RESUMEN

Introduction: AXL receptor expression is proposed to confer immune-checkpoint inhibitor (ICI)-resistance in non-small cell lung cancer (NSCLC) patients. We sought to interrogate AXL expression in conjunction with mutational and tumor-microenvironmental features to uncover predictive mechanisms of resistance in ICI-treated NSCLC patients. Methods: Tumor samples from 111 NSCLC patients treated with ICI-monotherapy were analyzed by immunohistochemistry for tumor- and immune-AXL expression. Subsets of patients were analyzed by whole-exome sequencing (n = 44) and imaging mass cytometry (n = 14). Results were related to ICI-outcome measurements. Results: Tumor-cell AXL expression correlated with aggressive phenotypic features including reduced OS in patients treated with ICIs (P = 0.04) after chemotherapy progression, but conversely associated with improved disease control (P = 0.045) in ICI-treated, PD-L1 high first-line patients. AXL+ immune-cell infiltration correlated with total immune-cell infiltration and improved overall outcomes (PFS: P = 0.044, OS: P = 0.054). Tumor-cell AXL-upregulation showed enrichment in mutations associated with PD-L1-upregulation and ICI-response such as MUC4 and ZNF469, as well as adverse mutations including CSMD1 and LRP1B which associated with an immune-suppressed tumor phenotype and poor ICI prognosis particularly within chemotherapy-treated patients. Tumor mutational burden had no effect on ICI-outcomes and was associated with a lack of tumor-infiltrating immune cells. Spatial-immunophenotyping provided evidence that tumor-cell AXL-upregulation and adverse mutations modulate the tumor microenvironment in favor of infiltrating, activated neutrophils over anti-tumor immune-subsets including CD4 and CD8 T-cells. Conclusion: Tumor-cell AXL-upregulation correlated with distinct oncotypes and microenvironmental immune-profiles that define chemotherapy-induced mechanisms of ICI-resistance, which suggests the combination of AXL inhibitors with current chemoimmunotherapy regimens can benefit NSCLC patients.

Asunto(s)

Tirosina Quinasa del Receptor Axl , Carcinoma de Pulmón de Células no Pequeñas , Inhibidores de Puntos de Control Inmunológico , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras , Microambiente Tumoral , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Proto-Oncogénicas/genética , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Masculino , Femenino , Microambiente Tumoral/inmunología , Anciano , Persona de Mediana Edad , Biomarcadores de Tumor , Mutación , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Resultado del Tratamiento , Anciano de 80 o más Años , Resistencia a Antineoplásicos/genética , Adulto

RESUMEN

In the realm of this study, obtaining a comprehensive understanding of ischemic brain injury and its molecular foundations is of paramount importance. Our study delved into single-cell data analysis, with a specific focus on sub-celltypes and differentially expressed genes in the aftermath of ischemic injury. Notably, we observed a significant enrichment of the "ATP METABOLIC PROCESS" and "ATP HYDROLYSIS ACTIVITY" pathways, featuring pivotal genes such as Pbx3, Dguok, and Kif21b. A remarkable finding was the consistent upregulation of genes like Fabp7 and Bcl11a within the MCAO group, highlighting their crucial roles in regulating the pathway of mitochondrial ATP synthesis coupled proton transport. Furthermore, our network analysis unveiled pathways like "Neuron differentiation" and "T cell differentiation" as central in the regulatory processes of sub-celltypes. These findings provide valuable insights into the intricate molecular responses and regulatory mechanisms that govern brain injury. The shared differentially expressed genes among sub-celltypes emphasize their significance in orchestrating responses post-ischemic injury. Our research, viewed from the perspective of a medical researcher, contributes to the evolving understanding of the molecular landscape underlying ischemic brain injury, potentially paving the way for targeted therapeutic strategies and improved patient outcomes.

