Mutational analysis of non-coding genome in prostate cancer using whole genome sequencing

Prostate Cancer is a lethal disease characterized as progressive and possessing distinct molecular heterogeneity during its timespan. Vast number of abnormalities has been reported till now with number of somatic mutations and germline risk factors in addition to rearrangements in the chromatin. All of them together makes the architecture of prostate genome very complex and hard to understand. Transcription machinery is central to gene regulation, with a considerable vacuum in information till date related to abnormalities in the regulatory regions in prostate genome we have tried to explore the non-coding genome and detect mutations in regulatory regions to find if they hold any significance in prostate cancer. Using prostate cancer sample data from the whole genome sequencing study by Berger et al., 2011 which were seven matched normal-tumor genomes, encouraging results were produced by applying a pipeline of independently selected tools for variant analysis. DNase I hypersensitivity sites (DNaseI HSs) for LNCaP cell lines obtained from The Encyclopedia of DNA Elements (ENCODE) Consortium were used as markers for regulatory region in the genome and from initial 54679 SNVs detected across 7 samples, 21 promoter and 621 enhancer mutations were detected overlapping the DNaseI HS peaks. Out of which 4 and 21 mutations in promoters and enhancers respectively were the finally filtered out whose genes where found relevant directly or indirectly in prostate and other cancers by performing extensive search in the published literature. For concrete evidence validation would be the next step as results were mere implications, but our study is a diligent effort in delineating the intricate genomics involved in prostate cancer outside the gene coding regions

The basic helix-loop-helix transcription factor TCF4 impacts brain architecture as well as neuronal morphology and differentiation

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt–Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS

Mutational analysis of non-coding genome in prostate cancer using whole genome sequencing

Prostate Cancer is a lethal disease characterized as progressive and possessing distinct molecular heterogeneity during its timespan. Vast number of abnormalities has been reported till now with number of somatic mutations and germline risk factors in addition to rearrangements in the chromatin. All of them together makes the architecture of prostate genome very complex and hard to understand. Transcription machinery is central to gene regulation, with a considerable vacuum in information till date related to abnormalities in the regulatory regions in prostate genome we have tried to explore the non-coding genome and detect mutations in regulatory regions to find if they hold any significance in prostate cancer. Using prostate cancer sample data from the whole genome sequencing study by Berger et al., 2011 which were seven matched normal-tumor genomes, encouraging results were produced by applying a pipeline of independently selected tools for variant analysis. DNase I hypersensitivity sites (DNaseI HSs) for LNCaP cell lines obtained from The Encyclopedia of DNA Elements (ENCODE) Consortium were used as markers for regulatory region in the genome and from initial 54679 SNVs detected across 7 samples, 21 promoter and 621 enhancer mutations were detected overlapping the DNaseI HS peaks. Out of which 4 and 21 mutations in promoters and enhancers respectively were the finally filtered out whose genes where found relevant directly or indirectly in prostate and other cancers by performing extensive search in the published literature. For concrete evidence validation would be the next step as results were mere implications, but our study is a diligent effort in delineating the intricate genomics involved in prostate cancer outside the gene coding regions

Gazette de Bayonne, de Biarritz et du Pays basque

04 septembre 19241924/09/04.Appartient à l’ensemble documentaire : Aquit

Cancer-Associated Fibroblasts Exert Proangiogenic Activity in Merkel Cell Carcinoma

: The tumor microenvironment is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although tumor microenvironment is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. In this study, we provide a molecular and functional characterization of cancer-associated fibroblasts (CAFs), the major tumor microenvironment component, in patient-derived xenografts of patients with MCC. We show that subcutaneous coinjection of patient-derived CAFs and human MCC MKL-1 cells into severe combined immunodeficient mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the aminopeptidase A/angiotensin II and III/angiotensin II type 1 receptor axis, with the expression of aminopeptidase A in CAFs being a triggering event. Together, our findings point to aminopeptidase A as a potential marker for MCC prognostic stratification and as a candidate for therapeutic intervention

Transcriptional behavior of the HIV-1 promoter in context of the BACH2 prominent proviral integration gene

Chronic infection with human immunodeficiency virus (HIV)-1 is characterized by accumulation of proviral sequences in the genome of target cells. Integration of viral DNA in patients on long-term antiretroviral therapy selectively persists at preferential loci, suggesting site-specific crosstalk of viral sequences and human genes. This crosstalk likely contributes to chronic HIV disease through modulation of host immune pathways and emergence of clonal infected cell populations. To systematically interrogate such effects, we undertook genome engineering to generate Jurkat cell models that replicate integration of HIV-1 long terminal repeat (LTR) sequences at the BTB and CNC Homolog 2 (BACH2) integration locus. This locus is a prominent HIV-1 integration gene in chronic infection, found in 30 % of long-term treated patients with mapped proviral integrations. Using five clonal models carrying an LTR-driven reporter at different BACH2 intergenic regions, we here show that LTR transcriptional activity is repressed in BACH2 regions associated with proviral-DNA integrations in vivo but not in a control region. Our data indicates that this repression is in part epigenetically regulated, particularly through DNA methylation. Importantly, we demonstrate that transcriptional activity of the LTR is independent of BACH2 gene transcription and vice versa in our models. This suggests no transcriptional interference of endogenous and HIV-1 promoters. Taken together, our study provides first insights into how activity of HIV-1 LTR sequences is regulated at the BACH2 locus as prominent example for a recurrently-detected integration gene in chronic infection. Given the importance of integration-site dependent virus/host crosstalk for chronic HIV disease, our findings for the BACH2 locus have potential implications for future therapeutic strategies.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Epstein Barr virus-mediated transformation of B cells from XIAP-deficient patients leads to increased expression of the tumor suppressor CADM1

X-linked lymphoproliferative disease (XLP) is either caused by loss of the SLAM-associated protein (SAP; XLP-1) or the X-linked inhibitor of apoptosis (XIAP; XLP-2). In both instances, infection with the oncogenic human Epstein Barr virus (EBV) leads to pathology, but EBV-associated lymphomas only emerge in XLP-1 patients. Therefore, we investigated the role of XIAP during B cell transformation by EBV. Using humanized mice, IAP inhibition in EBV-infected mice led to a loss of B cells and a tendency to lower viral titers and lymphomagenesis. Loss of memory B cells was also observed in four newly described patients with XIAP deficiency. EBV was able to transform their B cells into lymphoblastoid cell lines (LCLs) with similar growth characteristics to patient mothers' LCLs in vitro and in vivo. Gene expression analysis revealed modest elevated lytic EBV gene transcription as well as the expression of the tumor suppressor cell adhesion molecule 1 (CADM1). CADM1 expression on EBV-infected B cells might therefore inhibit EBV-associated lymphomagenesis in patients and result in the absence of EBV-associated malignancies in XLP-2 patients

ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies

Host KIR/HLA-C Genotypes Determine HIV-Mediated Changes of the NK Cell Repertoire and Are Associated With Vpu Sequence Variations Impacting Downmodulation of HLA-C

NK cells play a pivotal role in viral immunity, utilizing a large array of activating and inhibitory receptors to identify and eliminate virus-infected cells. Killer-cell immunoglobulin-like receptors (KIRs) represent a highly polymorphic receptor family, regulating NK cell activity and determining the ability to recognize target cells. Human leukocyte antigen (HLA) class I molecules serve as the primary ligand for KIRs. Herein, HLA-C stands out as being the dominant ligand for the majority of KIRs. Accumulating evidence indicated that interactions between HLA-C and its inhibitory KIR2DL receptors (KIR2DL1/L2/L3) can drive HIV-1-mediated immune evasion and thus may contribute to the intrinsic control of HIV-1 infection. Of particular interest in this context is the recent observation that HIV-1 is able to adapt to host HLA-C genotypes through Vpu-mediated downmodulation of HLA-C. However, our understanding of the complex interplay between KIR/HLA immunogenetics, NK cell-mediated immune pressure and HIV-1 immune escape is still limited. Therefore, we investigated the impact of specific KIR/HLA-C combinations on the NK cell receptor repertoire and HIV-1 Vpu protein sequence variations of 122 viremic, untreated HIV-1(+) individuals. Compared to 60 HIV-1(-) controls, HIV-1 infection was associated with significant changes within the NK cell receptor repertoire, including reduced percentages of NK cells expressing NKG2A, CD8, and KIR2DS4. In contrast, the NKG2C(+) and KIR3DL2(+) NK cell sub-populations from HIV-1(+) individuals was enlarged compared to HIV-1(-) controls. Stratification along KIR/HLA-C genotypes revealed a genotype-dependent expansion of KIR2DL1(+) NK cells that was ultimately associated with increased binding affinities between KIR2DL1 and HLA-C allotypes. Lastly, our data hinted to a preferential selection of Vpu sequence variants that were associated with HLA-C downmodulation in individuals with high KIR2DL/HLA-C binding affinities. Altogether, our study provides evidence that HIV-1-associated changes in the KIR repertoire of NK cells are to some extent predetermined by host KIR2DL/HLA-C genotypes. Furthermore, analysis of Vpu sequence polymorphisms indicates that differential KIR2DL/HLA-C binding affinities may serve as an additional mechanism how host genetics impact immune evasion by HIV-1