A DNA hypermethylation profile reveals new potential biomarkers for the evaluation of prognosis in urothelial bladder cancer

DNA hypermethylation has emerged as a molecular biomarker for the evaluation of cancer diagnosis and prognosis. We define a methylation signature of bladder cancer and evaluate whether this profile assesses prognosis of patients. Genome-wide methylation analysis was performed on 70 tumor and 10 normal bladder samples. Hypermethylation status of 1505 CpGs present in the promoter region of 807 genes was studied. Thirty-three genes were significantly hypermethylated in >= 10% of the tumors. Three clusters of patients were characterized by their DNA methylation profile, one at higher risk of dead of disease (p = 0.0012). Association between cluster distribution and stage (p = 0.02) or grade (p = 0.02) was demonstrated. Hypermethylation of JAK3 and absence of hypermethylation of EYA4, GAT6, and SOX1 were associated with low-grade non-invasive disease. On the other hand, in high-grade invasive disease hypermethylation of CSPG2, HOXA11, HOXA9, HS3ST2, SOX1, and TWIST1 was associated with muscle invasiveness. A panel of hypermethylated genes including APC, CSPG2, EPHA5, EYA4, HOXA9, IPF1, ISL1, JAK3, PITX2, SOX1, and TWIST1 predicted cancer-specific survival and SOX1 (HR = 3.46), PITX2 (HR = 4.17), CSPG2 (HR = 5.35), and JAK3 hypermethylation (HR = 0.19) did so independently. Silencing of genes by hypermethylation is a common event in bladder cancer and could be used to develop diagnostic and prognostic markers. Combined hypermethylation of SOX1, PITX2, or CSPG2 signals patients at higher risk of death from bladder cancer

CryoEM of RUVBL1-RUVBL2-ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8.

Biogenesis of the U5 small nuclear ribonucleoprotein (snRNP) is an essential and highly regulated process. In particular, PRPF8, one of U5 snRNP main components, requires HSP90 working in concert with R2TP, a cochaperone complex containing RUVBL1 and RUVBL2 AAA-ATPases, and additional factors that are still poorly characterized. Here, we use biochemistry, interaction mapping, mass spectrometry and cryoEM to study the role of ZNHIT2 in the regulation of the R2TP chaperone during the biogenesis of PRPF8. ZNHIT2 forms a complex with R2TP which depends exclusively on the direct interaction of ZNHIT2 with the RUVBL1-RUVBL2 ATPases. The cryoEM analysis of this complex reveals that ZNHIT2 alters the conformation and nucleotide state of RUVBL1-RUVBL2, affecting its ATPase activity. We characterized the interactions between R2TP, PRPF8, ZNHIT2, ECD and AAR2 proteins. Interestingly, PRPF8 makes a direct interaction with R2TP and this complex can incorporate ZNHIT2 and other proteins involved in the biogenesis of PRPF8 such as ECD and AAR2. Together, these results show that ZNHIT2 participates in the assembly of the U5 snRNP as part of a network of contacts between assembly factors required for PRPF8 biogenesis and the R2TP-HSP90 chaperone, while concomitantly regulating the structure and nucleotide state of R2TP.Agencia Estatal de Investigación (AEI/10.13039/501100011033), Ministerio de Ciencia e Innovación and co-funded by the European Regional Development Fund (ERDF-UE) [SAF2017-82632-P and PID2020-114429RB-I00 to O.L.]; Autonomous Region of Madrid and co-funded by the European Social Fund and the European Regional Development Fund [Y2018/BIO4747 and P2018/NMT4443 to O.L., and which support the contracts of S.C. and A.G-C.]; Funding for open access charge: Agencia Estatal de Investigación (AEI/10.13039/501100011033), Ministerio de Ciencia e Innovación, co-funded by the European Regional Development Fund (ERDF-UE) [SAF2017-82632-P to O.L.]; S.C. contract is funded by the CNIO Friends Program philanthropic initiative since June 2021.S

