The paralogues MAGOH and MAGOHB are oncogenic factors in high-grade gliomas and safeguard the splicing of cell division and cell cycle genes.

The exon junction complex (EJC) plays key roles throughout the lifespan of RNA and is particularly relevant in the nervous system. We investigated the roles of two EJC members, the paralogs MAGOH and MAGOHB, with respect to brain tumour development. High MAGOH/MAGOHB expression was observed in 14 tumour types; glioblastoma (GBM) showed the greatest difference compared to normal tissue. Increased MAGOH/MAGOHB expression was associated with poor prognosis in glioma patients, while knockdown of MAGOH/MAGOHB affected different cancer phenotypes. Reduced MAGOH/MAGOHB expression in GBM cells caused alterations in the splicing profile, including re-splicing and skipping of multiple exons. The binding profiles of EJC proteins indicated that exons affected by MAGOH/MAGOHB knockdown accumulated fewer complexes on average, providing a possible explanation for their sensitivity to MAGOH/MAGOHB knockdown. Transcripts (genes) showing alterations in the splicing profile are mainly implicated in cell division, cell cycle, splicing, and translation. We propose that high MAGOH/MAGOHB levels are required to safeguard the splicing of genes in high demand in scenarios requiring increased cell proliferation (brain development and GBM growth), ensuring efficient cell division, cell cycle regulation, and gene expression (splicing and translation). Since differentiated neuronal cells do not require increased MAGOH/MAGOHB expression, targeting these paralogs is a potential option for treating GBM.

RNA processing as an alternative route to attack glioblastoma.

Genomic analyses have become an important tool to identify new avenues for therapy. This is especially true for cancer types with extremely poor outcomes, since our lack of effective therapies offers no tangible clinical starting point to build upon. The highly malignant brain tumor glioblastoma (GBM) exemplifies such a refractory cancer, with only 15 month average patient survival. Analyses of several hundred GBM samples compiled by the TCGA (The Cancer Genome Atlas) have produced an extensive transcriptomic map, identified prevalent chromosomal alterations, and defined important driver mutations. Unfortunately, clinical trials based on these results have not yet delivered an improvement on outcome. It is, therefore, necessary to characterize other regulatory routes known for playing a role in tumor relapse and response to treatment. Alternative splicing affects more than 90% of the human coding genes and it is an important source for transcript variation and gene regulation. Mutations and alterations in splicing factors are highly prevalent in multiple cancers, demonstrating the potential for splicing to act as a tumor driver. As a result, numerous genes are expressed as cancer-specific splicing isoforms that are functionally distinct from the canonical isoforms found in normal tissue. These include genes that regulate cancer-critical pathways such as apoptosis, DNA repair, cell proliferation, and migration. Splicing defects can even induce genomic instability, a common characteristic of cancer, and a driver of tumor evolution. Importantly, components of the splicing machinery are targetable; multiple drugs can inhibit splicing factors or promote changes in splicing which could be exploited to begin improving clinical outcomes. Here, we review the current literature and present a case for exploring RNA processing as therapeutic route for the treatment of GBM.

A Benchmark of In-House Homologous Recombination Repair Deficiency Testing Solutions for High-Grade Serous Ovarian Cancer Diagnosis.

Homologous recombination deficiency (HRD) has become an important prognostic and predictive biomarker for patients with high-grade serous ovarian cancer who may benefit from poly-ADP ribose polymerase inhibitors (PARPi) and platinum-based therapies. HRD testing provides relevant information to personalize patients' treatment options and has been progressively incorporated into diagnostic laboratories. Here, we assessed the performance of an in-house HRD testing system deployable in a diagnostic clinical setting, comparing results from two commercially available next-generation sequencing (NGS)-based tumor tests (SOPHiA DDMTM HRD Solution and AmoyDx® (HRD Focus Panel)) with the reference assay from Myriad MyChoice® (CDx). A total of 85 ovarian cancer samples were subject to HRD testing. An overall strong correlation was observed across the three assays evaluated, regardless of the different underlying methods employed to assess genomic instability, with the highest pairwise correlation between Myriad and SOPHiA (R = 0.87, p-value = 3.39 × 10-19). The comparison of the assigned HRD status to the reference Myriad's test revealed a positive predictive value (PPV) and negative predictive value (NPV) of 90.9% and 96.3% for SOPHiA's test, while AmoyDx's test achieved 75% PPV and 100% NPV. This is the largest HRD testing evaluation using different methodologies and provides a clear picture of the robustness of NGS-based tests currently offered in the market. Our data shows that the implementation of in-house HRD testing in diagnostic laboratories is technically feasible and can be reliably performed with commercial assays. Also, the turnaround time is compatible with clinical needs, making it an ideal alternative to offer to a broader number of patients while maintaining high-quality standards at more accessible price tiers.

The RNA-binding protein SERBP1 functions as a novel oncogenic factor in glioblastoma by bridging cancer metabolism and epigenetic regulation.

BACKGROUND: RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy. RESULTS: We identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites. CONCLUSIONS: SERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state.

SIRT1 regulates Mxd1 during malignant melanoma progression.

In a murine melanoma model, malignant transformation promoted by a sustained stress condition was causally related to increased levels of reactive oxygen species resulting in DNA damage and massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, γH2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. Mxd1 was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, Mxd1 was found downregulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the Mxd1 expression. Sirt1 stable silencing increased Mxd1 mRNA expression and led to down-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, whose upregulation is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. Mxd1 was identified as a new SIRT1 target gene. SIRT1 promoted Mxd1 silencing, which led to increased activity of MYC oncogene contributing to melanoma progression.

Epigenetic modulations in activated cells early after HIV-1 infection and their possible functional consequences.

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either, replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array, we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition, activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2, IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely, based on epigenetic markers studied here, non-stimulated cells infected by HIV-1, showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover, non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations, it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection.

Epigenetic reprogramming as a key contributor to melanocyte malignant transformation.

Melanoma progression requires deregulation of gene expression by currently uncharacterized epigenetic mechanisms. A mouse model based on changes in cell microenvironment was developed by our group to study melanocyte malignant transformation. Melanoma cell lines (4C11- and 4C11+) were obtained as result of 5 sequential anchorage blockades of non-tumorigenic melan-a melanocytes. Melan-a cells submitted to 4 de-adhesion cycles were also established (4C), are non-tumorigenic and represent an intermediary phase of tumor progression. The aim of this work was to identify factors contributing to epigenetic modifications in early and later phases of malignant transformation induced by anchorage impediment. Epigenetic alterations occur early in tumorigenesis; 4C cell line shows changes in global and gene-specific DNA methylation and histone marks. Many histone modifications differ between melan-a, 4C, 4C11- (non-metastatic melanoma cell line) and 4C11+ (metastatic melanoma cell line) which could be associated with changes in gene and microRNA expression. These epigenetic alterations seem to play a key role in malignant transformation since melanocytes treated with 5-Aza-2'-deoxycytidine before each anchorage blockade do not transform. Some epigenetic changes seem to be also responsible for the maintenance of malignant phenotype, since melanoma cell lines (4C11- and 4C11+) treated in vitro with 5-Aza-2'-deoxycytidine or Trichostatin A showed reduction of tumor growth in vivo. Changes in gene expression reflecting cell adaptation to new environment were also observed. We propose a model in which sustained microenvironmental stress in melanocytes results in epigenetic reprogramming. Thus, after adaptation, cells may acquire epigenetic marks that could contribute to the establishment of a malignant phenotype.