Dual targeting of histone methyltransferase G9a and DNMT1 for the treatment of experimental hepatocellular carcinoma

Las modificaciones epigenéticas, como la metilación del ADN e histonas, cooperan funcionalmente para fomentar el crecimiento tumoral, incluido el carcinoma hepatocelular (HCC). La inhibición farmacológica de estos mecanismos puede abrir nuevas vías terapéuticas. El objetivo del trabajo desarrollado en la presente tesis doctoral fue determinar la eficacia terapéutica y el potencial mecanismo de acción de nuevos inhibidores duales de la histona-metiltransferasa G9a y la ADN-metiltransferasa 1 (DNMT1) en células HCC humanas y su interferencia con células fibrogénicas. Estos inhibidores han sido desarrollados en la propia Institución y sujetos a patente (EPOPatent application #14-382230.2-1462). La expresión de G9a y DNMT1, junto con la de su adaptador molecular UHRF1, se midió en muestras de HCC humanas (n = 268), tejidos peritumorales (n = 154) y líneas células de HCC (n = 32). Se evaluó el efecto de la inhibición individual y combinada de G9a y DNMT1 en el crecimiento de células HCC mediante enfoques farmacológicos y genéticos. La actividad de nuestro compuesto principal, CM-272, se examinó en células HCC bajo normoxia e hipoxia, células estelares hepáticas humanas y células LX2, y tumores de xenoinjerto formados por células HCC o células HCC + LX2 combinadas. Encontramos una sobreexpresión significativa y correlativa de G9a, DNMT1 y UHRF1 en HCC en asociación con un mal pronóstico. La inhibición farmacológica independiente de G9a y DNMT1 redujo sinérgicamente el crecimiento de las células HCC. CM-272 inhibió potentemente la proliferación de células HCC y LX2, y detuvo el crecimiento del tumor en xenoinjertos de HCC y HCC combinadas con LX2. CM-272 impidió la adaptación metabólica de las células HCC a la hipoxia, e indujo un fenotipo diferenciado en las células HCC y fibrogénicas. La expresión del gen supresor de tumores metabólicos fructosa-1,6-bifosfatasa (FBP1), reprimido epigenéticamente en el HCC, fue restaurada por CM-272. Como conclusión del trabajo, la inhibición combinada de G9a / DNMT1 con compuestos como CM-272 es una estrategia prometedora para el tratamiento del HCC. Nuestros hallazgos también subrayan el potencial de la terapia de diferenciación en el HCC

Epigenetic mechanisms in hepatic stellate cell activation during liver fibrosis and carcinogenesis

Liver fibrosis is an essential component of chronic liver disease (CLD) and hepatocarcinogenesis. The fibrotic stroma is a consequence of sustained liver damage combined with exacerbated extracellular matrix (ECM) accumulation. In this context, activation of hepatic stellate cells (HSCs) plays a key role in both initiation and perpetuation of fibrogenesis. These cells suffer profound remodeling of gene expression in this process. This review is focused on the epigenetic alterations participating in the transdifferentiation of HSCs from the quiescent to activated state. Recent advances in the field of DNA methylation and post-translational modifications (PTM) of histones (acetylation and methylation) patterns are discussed here, together with altered expression and activity of epigenetic remodelers. We also consider recent advances in translational approaches, including the use of epigenetic marks as biomarkers and the promising antifibrotic properties of epigenetic drugs that are currently being used in patients

Splicing events in the control of genome integrity: role of SLU7 and truncated SRSF3 proteins

Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA–DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis

Splicing events in the control of genome integrity: role of SLU7 and truncated SRSF3 proteins

Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA–DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis