RESUMEN

The contribution of splicing variants to molecular diagnostics of inherited diseases is reported to be less than 10%. This figure is likely an underestimation due to several factors including difficulty in predicting the effect of such variants, the need for functional assays, and the inability to detect them (depending on their locations and the sequencing technology used). The aim of this study was to assess the utility of Nanopore sequencing in characterizing and quantifying aberrant splicing events. For this purpose, we selected 19 candidate splicing variants that were identified in patients affected by inherited retinal dystrophies. Several in silico tools were deployed to predict the nature and estimate the magnitude of variant-induced aberrant splicing events. Minigene assay or whole blood-derived cDNA was used to functionally characterize the variants. PCR amplification of minigene-specific cDNA or the target gene in blood cDNA, combined with Nanopore sequencing, was used to identify the resulting transcripts. Thirteen out of nineteen variants caused aberrant splicing events, including cryptic splice site activation, exon skipping, pseudoexon inclusion, or a combination of these. Nanopore sequencing allowed for the identification of full-length transcripts and their precise quantification, which were often in accord with in silico predictions. The method detected reliably low-abundant transcripts, which would not be detected by conventional strategies, such as RT-PCR followed by Sanger sequencing.

Asunto(s)

Secuenciación de Nucleótidos de Alto Rendimiento , Secuenciación de Nanoporos , Distrofias Retinianas , Humanos , Distrofias Retinianas/genética , Distrofias Retinianas/diagnóstico , Secuenciación de Nanoporos/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Empalme Alternativo/genética , Empalme del ARN/genética , Exones/genética

RESUMEN

In advanced prostate cancer (PC), in particular after acquisition of resistance to androgen receptor (AR) signaling inhibitors (ARSI), upregulation of AR splice variants compromises endocrine therapy efficiency. Androgen receptor splice variant-7 (ARV7) is clinically the most relevant and has a distinct 3' untranslated region (3'UTR) compared to the AR full-length variant, suggesting a unique post-transcriptional regulation. Here, we set out to evaluate the applicability of the ARV7 3'UTR as a therapy target. A common single nucleotide polymorphism, rs5918762, was found to affect the splicing rate and thus the expression of ARV7 in cellular models and patient specimens. Serine/arginine-rich splicing factor 9 (SRSF9) was found to bind to and increase the inclusion of the cryptic exon 3 of ARV7 during the splicing process in the alternative C allele of rs5918762. The dual specificity protein kinase CLK2 interferes with the activity of SRSF9 by regulating its expression. Inhibition of the Cdc2-like kinase (CLK) family by the small molecules cirtuvivint or lorecivivint results in the decreased expression of ARV7. Both inhibitors show potent anti-proliferative effects in enzalutamide-treated or -naive PC models. Thus, targeting aberrant alternative splicing at the 3'UTR of ARV7 by disturbing the CLK2/SRSF9 axis might be a valuable therapeutic approach in late stage, ARSI-resistant PC.

RESUMEN

Castration-resistant prostate cancer (CRPC) remains a significant therapeutic challenge due to its resistance to standard androgen deprivation therapy (ADT). The emergence of androgen receptor splice variant 7 (AR-V7) has been implicated in CRPC progression, contributing to treatment resistance. Current treatments, including first-generation chemotherapy, androgen receptor blockers, radiation therapy, immune therapy, and PARP inhibitors, often come with substantial side effects and limited efficacy. Natural compounds, particularly those derived from herbal medicine, have garnered increasing interest as adjunctive therapeutic agents against CRPC. This review explores the role of AR-V7 in CRPC and highlights the promising benefits of natural compounds as complementary treatments to conventional drugs in reducing CRPC and overcoming therapeutic resistance. We delve into the mechanisms of action underlying the anti-CRPC effects of natural compounds, showcasing their potential to enhance therapeutic outcomes while mitigating the side effects associated with conventional therapies. The exploration of natural compounds offers promising avenues for developing novel treatment strategies that enhance therapeutic outcomes and reduce the adverse effects of conventional CRPC therapies. These compounds provide a safer, more effective approach to managing CRPC, representing a significant advancement in improving patient care.

RESUMEN

Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.

Asunto(s)

Hormona Liberadora de Hormona del Crecimiento , Neoplasias , Receptores de Neuropéptido , Receptores de Hormona Reguladora de Hormona Hipofisaria , Humanos , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Niño , Masculino , Femenino , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Proyectos Piloto , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Preescolar , Adolescente , Neoplasias/genética , Neoplasias/metabolismo , Hungría , Lactante , ARN Mensajero/genética , ARN Mensajero/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/metabolismo , Estudios de Cohortes

RESUMEN

BACKGROUND: Colorectal cancer (CRC) remains a global health concern with persistently high incidence and mortality rates. However, the specific pathogenesis of CRC remains poorly understood. This study aims to investigate the role and pathogenesis of serine and arginine rich splicing factor 10 (SRSF10) in colorectal cancer. METHODS: Bioinformatics analysis was employed to predict SRSF10 gene expression in CRC patients. Functional experiments involving SRSF10 knockdown and overexpression were conducted using CCK8, transwell, scratch assay, and flow cytometry. Additionally, the PRIdictor website was utilized to predict the SRSF10 interaction site with RFC5. The identification of different transcripts of SRSF10-acting RFC5 pre-mRNA was achieved through agarose gel electrophoresis. RESULT: The knockdown of SRSF10 inhibited the proliferation and migration ability of CRC cells, while promoting apoptosis and altering the DNA replication of CRC cells. Conversely, when SRSF10 was highly expressed, it enhanced the proliferation and migration ability of CRC cells and caused changes in the cell cycle of colorectal cancer cells. This study revealed a change in the replicating factor C subunit 5 (RFC5) gene in colorectal cancer cells following SRSF10 knockdown. Furthermore, it was confirmed that SRSF10 increased RFC5 exon2-AS1(S) transcription variants, thereby promoting the development of colorectal cancer through AS1 exclusion to exon 2 of RFC5. CONCLUSION: In summary, this study demonstrates that SRSF10 promotes the progression of colorectal cancer by generating an aberrantly spliced exclusion isoform of AS1 within RFC5 exon 2. These findings suggest that SRSF10 could serve as a crucial target for the clinical diagnosis and treatment of CRC.

Asunto(s)

Empalme Alternativo , Apoptosis , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Proteína de Replicación C , Factores de Empalme Serina-Arginina , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Proliferación Celular/genética , Movimiento Celular/genética , Apoptosis/genética , Línea Celular Tumoral , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Represoras , Proteínas de Ciclo Celular

RESUMEN

PURPOSE: The contribution of androgen receptors (AR) on bladder cancer has been demonstrated in pre-clinical studies, however in clinical studies, only the canonical AR (AR-FL) protein was measured by immunohistochemistry and conflicting results were obtained. To get better insight into the alterations of AR signalling, we used western blotting (WB) method and simultaneously measured both mRNA and protein levels of AR-FL and AR-V7. METHODS: 23 naive non-muscle invasive bladder cancer patients and 12 healthy individuals were included. AR-FL protein, AR-FL mRNA, AR-V7 protein and AR-V7 mRNA levels were quantitatively measured by WB and qRT-PCR. RESULTS: While AR-FL protein and AR-V7 mRNA were significantly higher in bladder cancer, AR-FL mRNA and AR-V7 protein were lower. AR-V7 mRNA level was higher in patients with tumour size over 3 cm and AR-FL protein was higher in single tumours (p < 0,005). The small sampling size and the inclusion of only male participants were the main limitations. CONCLUSIONS: The increase of AR-FL protein in bladder cancer supports the contribution of the AR pathway in bladder cancer. The presence of high AR-FL protein despite low mRNA levels may be due to a disruption in post-transcriptional regulatory mechanisms. AR-V7 was demonstrated for the first time in bladder tissue and found significantly different in bladder cancer tissues. Our study reached new and valuable findings and will shed light on the studies that aim to clarify the role of the AR pathway in bladder cancer.