Asunto(s)

Adenosina Trifosfato , Infarto de la Arteria Cerebral Media , Cinesinas , Mitocondrias , Células Precursoras de Oligodendrocitos , Transducción de Señal , Animales , Transducción de Señal/genética , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/metabolismo , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/biosíntesis , Cinesinas/genética , Cinesinas/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Humanos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Ratas , Proteínas Proto-Oncogénicas

RESUMEN

Lidamycin (LDM) has been confirmed to have a strong anti-pancreatic cancer effect and can affect the mitochondrial function of pancreatic cancer cells. Mitofusin-2 (Mfn2) is located in the outer membrane of mitochondria, and Mfn2 is currently believed to play a role in cancer inhibition in pancreatic cancer. In order to explore whether the anti-pancreatic cancer effect of LDM is related to Mfn2-mediated mitophagy, Bioinformatics and in vitro cell experiments are used for experimental research. The experimental results demonstrated that Mfn2 is correlated with mitochondrial autophagy in pancreatic cancer. Lidamycin can increase the expression of Mfn2 in pancreatic cancer and affect the process of EMT, affect the level of reactive oxygen species and mitochondrial membrane potential, and increase the expression of mitochondrial autophagy marker proteins BNIP3L and Beclin1. These results demonstrate that Mfn2 affects mitophagy in pancreatic cancer cells by regulating the expression of Mfn2.

Asunto(s)

GTP Fosfohidrolasas , Proteínas de la Membrana , Proteínas Mitocondriales , Mitofagia , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Mitofagia/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Línea Celular Tumoral , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Aminoglicósidos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Beclina-1/metabolismo , Beclina-1/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor

RESUMEN

Epigenetic regulation plays a role in Parkinson's disease (PD), and ten-eleven translocation methylcytosine dioxygenase 1 (TET1) catalyzes the first step in DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine. We investigated whether TET1 binds to the promoter of the transient receptor potential cation channel subfamily V member 1 (TRPV1) and regulates its expression, thereby controlling oxidative stress in PD. TRPV1 was identified as an oxidative stress-associated gene in the GSE20186 dataset including substantia nigra from 14 patients with PD and 14 healthy controls and the Genecards database. Lentiviral vectors were used to manipulate Trpv1 expression in rats, followed by 6-hydroxydopamine hydrochloride (6-OHDA) injection for modeling. Behavioral tests, immunofluorescence, Nissl staining, western blot assays, DHE fluorescent probe, biochemical analysis, and ELISA were conducted to assess oxidative stress and neurotoxicity. Trpv1 expression was significantly reduced in the brain tissues of 6-OHDA-treated Parkinsonian rats. Trpv1 alleviated behavioral dysfunction, oxidative stress, and dopamine neuron loss in rats. TET1 mediated TRPV1 hydroxymethylation to promote its expression, and Trpv1 inhibition reversed the mitigating effect of Tet1 on oxidative stress and behavioral dysfunction in PD. TRPV1 activated the AMPK signaling by promoting AMPK phosphorylation to alleviate neurotoxicity and oxidative stress in SH-SY5Y cells. Tet1-mediated Trpv1 hydroxymethylation modification promotes the Ampk signaling activation, thereby eliciting neuroprotection in 6-OHDA-treated Parkinsonian rats. These findings provide experimental evidence that targeting the TET1/TRPV1 axis may be neuroprotective for PD by acting on the AMPK signaling.

Asunto(s)

Metilación de ADN , Estrés Oxidativo , Enfermedad de Parkinson , Ratas Sprague-Dawley , Transducción de Señal , Canales Catiónicos TRPV , Animales , Ratas , Estrés Oxidativo/efectos de los fármacos , Masculino , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Humanos , Modelos Animales de Enfermedad , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Oxidopamina , Epigénesis Genética , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/genética , Fármacos Neuroprotectores/farmacología , Dioxigenasas

RESUMEN

BACKGROUND: Breast tumorigenesis is a complex and multistep process accompanied by both genetic and epigenetic dysregulation. In contrast to the extensive studies on DNA epigenetic modifications 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in malignant breast tumors, their roles in the early phases of breast tumorigenesis remain ambiguous. RESULTS: DNA 5hmC and 5mC exhibited a consistent and significant decrease from usual ductal hyperplasia to atypical ductal hyperplasia and subsequently to ductal carcinoma in situ (DCIS). However, 5hmC showed a modest increase in invasive ductal breast cancer compared to DCIS. Genomic analyses showed that the changes in 5hmC and 5mC levels occurred around the transcription start sites (TSSs), and the modification levels were strongly correlated with gene expression levels. Meanwhile, it was found that differentially hydroxymethylated regions (DhMRs) and differentially methylated regions (DMRs) were overlapped in the early phases and accompanied by the enrichment of active histone marks. In addition, TET2-related DNA demethylation was found to be involved in breast tumorigenesis, and four transcription factor binding sites (TFs: ESR1, FOXA1, GATA3, FOS) were enriched in TET2-related DhMRs/DMRs. Intriguingly, we also identified a certain number of common DhMRs between tumor samples and cell-free DNA (cfDNA). CONCLUSIONS: Our study reveals that dynamic changes in DNA 5hmC and 5mC play a vital role in propelling breast tumorigenesis. Both TFs and active histone marks are involved in TET2-related DNA demethylation. Concurrent changes in 5hmC signals in primary breast tumors and cfDNA may play a promising role in breast cancer screening.

Asunto(s)

5-Metilcitosina , Neoplasias de la Mama , Proteínas de Unión al ADN , Dioxigenasas , Proteínas Proto-Oncogénicas , Humanos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Femenino , Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Carcinogénesis/genética , Metilación de ADN/genética , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica , Desmetilación del ADN

RESUMEN

BACKGROUD: Supernatants from various cytological samples, including body cavity effusion, sputum, bronchoalveolar lavage fluid (BALF), and needle aspiration, have been validated for detecting genetic alterations using cell-free DNA (cfDNA) in patients with non-small cell lung cancer (NSCLC). However, the sensitivity of fusion variations detection remains challenging. The protection of cell-free RNA (cfRNA) is critical for resolving the issue. METHODS: A protective solution (PS) was applied for preserving cfRNA in cytological supernatant (CS), and the quality of protected cfRNA was assessed by cycle threshold (CT) values from reverse transcription quantitative polymerase chain reaction (RT-qPCR). Furthermore, we collected an additional set of malignant cytological and matched tumor samples from 84 NSCLC patients, cfDNA & cfRNA extraction and double detection for driver gene mutations was validated using the multi-gene mutations detection by RT-qPCR. RESULTS: Under the optimal protection system, 91.0% (101/111) of cfRNA were protected effectively. Among the 84 NSCLC patient samples, seven cytological samples failed the tests. In comparison with tumor samples, the overall sensitivity and specificity of detecting driver genes of supernatant cfDNA and cfRNA were 93.8% (74/77) and 100% (77/77), respectively. Notably, when focusing exclusively on patients with fusion gene changes, both sensitivity and specificity reached 100% (11/11) for EML4-ALK, ROS1, RET fusions, and MET ex14 skipping. CONCLUSION: These findings suggest that cfDNA & cfRNA extraction and double detection strategy recommended in this study improve the accuracy of driver genes mutations test, especially for RNA-based assay.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/diagnóstico , Ácidos Nucleicos Libres de Células/genética , Mutación , Masculino , Femenino , Biomarcadores de Tumor/genética , Sensibilidad y Especificidad , Persona de Mediana Edad , Anciano , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas

RESUMEN

The centromere, a chromosome locus defined by the histone H3-like protein centromeric protein A (CENP-A), promotes assembly of the kinetochore to bind microtubules during cell division. Centromere maintenance requires CENP-A to be actively replenished by dedicated protein machinery in the early G1 phase of the cell cycle to compensate for its dilution after DNA replication. Cyclin-dependent kinases (CDKs) limit CENP-A deposition to once per cell cycle and function as negative regulators outside of early G1. Antithetically, Polo-like kinase 1 (PLK1) promotes CENP-A deposition in early G1, but the molecular details of this process are still unknown. We reveal here a phosphorylation network that recruits PLK1 to the deposition machinery to control a conformational switch required for licensing the CENP-A deposition reaction. Our findings clarify how PLK1 contributes to the epigenetic maintenance of centromeres.

Asunto(s)

Proteínas de Ciclo Celular , Proteína A Centromérica , Centrómero , Proteínas Cromosómicas no Histona , Epigénesis Genética , Quinasa Tipo Polo 1 , Humanos , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Fase G1 , Células HeLa , Cinetocoros/metabolismo , Fosforilación , Quinasa Tipo Polo 1/genética , Quinasa Tipo Polo 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética

RESUMEN

Functional genomic screens in two-dimensional cell culture models are limited in identifying therapeutic targets that influence the tumor microenvironment. By comparing targeted CRISPR-Cas9 screens in a two-dimensional culture with xenografts derived from the same cell line, we identified MEN1 as the top hit that confers differential dropout effects in vitro and in vivo. MEN1 knockout in multiple solid cancer types does not impact cell proliferation in vitro but significantly promotes or inhibits tumor growth in immunodeficient or immunocompetent mice, respectively. Mechanistically, MEN1 knockout redistributes MLL1 chromatin occupancy, increasing H3K4me3 at repetitive genomic regions, activating double-stranded RNA expression and increasing neutrophil and CD8+ T cell infiltration in immunodeficient and immunocompetent mice, respectively. Pharmacological inhibition of the menin-MLL interaction reduces tumor growth in a CD8+ T cell-dependent manner. These findings reveal tumor microenvironment-dependent oncogenic and tumor-suppressive functions of MEN1 and provide a rationale for targeting MEN1 in solid cancers.

Asunto(s)

Linfocitos T CD8-positivos , Sistemas CRISPR-Cas , N-Metiltransferasa de Histona-Lisina , Proteínas Proto-Oncogénicas , Microambiente Tumoral , Microambiente Tumoral/genética , Animales , Humanos , Ratones , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Neoplasias/genética , Neoplasias/patología , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Femenino

RESUMEN

Overcoming temozolomide (TMZ)-resistance is a major challenge in glioblastoma therapy. Therefore, identifying the key molecular player in chemo-resistance becomes urgent. We previously reported the downregulation of PDCD10 in primary glioblastoma patients and its tumor suppressor-like function in glioblastoma cells. Here, we demonstrate that the loss of PDCD10 causes a significant TMZ-resistance during treatment and promotes a rapid regrowth of tumor cells after treatment. PDCD10 knockdown upregulated MGMT, a key enzyme mediating chemo-resistance in glioblastoma, accompanied by increased expression of DNA mismatch repair genes, and enabled tumor cells to evade TMZ-induced cell-cycle arrest. These findings were confirmed in independent models of PDCD10 overexpressing cells. Furthermore, PDCD10 downregulation led to the dedifferentiation of glioblastoma cells, as evidenced by increased clonogenic growth, the upregulation of glioblastoma stem cell (GSC) markers, and enhanced neurosphere formation capacity. GSCs derived from PDCD10 knockdown cells displayed stronger TMZ-resistance and regrowth potency, compared to their parental counterparts, indicating that PDCD10-induced stemness may independently contribute to tumor malignancy. These data provide evidence for a dual role of PDCD10 in tumor suppression by controlling both chemo-resistance and dedifferentiation, and highlight PDCD10 as a potential prognostic marker and target for combination therapy with TMZ in glioblastoma.

Asunto(s)

Proteínas Reguladoras de la Apoptosis , Resistencia a Antineoplásicos , Glioblastoma , Temozolomida , Humanos , Glioblastoma/patología , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológico , Temozolomida/farmacología , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proliferación Celular/efectos de los fármacos , Metilasas de Modificación del ADN/metabolismo , Metilasas de Modificación del ADN/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética

RESUMEN

MDM4 is upregulated in the majority of melanoma cases and has been described as a "key therapeutic target in cutaneous melanoma". Numerous isoforms of MDM4 exist, with few studies examining their specific expression in human tissues. The changes in splicing of MDM4 during human melanomagenesis are critical to p53 activity and represent potential therapeutic targets. Compounding this, studies relying on short reads lose "connectivity" data, so full transcripts are frequently only inferred from the presence of splice junction reads. To address this problem, long-read nanopore sequencing was utilized to read the entire length of transcripts. Here, MDM4 transcripts, both alternative and canonical, are characterized in a pilot cohort of human melanoma specimens. RT-PCR was first used to identify the presence of novel splice junctions in these specimens. RT-qPCR then quantified the expression of major MDM4 isoforms observed during sequencing. The current study both identifies and quantifies MDM4 isoforms present in melanoma tumor samples. In the current study, we observed high expression levels of MDM4-S, MDM4-FL, MDM4-A, and the previously undescribed Ensembl transcript MDM4-209. A novel transcript lacking both exons 6 and 9 is observed and named MDM4-A/S for its resemblance to both MDM4-A and MDM4-S isoforms.

Asunto(s)

Melanoma , Isoformas de Proteínas , Humanos , Melanoma/genética , Melanoma/patología , Melanoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Empalme Alternativo , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nanoporos/métodos

RESUMEN

Radiation therapy continues to be the cornerstone treatment for malignant brain tumors, the majority of which express wild-type p53. Therefore, the identification of drugs that promote the ionizing radiation (IR)-induced activation of p53 is expected to increase the efficacy of radiation therapy for these tumors. The growth inhibitory effects of CEP-1347, a known inhibitor of MDM4 expression, on malignant brain tumor cell lines expressing wild-type p53 were examined, alone or in combination with IR, by dye exclusion and/or colony formation assays. The effects of CEP-1347 on the p53 pathway, alone or in combination with IR, were examined by RT-PCR and Western blot analyses. The combination of CEP-1347 and IR activated p53 in malignant brain tumor cells and inhibited their growth more effectively than either alone. Mechanistically, CEP-1347 and IR each reduced MDM4 expression, while their combination did not result in further decreases. CEP-1347 promoted IR-induced Chk2 phosphorylation and increased p53 expression in concert with IR in a Chk2-dependent manner. The present results show, for the first time, that CEP-1347 is capable of promoting Chk2-mediated p53 activation by IR in addition to inhibiting the expression of MDM4 and, thus, CEP-1347 has potential as a radiosensitizer for malignant brain tumors expressing wild-type p53.

Asunto(s)

Neoplasias Encefálicas , Quinasa de Punto de Control 2 , Radiación Ionizante , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Quinasa de Punto de Control 2/metabolismo , Quinasa de Punto de Control 2/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Fosforilación/efectos de los fármacos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación

RESUMEN

BACKGROUND: The Axl gene is a receptor tyrosine kinase essential for male fertility. With other Tyro3 family members, it regulates cell apoptosis and preserves the organization of seminiferous tubules. However, the regulation of the expression of Axl in testicular Sertoli cells is not entirely understood. The transcription factors NR5A1 and JUNB are involved in several male fertility mechanisms such as sex development and steroidogenesis. We hypothesize that Axl promoter activity is regulated by cooperation between JUNB and NR5A1 in Sertoli cells. METHODS AND RESULTS: Following transfections of TM4 Sertoli cells with DsiRNA interference against Axl, our results show that cell morphology may be regulated by AXL. Using transfections of expression plasmids and reporter plasmids containing the Axl promoter, we report that Axl expression is highly activated by cooperation between NR5A1 and JUNB in TM4 and 15P-1 Sertoli cells. Chromatin immunoprecipitation and luciferase reporter assays with 5' promoter deletions demonstrate that JUNB and NR5A1 are being recruited to DNA regulatory elements in the proximal region of the Axl promoter. The fourth intronic region of Axl also participates in the recruitment of JUNB. CONCLUSION: Thus, Axl expression is regulated by a cooperation between the transcription factors JUNB and NR5A1 and influences the morphology of TM4 Sertoli cells.

Asunto(s)

Tirosina Quinasa del Receptor Axl , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras , Células de Sertoli , Factor Esteroidogénico 1 , Factores de Transcripción , Animales , Células de Sertoli/metabolismo , Masculino , Ratones , Regiones Promotoras Genéticas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Línea Celular , Factor Esteroidogénico 1/genética , Factor Esteroidogénico 1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica

RESUMEN

Split-hand/foot malformation is a serious congenital limb malformation characterized by syndactyly and underdevelopment of the phalanges and metatarsals. In this study, we reported a case of a fetus with hand-foot cleft deformity. Whole exome and Sanger sequencing were used to filter out candidate gene mutation sites and provide pre-implantation genetic testing(PGT) for family members. Genetic testing results showed that there was a homozygous mutation c.786G>A (p.Trp262*) in the fetal WNT10B, and both parents were carriers of heterozygous mutations. PGT results showed that out of the two blastocysts, one was a heterozygous mutant and the other was a homozygous mutant. All the embryos had diploid chromosomes. The heterozygous embryo was transferred, and a singleton pregnancy was successfully achieved. This study suggests that homozygous mutations in WNT10B are the likely cause of hand-foot clefts in this family. For families with monogenic diseases, preimplantation genetic testing can effectively prevent the birth of an affected child only after identifying the pathogenic mutation.

Asunto(s)

Pruebas Genéticas , Deformidades Congénitas de las Extremidades , Linaje , Diagnóstico Preimplantación , Humanos , Femenino , Deformidades Congénitas de las Extremidades/genética , Diagnóstico Preimplantación/métodos , Proteínas Wnt/genética , Embarazo , Mutación , Masculino , Adulto , Pueblo Asiatico/genética , Homocigoto , Pueblos del Este de Asia , Proteínas Proto-Oncogénicas

RESUMEN

Clonal hematopoiesis of indeterminate potential (CHIP) is linked to diverse aging-related diseases, including hematologic malignancy and atherosclerotic cardiovascular disease (ASCVD). While CHIP is common among older adults, the underlying factors driving its development are largely unknown. To address this, we performed whole-exome sequencing on 8,374 blood DNA samples collected from 4,187 Atherosclerosis Risk in Communities Study (ARIC) participants over a median follow-up of 21 years. During this period, 735 participants developed incident CHIP. Splicing factor genes (SF3B1, SRSF2, U2AF1, and ZRSR2) and TET2 CHIP grow significantly faster than DNMT3A non-R882 clones. We find that age at baseline and sex significantly influence the incidence of CHIP, while ASCVD and other traditional ASCVD risk factors do not exhibit such associations. Additionally, baseline synonymous passenger mutations are strongly associated with CHIP status and are predictive of new CHIP clone acquisition and clonal growth over extended follow-up, providing valuable insights into clonal dynamics of aging hematopoietic stem and progenitor cells. This study also reveals associations between germline genetic variants and incident CHIP. Our comprehensive longitudinal assessment yields insights into cell-intrinsic and -extrinsic factors contributing to the development and progression of CHIP clones in older adults.

Asunto(s)

Hematopoyesis Clonal , Dioxigenasas , Humanos , Hematopoyesis Clonal/genética , Masculino , Femenino , Anciano , Estudios Longitudinales , Persona de Mediana Edad , Dioxigenasas/genética , ADN Metiltransferasa 3A , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Aterosclerosis/genética , Factores de Riesgo , Secuenciación del Exoma , Proteínas de Unión al ADN/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Envejecimiento/genética , Incidencia , Mutación

RESUMEN

Recruitment of non-canonical BCOR-PRC1.1 to non-methylated CpG islands via KDM2B plays a fundamental role in transcription control during developmental processes and cancer progression. However, the mechanism is still largely unknown on how this recruitment is regulated. Here, we unveiled the importance of the Poly-D/E regions within the linker of BCOR for its binding to KDM2B. Interestingly, we also demonstrated that these negatively charged Poly-D/E regions on BCOR play autoinhibitory roles in liquid-liquid phase separation (LLPS) of BCORANK-linker-PUFD/PCGF1RAWUL. Through neutralizing negative charges of these Poly-D/E regions, Ca2+ not only weakens the interaction between BCOR/PCGF1 and KDM2B, but also promotes co-condensation of the enzymatic core of BCOR-PRC1.1 with KDM2B into liquid-like droplet. Accordingly, we propose that Ca2+ could modulate the compartmentation and recruitment of the enzymatic core of BCOR-PRC1.1 on KDM2B target loci. Thus, our finding advances the mechanistic understanding on how the tethering of BCOR-PRC1.1 enzymatic core to KDM2B is regulated.

Asunto(s)

Calcio , Histona Demetilasas con Dominio de Jumonji , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas , Proteínas Represoras , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Calcio/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/química , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/genética , Unión Proteica , Separación de Fases , Proteínas F-Box

RESUMEN

In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.

Asunto(s)

5-Metilcitosina , Proteínas de Unión al ADN , Dioxigenasas , Proteínas Proto-Oncogénicas , Dioxigenasas/metabolismo , Dioxigenasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análisis , 5-Metilcitosina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Pruebas de Enzimas/métodos , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/química , Metilación de ADN , Citosina/análogos & derivados , Citosina/análisis , Citosina/metabolismo , Citosina/química , Oxidación-Reducción

RESUMEN

Recent studies have highlighted the significant role of 5-hydroxymethylcytosine (5hmC) in carcinogenesis. However, the specific role of 5hmC in osteosarcoma (OS) remains largely unexplored. The-re-fore, this study aimed to investigate the function of 5hmC and TET3 in OS. In this study, we found a decreased total level of 5hmC in OS tissues. The expression of the TET3 protein was also decreased in OS. Importantly, the decreased levels of TET3 were associated with a decreased disease-free survival (DFS) rate in patients. To investigate the role of TET3 and 5hmC in OS, we manipulated the levels of TET3 in MG-63 cells. Silencing TET3 in these cells resulted in a twofold increase in proliferation. Additio-nally, the level of 5hmC decreased in these cells. Con-versely, over-expression of TET3 in MG-63 cells led to the expected inhibition of proliferation and invasion, accompanied by an increase in 5hmC levels. In conclusion, both 5hmC and TET3 protein levels were decreased in OS. Additionally, the over-expression of TET3 inhibited the proliferation of MG-63 cells, while the suppression of TET3 had the opposite effect. These findings suggest that decreased levels of 5hmC and TET3 may serve as potential markers for OS.

Asunto(s)

5-Metilcitosina , Proliferación Celular , Desmetilación del ADN , Dioxigenasas , Epigénesis Genética , Femenino , Humanos , Masculino , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Dioxigenasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética

Asunto(s)

Resistencia a Antineoplásicos , N-Metiltransferasa de Histona-Lisina , Proteína de la Leucemia Mieloide-Linfoide , Proteínas Proto-Oncogénicas , Humanos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Resistencia a Antineoplásicos/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Animales

RESUMEN

OBJECTIVE: To evaluate the frequency and prognostic significance of DTA (DNMT3A、TET2、ASXL1) gene mutation and co-occurring mutations in patients with myelodysplastic syndrome (MDS). METHODS: The clinical data of 102 newly diagnosed MDS patients who accepted Next Generation Sequencing (NGS) was retrospectively analyzed. According to whether the patients had DTA gene mutation, the patients were divided into DTA mutated (DTA-mut) group and wild type (DTA-wt) group, and the relationship between gene mutation and clinical characteristics and prognosis was analyzed. RESULTS: Among the 102 MDS patients, 96% (98/102) presented with mutation, while the mean number of mutations was 3.04 mutations/patient. DTA-mut was detected in 56.9% (58/102) patients. The most frequent co-mutated genes in DTA-mut group were SF3B1 (25.8%), RUNX1 (24.1%), U2AF1 (18.9%), SRSF2, EZH2, SETBP1 (17.2%), STAG2 (15.5%), IDH2 (12.1%) and BCOR, CBL (10.3%). The two groups showed no significant differences in ages, blood parameters, bone marrow blasts, WHO 2022 classification, IPSS-R risk category and rate of conversion to leukemia. Compared with the DTA-wt group, the mutation frequency of RUNX1 was higher (P = 0.02), while mutation frequency of TP53 was lower (P = 0.001) and the mutation frequency of ≥ 3 co-mutated genes was higher in DTA-mut group (P = 0.00). Survival analysis showed that the overall survivals (OS) of DTA-mut patients was significantly inferior to that of DTA-wt patients (P = 0.0332). According to IPSS-R classification, a statistically significant difference in OS was only observed in higher risk (IPSS-R > 3.5) group (P = 0.0058). In the context of DTA mutation, the OS of patients with RUNX1 mutation was shorter than that of patients without RUNX1 mutation significantly (P = 0.0074). The OS of patients with SF3B1 mutation was longer than that of patients without SF3B1 mutation, but there was no statistical difference (P = 0.0827). DTA mutations were not independent prognostic factors when DTA and co-mutated genes with frequency > 10% were considered in Cox regression model (P = 0.329). However, multivariate analysis confirmed an independently adverse prognosis of RUNX1 co-mutation (P = 0.042, HR = 2.426, 95% CI:1.031-5.711) in DTA-mut cohort. Moreover, our multivariable analysis suggests that SRSF2-mut was an independent poor prognostic factor for all MDS patients (P = 0.047), but lost significance (P = 0.103) for DTA-mut patients. CONCLUSIONS: DTA mutations are frequently observed in patients with MDS, often accompanied by genes involved in RNA splicing and transcription factors like SF3B1 and RUNX1. DTA and concomitant mutations affect prognosis in MDS patients and RUNX1 was identified as an independent poor prognostic factor in patients with DTA mutations.

Asunto(s)

ADN (Citosina-5-)-Metiltransferasas , ADN Metiltransferasa 3A , Proteínas de Unión al ADN , Dioxigenasas , Mutación , Síndromes Mielodisplásicos , Proteínas Proto-Oncogénicas , Proteínas Represoras , Humanos , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/mortalidad , Masculino , Femenino , Mutación/genética , Persona de Mediana Edad , Pronóstico , Anciano , Adulto , Proteínas Proto-Oncogénicas/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas Represoras/genética , Proteínas de Unión al ADN/genética , Estudios Retrospectivos , Anciano de 80 o más Años , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Adolescente , Adulto Joven

RESUMEN

Aim: To evaluate the urinary biomarkers related to sepsis in preterm newborns (NBs) and to investigate the predictive capacity of these biomarkers for a longer hospital stay.Methods: Serum and urine were collected from 27 healthy NBs, 24 NBs with neonatal infection without sepsis and 11 NBs with sepsis for the measurement of sindecan-1, lipocalin associated with urinary neutrophil gelatinase (uNGAL), urinary cystatin-C (uCysC) and urinary kidney injury molecule-1.Results: Levels of uNGAL and urinary cystatin-C were elevated in NBs with sepsis and neonatal infection, and uNGAL was significant predictor of hospital stay longer than 30 days (odds ratio: 1.052; 95% CI: 1.012-1.093; p = 0.01).Conclusion: uNGAL was associated with sepsis in preterm NBs and was useful to predict extended hospital stay.

---

[Box: see text].

Asunto(s)

Biomarcadores , Cistatina C , Recien Nacido Prematuro , Tiempo de Internación , Lipocalina 2 , Sepsis , Humanos , Recién Nacido , Cistatina C/sangre , Cistatina C/orina , Lipocalina 2/orina , Lipocalina 2/sangre , Biomarcadores/orina , Biomarcadores/sangre , Sepsis/orina , Sepsis/diagnóstico , Sepsis/sangre , Masculino , Femenino , Recien Nacido Prematuro/orina , Proteínas de Fase Aguda/orina , Proteínas Proto-Oncogénicas/orina , Proteínas Proto-Oncogénicas/sangre