Claudin-3 Loss of Expression Is a Prognostic Marker in Castration-Resistant Prostate Cancer

Castration-resistant prostate cancer (CRPC) development is the foremost concern after treatment of patients with high risk with locally advanced or metastatic prostate cancer. Androgen receptor (AR) is the main driver of CRPC development, through its interaction with epigenetic modifier genes, placing epigenetics modifications in the forefront of CRPC development. Comparing the DNA methylation and expression profile of androgen-sensitive and -refractory prostate cancer cells, we describe the epigenetic silencing of claudin-3 (CLDN3) in AR positive cells resistant to androgen deprivation (LNCaP-abl). CLDN3 silencing was associated with DNA methylation, loss of histone acetylation and H3K27 methylation, and was re-expressed by the combined treatment with the epigenetic modulators Aza and SAHA. From a functional point of view, CLDN3 loss was associated with increased cellular invasion. Immunohistochemical analysis showed decreased CLDN3 expression in samples from CRPC patients. Interestingly, CLDN3 expression was significantly decreased in samples from patients with high total Gleason score (>= 8) and locally advanced tumors. Finally, CLDN3 loss of expression was associated with worse disease-free survival and time to clinical progression. In conclusion, our findings strongly indicate that epigenetic silencing of CLDN3 is a common event in CRPC that could be useful as a molecular marker for the prognosis of prostate cancer patients and to discriminate aggressive from indolent prostate tumors

Epigenetic Regulation of Gfi1 in Endocrine-Related Cancers: A Role Regulating Tumor Growth

Prostate and breast cancer constitute the most common cancers among men and women worldwide. The aging population is one of the main risk factors for prostate and breast cancer development and accumulating studies link aging with epigenetic changes. Growth factor independence-1 (Gfi1) is a transcriptional repressor with an important role in human malignancies, including leukemia, colorectal carcinoma, and lung cancer, but its role in prostate and breast cancer is unknown. We have found that Gfi1 epigenetic silencing is a common event in prostate and breast cancer. Gfi1 re-expression in prostate and breast cancer cell lines displaying Gfi1 epigenetic silencing decreases cell proliferation, reduced colony formation density, and tumor growth in nude mice xenografts. In addition, we found that Gfi1 repress alpha 1-anti-trypsin (AAT) and alpha 1-anti-chymotrypsin (ACT) expression, two genes with important functions in cancer development, suggesting that Gfi1 silencing promotes tumor growth by increasing AAT and ACT expression in our system. Finally, Gfi1 epigenetic silencing could be a promising biomarker for prostate cancer progression because it is associated with shorter disease-free survival. In conclusion, our findings strongly indicate that Gfi1 epigenetic silencing in prostate and breast cancer could be a crucial step in the development of these two-well characterized endocrine related tumors.Instituto de Salud Carlos II

Epigenetics in schizophrenia: a pilot study of global DNA methylation in different brain regions associated with higher cognitive functions

Attempts to discover genes that are involved in the pathogenesis of major psychiatric disorders have been frustrating and often fruitless. Concern is building about the need to understand the complex ways in which nature and nurture interact to produce mental illness. We analyze the epigenome in several brain regions from schizophrenic patients with severe cognitive impairment using high-resolution (450K) DNA methylation array. We identified 139 differentially methylated CpG sites included in known and novel candidate genes sequences as well as in and intergenic sequences which functions remain unknown. We found that altered DNA methylation is not restricted to a particular region, but includes others such as CpG shelves and gene bodies, indicating the presence of different DNA methylation signatures depending on the brain area analyzed. Our findings suggest that epimutations are not relatables between different tissues or even between tissues' regions, highlighting the need to adequately study brain samples to obtain reliable data concerning the epigenetics of schizophrenia

Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.Spanish Ministry of Science and Innovation SAF2017-82632-P Andres Lopez-Perrote Carlos F Rodriguez Marina Serna Oscar Llorca. Autonomous Government of Madrid Y2018/BIO4747 Ana Gonzalez-Corpas Oscar Llorca. Autonomous Government of Madrid P2018/NMT4443 Ana Gonzalez-Corpas Oscar Llorca MRC Core funding Javier F Caceres Spanish Ministry of Science and Innovation BES-2015-071348 Carlos F Rodriguez The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.S

Epigenetics in schizophrenia: a pilot study of global DNA methylation in different brain regions associated with higher cognitive functions

Attempts to discover genes that are involved in the pathogenesis of major psychiatric disorders have been frustrating and often fruitless. Concern is building about the need to understand the complex ways in which nature and nurture interact to produce mental illness. We analyze the epigenome in several brain regions from schizophrenic patients with severe cognitive impairment using high-resolution (450K) DNA methylation array. We identified 139 differentially methylated CpG sites included in known and novel candidate genes sequences as well as in and intergenic sequences which functions remain unknown. We found that altered DNA methylation is not restricted to a particular region, but includes others such as CpG shelves and gene bodies, indicating the presence of different DNA methylation signatures depending on the brain area analyzed. Our findings suggest that epimutations are not relatables between different tissues or even between tissues' regions, highlighting the need to adequately study brain samples to obtain reliable data concerning the epigenetics of schizophrenia

The Bacterial Mucosal Immunotherapy MV130 Protects Against SARS-CoV-2 Infection and Improves COVID-19 Vaccines Immunogenicity

COVID-19-specific vaccines are efficient prophylactic weapons against SARS-CoV-2 virus. However, boosting innate responses may represent an innovative way to immediately fight future emerging viral infections or boost vaccines. MV130 is a mucosal immunotherapy, based on a mixture of whole heat-inactivated bacteria, that has shown clinical efficacy against recurrent viral respiratory infections. Herein, we show that the prophylactic intranasal administration of this immunotherapy confers heterologous protection against SARS-CoV-2 infection in susceptible K18-hACE2 mice. Furthermore, in C57BL/6 mice, prophylactic administration of MV130 improves the immunogenicity of two different COVID-19 vaccine formulations targeting the SARS-CoV-2 spike (S) protein, inoculated either intramuscularly or intranasally. Independently of the vaccine candidate and vaccination route used, intranasal prophylaxis with MV130 boosted S-specific responses, including CD8+-T cell activation and the production of S-specific mucosal IgA antibodies. Therefore, the bacterial mucosal immunotherapy MV130 protects against SARS-CoV-2 infection and improves COVID-19 vaccines immunogenicity.CF was supported by AECC Foundation (INVES192DELF) and is currently funded by the Miguel Servet program (ID: CP20/00106) (ISCIII). IH-M receives the support of a fellowship from la Caixa Foundation (ID 100010434, fellowship code: LCF/BQ/IN17/11620074) and from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 713673. AJ-C is a postgraduate fellow of the City Council of Madrid at the Residencia de Estudiantes (2020–2021). GD is supported by an European Molecular Biology Organization (EMBO) Long-term fellowship (ALTF 379-2019). This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. Project number 892965. OL and JA-C acknowledge Comunidad de Madrid (Tec4Bio-CM, S2018/NMT-4443, FEDER). Work in OL laboratory was funded by CNIO with the support of the projects Y2018/BIO4747 and P2018/NMT4443 from Comunidad de Madrid and co-funded by the European Social Fund and the European Regional Development Fund. The CNIO is supported by the Instituto de Salud Carlos III (ISCIII). Work at CNB and CISA is funded by the Spanish Health Ministry, Instituto de Salud Carlos III (ISCIII), Fondo COVID-19 grant COV20/00151, and Fondo Supera COVID-19 (Crue Universidades-Banco Santander) (to JG-A). Work in the DS laboratory is funded by the CNIC; by the European Research Council (ERC-2016-Consolidator Grant 725091); by Agencia Estatal de Investigación (PID2019-108157RB); by Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); by Fondo Solidario Juntos (Banco Santander); by a research agreement with Inmunotek S.L.; and by Fundació La Marató de TV3 (201723). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MICINN, and the Pro CNIC Foundation.Peer reviewe