Asunto(s)

Receptores Androgénicos , Neoplasias de la Vejiga Urinaria , Humanos , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Receptores Androgénicos/genética , Masculino , Persona de Mediana Edad , Anciano , Femenino , ARN Mensajero/metabolismo , ARN Mensajero/genética , Isoformas de Proteínas/genética , Anciano de 80 o más Años

RESUMEN

Hereditary spastic paraplegia (HSP) is a group of genetically heterogenous neurodegenerative disorders characterized by progressive spasticity and weakness of lower limbs. We report a novel splicing variant (c.1617-2A>C) of the SPAST gene in a heterozygous carrier from an Italian family with autosomal dominant HSP. The case study describes a pure form of spastic paraparesis with the cardinal clinical features of SPG4. The novel variant affects a canonical splice site and is likely to disrupt RNA splicing. We conclude that the c.1617-2A>C substitution is a null variant, which could be classified as pathogenic; its penetrance should be further investigated.

RESUMEN

Albinism is a phenotypically and genetically heterogeneous condition characterized by a variable degree of hypopigmentation and by ocular features leading to reduced visual acuity. Whereas numerous genotypic studies have been conducted throughout the world, very little is known about the genotypic spectrum of albinism in Africa and especially in sub-Saharan Western Africa. Here we report the analysis of all known albinism genes in a series a 23 patients originating from Mali. Four were diagnosed with OCA 1 (oculocutaneous albinism type 1), 17 with OCA 2, and two with OCA 4. OCA2 variant NM_000275.3:c.819_822delinsGGTC was most frequently encountered. Four novel variants were identified (two in TYR, two in OCA2). A deep intronic variant was found to alter splicing of the OCA2 RNA by inclusion of a pseudo exon. Of note, the OCA2 exon 7 deletion commonly found in eastern, central, and southern Africa was absent from this series. African patients with OCA 1 and OCA 4 had only been reported twice and once, respectively, in previous publications. This study constitutes the first report of the genotypic spectrum of albinism in a western sub-Saharan country.

RESUMEN

It has been well documented that mutations in coding DNA or cis-regulatory elements underlie natural phenotypic variation in many organisms. However, the development of sophisticated functional tools in recent years in a wide range of traditionally non-model systems have revealed many 'unusual suspects' in the molecular bases of phenotypic evolution, including upstream open reading frames (uORFs), cryptic splice sites, and small RNAs. Furthermore, large-scale genome sequencing, especially long-read sequencing, has identified a cornucopia of structural variation underlying phenotypic divergence and elucidated the composition of supergenes that control complex multi-trait polymorphisms. In this review article we highlight recent studies that demonstrate this great diversity of molecular mechanisms producing adaptive genetic variation and the panoply of evolutionary paths leading to the 'grandeur of life'.

Asunto(s)

Evolución Molecular , Sistemas de Lectura Abierta , Fenotipo , Sistemas de Lectura Abierta/genética , Humanos , Animales , Variación Genética/genética , Mutación

RESUMEN

OBJECTIVE: ACAN gene variants, prevalent monogenic defects linked to short stature, are characterized by impaired cartilage generation in growth plates. We aimed to unravel the genetic basis of short stature in a specific pedigree by investigating the role of a novel non-canonical splicing-site variant, c.630-13G > A, within the ACAN gene. METHOD: Sanger sequencing was used for pedigree verification, and the effects of this variant on mRNA splicing were analyzed through minigene assay. RESULTS: The study revealed that this variant led to the creation of a previously unreported splice site in the fourth intron, resulting in the incorporation of an 11 bp sequence from the intron into the final transcript. This alteration led to a frameshift and formation of a premature termination codon, impacting the structure of the aggrecan protein. CONCLUSIONS: We document the pathogenicity of an ACAN non-canonical splicing-site variant, emphasizing the significance of considering intronic variants during genetic testing.

