformin action. GWA analyses in LCLs identified 198 mRNA expression probe sets, 12 SNP loci, and 5 DNA methylation loci associated with metformin IC50 with P-values <10−4 or <10−5. Integrated SNP/methylation loci-expression-IC50 analyses found 3 SNP loci or 5 DNA methylation loci associated with metformin IC50 through trans-regulation of expression of 11 or 26 genes with P-value <10−4. Functional validation of top 61 candidate genes in 4 IPA networks indicated down regulation of 14 genes significantly altered metformin sensitivity in two breast cancer cell lines. Mechanistic studies revealed that the E3 ubiquitin ligase, STUB1, could influence metformin response by facilitating proteasome-mediated degradation of cyclin A. GWAS using a genomic data-enriched LCL model system, together with functional and mechanistic studies using cancer cell lines, help us to identify novel genetic and epigenetic biomarkers involved in metformin anticancer response.
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Metformina/metabolismo , Metformina/farmacología , Antineoplásicos/metabolismo , Biomarcadores Farmacológicos/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Metilación de ADN , Epigénesis Genética/genética , Epigenómica , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Farmacogenética/métodos , Polimorfismo de Nucleótido Simple/genética , ARN Interferente Pequeño/metabolismo , Transcriptoma/genética , Ubiquitina-Proteína Ligasas/efectos de los fármacos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Neoadjuvant chemotherapy (NAC) for breast cancer is widely utilized, and we performed genome-wide association studies (GWAS) to determine whether germ-line genetic variability was associated with benefit in terms of pathological complete response (pCR), disease-free survival, and overall survival in patients entered on the NSABP B-40 NAC trial, wherein patients were randomized to receive, or not, bevacizumab in addition to chemotherapy. Patient DNA samples were genotyped with the Illumina OmniExpress BeadChip. Replication was attempted with genotyping data from 1398 HER2-negative patients entered on the GeparQuinto NAC study in which patients were also randomized to receive, or not, bevacizumab in addition to chemotherapy. A total of 920 women from B-40 were analyzed, and 237 patients achieved a pCR. GWAS with three phenotypes (pCR, disease-free survival, overall survival) revealed no single nucleotide polymorphisms (SNPs) that were genome-wide significant (i.e. P≤5E-08) signals; P values for top SNPs were 2.04E-07, 5.61E-08, and 5.63E-08, respectively, and these SNPs were not significant in the GeparQuinto data. An ad-hoc GWAS was performed in the patients randomized to bevacizumab (457 patients with 128 pCR) who showed signals on chromosome 6, located within a gene, CDKAL1, that approached, but did not reach, genome-wide significance (top SNP rs7453577, P=2.97E-07). However, this finding was significant when tested in the GeparQuinto data set (P=0.04). In conclusion, we identified no SNPs significantly associated with NAC. The observation, in a hypothesis-generating GWAS, of an SNP in CDKAL1 associated with pCR in the bevacizumab arm of both B-40 and GeparQuinto requires further validation and study.