Papel de SHP-1 en la regulación de la expresión génica y su implicación en la progresión del cáncer de próstata

El cáncer de próstata se desarrolla por alteraciones genéticas y epigenéticas, que conducen a una desregulación de las vías de transducción de señales que controlan el comportamiento normal de las células de nuestro organismo. Por lo que conocer la interrelación entre las alteraciones epigenéticas y reguladores de la señalización celular en cáncer de próstata ayudará a encontrar nuevos biomarcadores, y a desarrollar terapias efectivas, uno de los mayores retos de la ciencia en nuestros días. SHP-1 es una tirosina fosfatasa que regula la actividad de vías de transducción de señales implicadas en procesos celulares fundamentales, y está presente en próstata humana normal y tumoral, pero se pierde su expresión en los tumores de próstata más agresivos e indiferenciados. Esto unido a la presencia de SHP-1 en el núcleo y su papel en el control del ciclo celular mediante cambios en la expresión de reguladores de este, nos hizo plantearnos el posible papel de SHP-1 en la regulación de la expresión mediante el control de las modificaciones epigenéticas. En este trabajo hemos encontrado que la anulación de la expresión de SHP-1, en células de cáncer de próstata, modifica la expresión de un grupo de genes inducido, al menos en parte, por cambios en la metilación de su región promotora. En concreto, se observa una correlación entre la pérdida de metilación del DNA de RunX1T1, Gstp1 y Kit con un aumento de su expresión; y un aumento de los niveles de metilación de CEBPD que coincide con una disminución de su expresión. Por otro lado, se estudiaron los mecanismos por los que SHP-1 podría regular la expresión, y más concretamente las posibles interacciones de esta fosfotirosina fosfatasa con proteínas responsables de las modificaciones epigenéticas. En este sentido, se identificó la interacción entre SHP-1 y JAK2, una quinasa que fosforila a la tirosina 41 de la histona H3 (H3Y41), una marca relacionada con promotores transcripcionalmente activos. También hemos demostrado por primera vez, que SHP-1 interacciona con HDAC2, y que esto influye en su actividad represora, ya que cuando se anula la expresión de SHP-1 se produce un aumento de los niveles de acetilación globales, aumentó la acetilación en los promotores de los genes cuya expresión estaba aumentada (Gstp1 y Kit). Por último, este trabajo demuestra que la anulación de SHP-1 en las células LNCaP promueve la progresión tumoral favoreciendo que las células se comporten de manera similar a las resistentes al tratamiento hormonal. Del mismo modo, también hemos demostrado que la ausencia de SHP-1 incrementa su capacidad migratoria e invasora en células de cáncer de próstata sensibles a andrógenos (LNCaP). En conclusión, hemos demostrado que SHP-1 está implicada en la regulación de la expresión génica mediante la metilación del DNA y la regulación de la acetilación de histonas. Además de demostrar que SHP-1 está implicada en la progresión tumora

DNA Methylation, Histone Modifications, and Signal Transduction Pathways: A Close Relationship in Malignant Gliomas Pathophysiology

Gliomas are the most common type of primary brain tumor. Although tremendous progress has been achieved in the recent years in the diagnosis and treatment, its molecular etiology remains unknown. In this regard, epigenetics represents a new approach to study the mechanisms that control gene expression and function without changing the sequence of the genome. In the present paper we describe the main findings about the alterations of cell signaling pathways in the most aggressive glioma in the adult population, namely, glioblastoma, in which epigenetic mechanisms and the emerging role of cancer stem cell play a crucial function in the development of new biomarkers for its detection and prognosis and the corresponding development of new pharmacological strategies