Asunto(s)

Agrecanos , Intrones , Linaje , Empalme del ARN , Humanos , Agrecanos/genética , Agrecanos/metabolismo , Femenino , Masculino , Enanismo/genética , Sitios de Empalme de ARN/genética

RESUMEN

Pituitary neuroendocrine tumors (PitNETs) are generally benign but comprise an aggressive, invasive, therapy-resistant, metastatic subset, underpinning a need for novel therapeutic targets. PitNETs exhibit low mutation rates but are associated with conditions linked to alternative splicing, an alternative oncogene pathway activation mechanism. PitNETs express the neurotrophin receptor TrkA, which exhibits oncogenic alternative TrkAIII splicing in other neuroendocrine tumors. We, therefore, assessed whether TrkAIII splicing represents a potential oncogenic participant in PitNETs. TrkAIII splicing was RT-PCR assessed in 53 PitNETs and TrkA isoform(s) expression and activation were assessed by confocal immunofluorescence. TrkAIII splicing was also compared to HIF1α, HIF2α, SF3B1, SRSF2, U2AF1, and JCPyV large T antigen mRNA expression, Xbp1 splicing, and SF3B1 mutation. TrkAIII splicing was detected in all invasive and most non-invasive PitNETs and was significantly elevated in invasive cases. In PitNET lineages, TrkAIII splicing was significantly elevated in invasive PIT1 PitNETs and high in invasive and non-invasive SF1 and TPIT lineages. Immunoreactivity consistent with TrkAIII activation characterized PitNET expressing TrkAIII mRNA, and invasive Pit1 PitNETs exhibited elevated HIF2α expression. TrkAIII splicing did not associate with SF3B1 mutations, altered SF3B1, SRSF2, and U2AF1 or JCPyV large T antigen expression, or Xbp1 splicing. Therefore, TrkAIII splicing is common in PitNETs, is elevated in invasive, especially PIT1 tumors, can result in intracellular TrkAIII activation, and may involve hypoxia. The data support a role for TrkAIII splicing in PitNET pathogenesis and progression and identify TrkAIII as a novel potential target in refractory PitNETs.

RESUMEN

Background and objective: Previous germline studies on renal cell carcinoma (RCC) have usually pooled clear and non-clear cell RCCs and have not adequately accounted for population stratification, which might have led to an inaccurate estimation of genetic risk. Here, we aim to analyze the major germline drivers of RCC risk and clinically relevant but underexplored germline variant types. Methods: We first characterized germline pathogenic variants (PVs), cryptic splice variants, and copy number variants (CNVs) in 1436 unselected RCC patients. To evaluate the enrichment of PVs in RCC, we conducted a case-control study of 1356 RCC patients ancestry matched with 16 512 cancer-free controls using approaches accounting for population stratification and histological subtypes, followed by characterization of secondary somatic events. Key findings and limitations: Clear cell RCC patients (n = 976) exhibited a significant burden of PVs in VHL compared with controls (odds ratio [OR]: 39.1, p = 4.95e-05). Non-clear cell RCC patients (n = 380) carried enrichment of PVs in FH (OR: 77.9, p = 1.55e-08) and MET (OR: 1.98e11, p = 2.07e-05). In a CHEK2-focused analysis with European participants, clear cell RCC (n = 906) harbored nominal enrichment of low-penetrance CHEK2 variants-p.Ile157Thr (OR: 1.84, p = 0.049) and p.Ser428Phe (OR: 5.20, p = 0.045), while non-clear cell RCC (n = 295) exhibited nominal enrichment of CHEK2 loss of function PVs (OR: 3.51, p = 0.033). Patients with germline PVs in FH, MET, and VHL exhibited significantly earlier age of cancer onset than patients without germline PVs (mean: 46.0 vs 60.2 yr, p < 0.0001), and more than half had secondary somatic events affecting the same gene (n = 10/15, 66.7%). Conversely, CHEK2 PV carriers exhibited a similar age of onset to patients without germline PVs (mean: 60.1 vs 60.2 yr, p = 0.99), and only 30.4% carried somatic events in CHEK2 (n = 7/23). Finally, pathogenic germline cryptic splice variants were identified in SDHA and TSC1, and pathogenic germline CNVs were found in 18 patients, including CNVs in FH, SDHA, and VHL. Conclusions and clinical implications: This analysis supports the existing link between several RCC risk genes and RCC risk manifesting in earlier age of onset. It calls for caution when assessing the role of CHEK2 due to the burden of founder variants with varying population frequency. It also broadens the definition of the RCC germline landscape of pathogenicity to incorporate previously understudied types of germline variants. Patient summary: In this study, we carefully compared the frequency of rare inherited mutations with a focus on patients' genetic ancestry. We discovered that subtle variations in genetic background may confound a case-control analysis, especially in evaluating the cancer risk associated with specific genes, such as CHEK2. We also identified previously less explored forms of rare inherited mutations, which could potentially increase the risk of kidney cancer.

RESUMEN

A specific splicing isoform of RNASET2 is associated with worse oncologic outcomes in clear cell renal cell carcinoma (ccRCC). However, the interplay between wild-type RNASET2 and its splice variant and how this might contribute to the pathogenesis of ccRCC remains poorly understood. We sought to better understand the relationship of RNASET2 in the pathogenesis of ccRCC and the interplay with a pathogenic splicing isoform (RNASET2-SV) and the tumor immune microenvironment. Using data from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium, we correlated clinical variables to RNASET2 expression and the presence of a specific RNASET2-SV. Immunohistochemical staining with matched RNA sequencing of ccRCC patients was then utilized to understand the spatial relationships of RNASET2 with immune cells. Finally, in vitro studies were performed to demonstrate the oncogenic role of RNASET2 and highlight its potential mechanisms. RNASET2 gene expression is associated with higher grade tumors and worse overall survival in The Cancer Genome Atlas cohort. The presence of the RNASET2-SV was associated with increased expression of the wild-type RNASET2 protein and epigenetic modifications of the gene. Immunohistochemical staining revealed increased intracellular accumulation of RNASET2 in patients with increased RNA expression of RNASET2-SV. In vitro experiments reveal that this accumulation results in increased cell proliferation, potentially from altered metabolic pathways. RNASET2 exhibits a tumor-promoting role in the pathogenesis of ccRCC that is increased in the presence of a specific RNASET2-SV and associated with changes in the cellular localization of the protein.

Asunto(s)

Carcinoma de Células Renales , Neoplasias Renales , Ribonucleasas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ribonucleasas/genética , Ribonucleasas/metabolismo , Microambiente Tumoral , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

Asunto(s)

Modelos Animales de Enfermedad , Fenilalanina Hidroxilasa , Fenilcetonurias , Animales , Ratones , Fenilcetonurias/genética , Fenilcetonurias/patología , Fenilcetonurias/metabolismo , Humanos , Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Sistemas CRISPR-Cas , Autofagia/genética , Mutación , Hígado/metabolismo , Hígado/patología

RESUMEN

Metastasis remains a major cause of morbidity and mortality in men with prostate cancer, and the functional impact of the genetic alterations, alone or in combination, driving metastatic disease remains incompletely understood. The proto-oncogene c-MYC, commonly deregulated in prostate cancer. Transgenic expression of c-MYC is sufficient to drive the progression to prostatic intraepithelial neoplasia and ultimately to moderately differentiated localized primary tumors, however, c-MYC-driven tumors are unable to progress through the metastatic cascade, suggesting that a "second-hit" is necessary in the milieu of aberrant c-MYC-driven signaling. Here, we identified cooperativity between c-MYC and KLF6-SV1, an oncogenic splice variant of the KLF6 gene. Transgenic mice that co-expressed KLF6-SV1 and c-MYC developed progressive and metastatic prostate cancer with a histological and molecular phenotype like human prostate cancer. Silencing c-MYC expression significantly reduced tumor burden in these mice supporting the necessity for c-MYC in tumor maintenance. Unbiased global proteomic analysis of tumors from these mice revealed significantly enriched vimentin, a dedifferentiation and pro-metastatic marker, induced by KLF6-SV1. c-MYC-positive tumors were also significantly enriched for KLF6-SV1 in human prostate cancer specimens. Our findings provide evidence that KLF6-SV1 is an enhancer of c-MYC-driven prostate cancer progression and metastasis, and a correlated genetic event in human prostate cancer with potential translational significance.

RESUMEN

Breast cancer is a major cause of death worldwide. The complexity of endocrine regulation in breast cancer may allow the cancer cells to escape from a particular treatment and result in resistant and aggressive disease. These breast cancers usually have fewer treatment options. Targeted therapies for cancer patients may offer fewer adverse side effects because of specificity compared to conventional chemotherapy. Signaling pathways of nuclear receptors, such as the estrogen receptor (ER), have been intensively studied and used as therapeutic targets. Recently, the role of the androgen receptor (AR) in breast cancer is gaining greater attention as a therapeutic target and as a prognostic biomarker. The expression of constitutively active truncated AR splice variants in breast cancer is a possible mechanism contributing to treatment resistance. Therefore, targeting both the full-length AR and AR variants, either through the activation or suppression of AR function, depending on the status of the ER, progesterone receptor, or human epidermal growth factor receptor 2, may provide additional treatment options. Studies targeting AR in combination with other treatment strategies are ongoing in clinical trials. The determination of the status of nuclear receptors to classify and identify patient subgroups will facilitate optimized and targeted combination therapies.

Asunto(s)

Neoplasias de la Mama , Neoplasias de la Próstata Resistentes a la Castración , Humanos , Masculino , Receptores Androgénicos/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Antagonistas de Receptores Androgénicos/farmacología , Antagonistas de Receptores Androgénicos/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico

RESUMEN

Growing evidence indicates that transposable elements (TEs) play important roles in evolution by providing genomes with coding and non-coding sequences. Identification of TE-derived functional elements, however, has relied on TE annotations in individual species, which limits its scope to relatively intact TE sequences. Here, we report a novel approach to uncover previously unannotated degenerate TEs (degTEs) by probing multiple ancestral genomes reconstructed from hundreds of species. We applied this method to the human genome and achieved a 10.8% increase in coverage over the most recent annotation. Further, we discovered that degTEs contribute to various cis-regulatory elements and transcription factor binding sites, including those of a known TE-controlling family, the KRAB zinc-finger proteins. We also report unannotated chimeric transcripts between degTEs and human genes expressed in embryos. This study provides a novel methodology and a freely available resource that will facilitate the investigation of TE co-option events on a full scale.

Asunto(s)

Elementos Transponibles de ADN , Secuencias Reguladoras de Ácidos Nucleicos , Humanos , Elementos Transponibles de ADN/genética , Genoma Humano/genética

RESUMEN

Rifampicin and rifabutin can activate the pregnane X receptor (PXR, NR1I2), thereby inducing pharmacokinetically important genes/proteins and reducing exposure to co-administered drugs. Because induction effects vary considerably between these antibiotics, differences could be due to unequal rifamycin-induced activation or tissue expression of the three major NR1I2 splice variants, PXR.1 (NM_003889), PXR.2 (NM_022002), and PXR.3 (NM_033013). Consequently, PXR activation (PXR reporter gene assays) and mRNA expression levels of total NR1I2, PXR.1, PXR.2, and PXR.3 were investigated by polymerase chain reaction in colon and liver samples from eleven surgical patients, in LS180 cells, and primary human hepatocytes. Compared to the colon, total NR1I2 mRNA expression was higher in the liver. Both tissues showed similar expression levels of PXR.1 and PXR.3, respectively. PXR.2 was not quantifiable in the colon samples. Rifampicin and rifabutin similarly enhanced PXR.1 and PXR.2 activity when transfected into LS180 cells, while PXR.3 could not be activated. In LS180 cells, rifampicin (10 µM) reduced total NR1I2 and PXR.3 expression 2-fold after 24 h, while rifabutin (10 µM) increased total NR1I2, PXR.1, PXR.2, and PXR.3 mRNA by approx. 50% after 96-h exposure. In primary human hepatocytes, rifampicin (10 µM) suppressed total NR1I2, PXR.1, and PXR.3 after 48-h exposure, and rifabutin (10 µM) had no significant impact on total NR1I2 or any of the splice variants studied. In conclusion, both antibiotics activated the studied PXR splice variants similarly but modified their expression differently. While rifampicin can suppress mRNA of PXR forms, rifabutin rather increases their expression levels.

Asunto(s)

Receptores de Esteroides , Rifampin , Humanos , Receptor X de Pregnano , Rifampin/farmacología , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Rifabutina , Antibacterianos , ARN Mensajero , Citocromo P-450 CYP3A

RESUMEN

N-type calcium channels (CaV2.2) are predominantly localized in presynaptic terminals, and are particularly important for pain transmission in the spinal cord. Furthermore, they have multiple isoforms, conferred by alternatively spliced or cassette exons, which are differentially expressed. Here, we have examined alternatively spliced exon47 variants that encode a long or short C-terminus in human CaV2.2. In the Ensembl database, all short exon47-containing transcripts were associated with the absence of exon18a, therefore, we also examined the effect of inclusion or absence of exon18a, combinatorially with the exon47 splice variants. We found that long exon47, only in the additional presence of exon18a, results in CaV2.2 currents that have a 3.6-fold greater maximum conductance than the other three combinations. In contrast, cell-surface expression of CaV2.2 in both tsA-201 cells and hippocampal neurons is increased ∼4-fold by long exon47, relative to short exon47, in either the presence or the absence of exon18a. This surprising discrepancy between trafficking and function indicates that cell-surface expression is enhanced by long exon47, independently of exon18a. However, in the presence of long exon47, exon18a mediates an additional permissive effect on CaV2.2 gating. We also investigated the single-nucleotide polymorphism in exon47 that has been linked to schizophrenia and Parkinson's disease, which we found is only non-synonymous in the short exon47 C-terminal isoform, resulting in two minor alleles. This study highlights the importance of investigating the combinatorial effects of exon inclusion, rather than each in isolation, in order to increase our understanding of calcium channel function.

Asunto(s)

Neuronas , Empalme del ARN , Humanos , Neuronas/metabolismo , Canales de Calcio Tipo N/genética , Isoformas de Proteínas/genética , Exones/genética

RESUMEN

Triple-negative breast cancer (TNBC) is an aggressive subtype with no targeted therapeutics. The luminal androgen receptor (LAR) subtype constitutes 15% of TNBC and is enriched for androgen receptor (AR) and AR target genes. Here, we show that a cohort of TNBC not only expresses AR at a much higher rate (∼80%) but also expresses AR splice variants (AR-SVs) (∼20%), further subclassifying LAR-TNBC. Higher AR and AR-SV expression and corresponding aggressive phenotypes are observed predominantly in specimens obtained from African American women. LAR TNBC specimens are enriched for interferon, Janus kinase (JAK)-signal activator and transducer (STAT), and androgen signaling pathways, which are exclusive to AR-expressing epithelial cancer cells. AR- and AR-SV-expressing TNBC cell proliferation and xenograft and patient-tumor explant growth are inhibited by AR N-terminal domain-binding selective AR degrader or by a JAK inhibitor. Biochemical analysis suggests that STAT1 is an AR coactivator. Collectively, our work identifies pharmacologically targetable TNBC subtypes and identifies growth-promoting interaction between AR and JAK-STAT signaling.

Asunto(s)

Neoplasias de la Mama Triple Negativas , Humanos , Femenino , Neoplasias de la Mama Triple Negativas/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Transducción de Señal/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica