Enhanced tumor response to adoptive T cell therapy with PHD2/3-deficient CD8 T cells.

While adoptive cell therapy has shown success in hematological malignancies, its potential against solid tumors is hindered by an immunosuppressive tumor microenvironment (TME). In recent years, members of the hypoxia-inducible factor (HIF) family have gained recognition as important regulators of T-cell metabolism and function. The role of HIF signalling in activated CD8 T cell function in the context of adoptive cell transfer, however, has not been explored in full depth. Here we utilize CRISPR-Cas9 technology to delete prolyl hydroxylase domain-containing enzymes (PHD) 2 and 3, thereby stabilizing HIF-1 signalling, in CD8 T cells that have already undergone differentiation and activation, modelling the T cell phenotype utilized in clinical settings. We observe a significant boost in T-cell activation and effector functions following PHD2/3 deletion, which is dependent on HIF-1α, and is accompanied by an increased glycolytic flux. This improvement in CD8 T cell performance translates into an enhancement in tumor response to adoptive T cell therapy in mice, across various tumor models, even including those reported to be extremely resistant to immunotherapeutic interventions. These findings hold promise for advancing CD8 T-cell based therapies and overcoming the immune suppression barriers within challenging tumor microenvironments.

Spatial transcriptomics profiling of gallbladder adenocarcinoma: a detailed two-case study of progression from precursor lesions to cancer.

BACKGROUND: Most studies on tumour progression from precursor lesion toward gallbladder adenocarcinoma investigate lesions sampled from distinct patients, providing an overarching view of pathogenic cascades. Whether this reflects the tumourigenic process in individual patients remains insufficiently explored. Genomic and epigenomic studies suggest that a subset of gallbladder cancers originate from biliary intraepithelial neoplasia (BilIN) precursor lesions, whereas others form independently from BilINs. Spatial transcriptomic data supporting these conclusions are missing. Moreover, multiple areas with precursor or adenocarcinoma lesions can be detected within the same pathological sample. Yet, knowledge about intra-patient variability of such lesions is lacking. METHODS: To characterise the spatial transcriptomics of gallbladder cancer tumourigenesis in individual patients, we selected two patients with distinct cancer aetiology and whose samples simultaneously displayed multiple areas of normal epithelium, BilINs and adenocarcinoma. Using GeoMx digital spatial profiling, we characterised the whole transcriptome of a high number of regions of interest (ROIs) per sample in the two patients (24 and 32 ROIs respectively), with each ROI covering approximately 200 cells of normal epithelium, low-grade BilIN, high-grade BilIN or adenocarcinoma. Human gallbladder organoids and cell line-derived tumours were used to investigate the tumour-promoting role of genes. RESULTS: Spatial transcriptomics revealed that each type of lesion displayed limited intra-patient transcriptomic variability. Our data further suggest that adenocarcinoma derived from high-grade BilIN in one patient and from low-grade BilIN in the other patient, with co-existing high-grade BilIN evolving via a distinct process in the latter case. The two patients displayed distinct sequences of signalling pathway activation during tumour progression, but Semaphorin 4 A (SEMA4A) expression was repressed in both patients. Using human gallbladder-derived organoids and cell line-derived tumours, we provide evidence that repression of SEMA4A promotes pseudostratification of the epithelium and enhances cell migration and survival. CONCLUSION: Gallbladder adenocarcinoma can develop according to patient-specific processes, and limited intra-patient variability of precursor and cancer lesions was noticed. Our data suggest that repression of SEMA4A can promote tumour progression. They also highlight the need to gain gene expression data in addition to histological information to avoid understimating the risk of low-grade preneoplastic lesions.

DNA Hypomethylation Underlies Epigenetic Swapping between AGO1 and AGO1-V2 Isoforms in Tumors.

Human tumors progress in part by accumulating epigenetic alterations, which include gains and losses of DNA methylation in different parts of the cancer cell genome. Recent work has revealed a link between these two opposite alterations by showing that DNA hypomethylation in tumors can induce the expression of transcripts that overlap downstream gene promoters and thereby induce their hypermethylation. Preliminary in silico evidence prompted us to investigate if this mechanism applies to the locus harboring AGO1, a gene that plays a central role in miRNA biogenesis and RNA interference. Inspection of public RNA-Seq datasets and RT-qPCR experiments show that an alternative transcript starting 13.4 kb upstream of AGO1 (AGO1-V2) is expressed specifically in testicular germ cells, and becomes aberrantly activated in different types of tumors, particularly in tumors of the esophagus, stomach, and lung. This expression pattern classifies AGO1-V2 into the group of "Cancer-Germline" (CG) genes. Analysis of transcriptomic and methylomic datasets provided evidence that transcriptional activation of AGO1-V2 depends on DNA demethylation of its promoter region. Western blot experiments revealed that AGO1-V2 encodes a shortened isoform of AGO1, corresponding to a truncation of 75 aa in the N-terminal domain, and which we therefore referred to as "∆NAGO1". Interestingly, significant correlations between hypomethylation/activation of AGO1-V2 and hypermethylation/repression of AGO1 were observed upon examination of tumor cell lines and tissue datasets. Overall, our study reveals the existence of a process of interdependent epigenetic alterations in the AGO1 locus, which promotes swapping between two AGO1 protein-coding mRNA isoforms in tumors.

Two Broad Categories Overlapping With Rheumatoid Arthritis Observed in Synovial Biopsies from Patients With Juvenile Idiopathic Arthritis.

OBJECTIVE: The objective is to characterize transcriptomic profiles and immune cell composition and distribution in juvenile idiopathic arthritis (JIA) synovial biopsies, assess for associations of these features with clinical parameters, and compare JIA and rheumatoid arthritis (RA) synovial features. METHODS: RNA sequencing (RNASeq) was performed on 24 samples, with pathway analysis and inference of relative abundance of immune cell subsets based on gene expression data. Two multiplex fluorescence immunohistochemistry (IHC) panels were performed on 28 samples (including 13 on which RNASeq was performed), staining for CD206- classical and CD206+ nonclassical macrophages, and CD8+ and CD4+ T and B lymphocytes. Data were compared to a published series of early RA synovial biopsies. RESULTS: Pathway analysis of the most variably expressed genes (n = 339) identified a B and plasma cell signature as the main driver of heterogeneity in JIA synovia, with strong overlap between JIA and RA synovitis. Multiplex IHC confirmed heterogeneity of immune cell infiltration. M1-like macrophage-rich synovial lining was associated with greater lining hypertrophy and higher (CD45+) pan-immune cell and CD8+ T cell infiltration. CONCLUSION: Our study indicates significant similarities between JIA and RA synovitis. Similar to RA, JIA synovia may be broadly categorized into two groups: (1) those with an inflammatory/adaptive immune transcriptomic signature, M1-like macrophage and CD8+ T cell infiltration, and thicker, M1-like macrophage-rich synovial lining, and (2) those with an M2-like macrophage transcriptomic signature, greater M2/M1-like macrophage ratios, and thinner, M2-like macrophage-rich synovial lining. Synovial features were not significantly associated with clinical parameters, likely because of group size and heterogeneity.

Tumor Microenvironment Modifications Induced by Afatinib in Squamous Cell Carcinoma of the Head and Neck: A Window-of-Opportunity Study (EORTC-90111-24111).

PURPOSE: The EORTC-90111-24111 phase II window study evaluated afatinib versus no preoperative treatment in patients with primary squamous cell carcinoma of the head and neck (HNSCC). We investigated afatinib-induced tumor and microenvironment modifications by comparing pre- and posttreatment tumor biopsies. PATIENTS AND METHODS: Thirty treatment-naïve patients with primary HNSCC were randomized. Twenty-five patients received afatinib for 14 days before surgery (40 mg 1×/day) and 5 patients were attributed to the control arm. Biopsies were taken at work-up and during surgery. Good quality RNA samples were used for omics analyses. The control arm was enlarged by samples coming from our previous similar window study. RESULTS: IHC analyses of afatinib-treated tumor biopsies showed a decrease in pEGFR (P ≤ 0.05) and pERK (P ≤ 0.05); and an increase in CD3+ (P ≤ 0.01) and CD8+ (P ≤ 0.01) T-cell infiltration, and in CD3+ (P ≤ 0.05) T-cell density. RNA sequencing analyses of afatinib-treated tumor samples showed upregulation of inflammatory genes and increased expression scores of signatures predictive of response to programmed cell death protein 1 blockade (P ≤ 0.05). In posttreatment biopsies of afatinib-treated patients, two clusters were observed. Cluster 1 showed a higher expression of markers and gene sets implicated in epithelial-to-mesenchymal transition (EMT) and activation of cancer-associated fibroblasts (CAF) compared with cluster 2 and controls. CONCLUSIONS: Short-term treatment with afatinib in primary HNSCC induces CD3+ and CD8+ tumor infiltration and, in some patients, EMT and CAF activation. These results open perspectives to overcome resistance mechanisms to anti-HER therapy and to potentiate the activity of immune checkpoint inhibitors.

Specific HIF-2α (Hypoxia-Inducible Factor-2) Inhibitor PT2385 Mitigates Placental Dysfunction In Vitro and in a Rat Model of Preeclampsia (RUPP).

BACKGROUND: Preeclampsia is one of the leading causes of maternal mortality worldwide and is strongly associated with long-term morbidity in mothers and newborns. Referred to as one of the deep placentation disorders, insufficient remodeling of the spiral arteries during the first trimester remains a major cause of placental dysfunction. Persisting pulsatile uterine blood flow causes abnormal ischemia/reoxygenation phenomenon in the placenta and stabilizes the HIF-2α (hypoxia-inducible factor-2α) in the cytotrophoblasts. HIF-2α signaling impairs trophoblast differentiation and increases sFLT-1 (soluble fms-like tyrosine kinase-1) secretion, which reduces fetal growth and causes maternal symptoms. This study aims to evaluate the benefits of using PT2385-an oral specific HIF-2α inhibitor-to treat severe placental dysfunction. METHODS: To evaluate its therapeutic potential, PT2385 was first studied in primary human cytotrophoblasts isolated from term placenta and exposed to 2.5% O2 to stabilize HIF-2α. Viability and luciferase assays, RNA sequencing, and immunostaining were used to analyze differentiation and angiogenic factor balance. The ability of PT2385 to mitigate maternal manifestations of preeclampsia was studied in the selective reduced uterine perfusion pressure model performed in Sprague-Dawley rats. RESULTS: In vitro, RNA sequencing analysis and conventional techniques showed that treated cytotrophoblast displayed an enhanced differentiation into syncytiotrophoblasts and normalized angiogenic factor secretion compared with vehicle-treated cells. In the selective reduced uterine perfusion pressure model, PT2385 efficiently decreased sFLT-1 production, thus preventing the onset of hypertension and proteinuria in pregnant dams. CONCLUSIONS: These results highlight HIF-2α as a new player in our understanding of placental dysfunction and support the use of PT2385 to treat severe preeclampsia in humans.

BRAFV600E Expression in Thyrocytes Causes Recruitment of Immunosuppressive STABILIN-1 Macrophages.

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy, and tumor progression. Histological follow-up by anatomo-pathologists revealed that two-thirds of surgically-removed thyroids do not present malignant lesions. Thus, continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains central to better understanding the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E was specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E-dependent TC. Lastly, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages in a PTC mouse model and the interest to further study this macrophage subpopulation in human thyroid tissues.

BRAFV600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment.

Identification of Tissue-Specific Gene Clusters Induced by DNA Demethylation in Lung Adenocarcinoma: More Than Germline Genes.

Genome-wide loss of DNA methylation is commonly observed in human cancers, but its impact on the tumor transcriptome remains ill-defined. Previous studies demonstrated that this epigenetic alteration causes aberrant activation of a germline-specific gene expression program. Here, we examined if DNA hypomethylation in tumors also leads to de-repression of gene clusters with other tissue specificities. To this end, we explored transcriptomic and methylomic datasets from human lung adenocarcinoma (LUAD) cell lines, normal lung, and lung alveolar type II cells, considered as the origin of LUAD. Interestingly, DNA demethylation in LUAD cell lines was associated with activation of not only germline-specific (CG) genes, but also gene clusters displaying specific expression in the gastrointestinal tract (GI), or in stratified epithelia (SE). Consistently, genes from all three clusters showed highly specific patterns of promoter methylation among normal tissues and cell types, and were generally sensitive to induction by a DNA demethylating agent. Analysis of TCGA datasets confirmed that demethylation and activation of CG, GI and SE genes also occurs in vivo in LUAD tumor tissues, in association with global genome hypomethylation. For genes of the GI cluster, we demonstrated that HNF4A is a necessary factor for transcriptional activation following promoter demethylation. Interestingly, expression of several SE genes, in particular FAM83A, correlated with both tumor grade and reduced patient survival. Together, our study uncovers novel cell-type specific gene clusters that become aberrantly activated in LUAD tumors in association with genome-wide hypomethylation.

Quantitative modeling identifies critical cell mechanics driving bile duct lumen formation.

Biliary ducts collect bile from liver lobules, the smallest functional and anatomical units of liver, and carry it to the gallbladder. Disruptions in this process caused by defective embryonic development, or through ductal reaction in liver disease have a major impact on life quality and survival of patients. A deep understanding of the processes underlying bile duct lumen formation is crucial to identify intervention points to avoid or treat the appearance of defective bile ducts. Several hypotheses have been proposed to characterize the biophysical mechanisms driving initial bile duct lumen formation during embryogenesis. Here, guided by the quantification of morphological features and expression of genes in bile ducts from embryonic mouse liver, we sharpened these hypotheses and collected data to develop a high resolution individual cell-based computational model that enables to test alternative hypotheses in silico. This model permits realistic simulations of tissue and cell mechanics at sub-cellular scale. Our simulations suggest that successful bile duct lumen formation requires a simultaneous contribution of directed cell division of cholangiocytes, local osmotic effects generated by salt excretion in the lumen, and temporally-controlled differentiation of hepatoblasts to cholangiocytes, with apical constriction of cholangiocytes only moderately affecting luminal size.

AG-205 Upregulates Enzymes Involved in Cholesterol Biosynthesis and Steroidogenesis in Human Endometrial Cells Independently of PGRMC1 and Related MAPR Proteins.

An inappropriate response to progestogens in the human endometrium can result in fertility issues and jeopardize progestin-based treatments against pathologies such as endometriosis. PGRMC1 can mediate progesterone response in the breast and ovaries but its endometrial functions remain unknown. AG-205 is an alleged PGRMC1 inhibitor but its specificity was recently questioned. We added AG-205 in the cultures of two endometrial cell lines and performed a transcriptomic comparison. AG-205 significantly increased expression of genes coding enzymes of the cholesterol biosynthetic pathway or of steroidogenesis. However, these observations were not reproduced with cells transfected with siRNA against PGRMC1 or its related proteins (MAPRs). Furthermore, AG-205 retained its ability to increase expression of selected target genes even when expression of PGRMC1 or all MAPRs was concomitantly downregulated, indicating that neither PGRMC1 nor any MAPR is required to mediate AG-205 effect. In conclusion, although AG-205 has attractive effects encouraging its use to develop therapeutic strategies, for instance against breast cancer, our study delivers two important warning messages. First, AG-205 is not specific for PGRMC1 or other MAPRs and its mechanisms of action remain unclear. Second, due to its effects on genes involved in steroidogenesis, its use may increase the risk for endometrial pathologies resulting from imbalanced hormones concentrations.

Transcriptional overlap links DNA hypomethylation with DNA hypermethylation at adjacent promoters in cancer.

Tumor development involves alterations in DNA methylation patterns, which include both gains (hypermethylation) and losses (hypomethylation) in different genomic regions. The mechanisms underlying these two opposite, yet co-existing, alterations in tumors remain unclear. While studying the human MAGEA6/GABRA3 gene locus, we observed that DNA hypomethylation in tumor cells can lead to the activation of a long transcript (CT-GABRA3) that overlaps downstream promoters (GABRQ and GABRA3) and triggers their hypermethylation. Overlapped promoters displayed increases in H3K36me3, a histone mark deposited during transcriptional elongation and known to stimulate de novo DNA methylation. Consistent with such a processive mechanism, increases in H3K36me3 and DNA methylation were observed over the entire region covered by the CT-GABRA3 overlapping transcript. Importantly, experimental induction of CT-GABRA3 by depletion of DNMT1 DNA methyltransferase, resulted in a similar pattern of regional DNA hypermethylation. Bioinformatics analyses in lung cancer datasets identified other genomic loci displaying this process of coupled DNA hypo/hypermethylation, and some of these included tumor suppressor genes, e.g. RERG and PTPRO. Together, our work reveals that focal DNA hypomethylation in tumors can indirectly contribute to hypermethylation of nearby promoters through activation of overlapping transcription, and establishes therefore an unsuspected connection between these two opposite epigenetic alterations.

Therapy-induced DNA methylation inactivates MCT1 and renders tumor cells vulnerable to MCT4 inhibition.

Metabolic plasticity in cancer cells makes use of metabolism-targeting agents very challenging. Drug-induced metabolic rewiring may, however, uncover vulnerabilities that can be exploited. We report that resistance to glycolysis inhibitor 3-bromopyruvate (3-BrPA) arises from DNA methylation in treated cancer cells and subsequent silencing of the monocarboxylate transporter MCT1. We observe that, unexpectedly, 3-BrPA-resistant cancer cells mostly rely on glycolysis to sustain their growth, with MCT4 as an essential player to support lactate flux. This shift makes cancer cells particularly suited to adapt to hypoxic conditions and resist OXPHOS inhibitors and anti-proliferative chemotherapy. In contrast, blockade of MCT4 activity in 3-BrPA-exposed cancer cells with diclofenac or genetic knockout, inhibits growth of derived spheroids and tumors in mice. This study supports a potential mode of collateral lethality according to which metabolic adaptation of tumor cells to a first-line therapy makes them more responsive to a second-line treatment.

A Mouse Model of Cholangiocarcinoma Uncovers a Role for Tensin-4 in Tumor Progression.

BACKGROUND AND AIMS: Earlier diagnosis and treatment of intrahepatic cholangiocarcinoma (iCCA) are necessary to improve therapy, yet limited information is available about initiation and evolution of iCCA precursor lesions. Therefore, there is a need to identify mechanisms driving formation of precancerous lesions and their progression toward invasive tumors using experimental models that faithfully recapitulate human tumorigenesis. APPROACH AND RESULTS: To this end, we generated a mouse model which combines cholangiocyte-specific expression of KrasG12D with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced inflammation to mimic iCCA development in patients with cholangitis. Histological and transcriptomic analyses of the mouse precursor lesions and iCCA were performed and compared with human analyses. The function of genes overexpressed during tumorigenesis was investigated in human cell lines. We found that mice expressing KrasG12D in cholangiocytes and fed a DDC diet developed cholangitis, ductular proliferations, intraductal papillary neoplasms of bile ducts (IPNBs), and, eventually, iCCAs. The histology of mouse and human IPNBs was similar, and mouse iCCAs displayed histological characteristics of human mucin-producing, large-duct-type iCCA. Signaling pathways activated in human iCCA were also activated in mice. The identification of transition zones between IPNB and iCCA on tissue sections, combined with RNA-sequencing analyses of the lesions supported that iCCAs derive from IPNBs. We further provide evidence that tensin-4 (TNS4), which is stimulated by KRASG12D and SRY-related HMG box transcription factor 17, promotes tumor progression. CONCLUSIONS: We developed a mouse model that faithfully recapitulates human iCCA tumorigenesis and identified a gene cascade which involves TNS4 and promotes tumor progression.

Dynamic Regulation of Expression of KRAS and Its Effectors Determines the Ability to Initiate Tumorigenesis in Pancreatic Acinar Cells.

Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations. SIGNIFICANCE: This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.

PAR-TERRA is the main contributor to telomeric repeat-containing RNA transcripts in normal and cancer mouse cells.

Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested to account for the vast majority of TERRA in embryonic stem cells (ESC). Here, we confirm the production of TERRA from both the chromosome 18q telomere and the PAR locus in mouse embryonic fibroblasts, ESC, and various mouse cancer and immortalized cell lines, and we identify two novel sources of TERRA on mouse chromosome 2 and X. Using various approaches, we show that PAR-TERRA molecules account for the majority of TERRA transcripts, displaying an increase of two to four orders of magnitude compared to the telomeric 18q transcript. Finally, we present a SILAC-based pull-down screen revealing a large overlap between TERRA-interacting proteins in human and mouse cells, including PRC2 complex subunits, chromatin remodeling factors, DNA replication proteins, Aurora kinases, shelterin complex subunits, Bloom helicase, Coilin, and paraspeckle proteins. Hence, despite originating from distinct genomic regions, mouse and human TERRA are likely to play similar functions in cells.

Epigenetic Coactivation of MAGEA6 and CT-GABRA3 Defines Orientation of a Segmental Duplication in the Human X Chromosome.

The human genome harbors many duplicated segments, which sometimes show very high sequence identity. This may complicate assignment during genome assembly. One such example is in Xq28, where the arrangement of 2 recently duplicated segments varies between genome assembly versions. The duplicated segments comprise highly similar genes, including MAGEA3 and MAGEA6, which display specific expression in testicular germline cells, and also become aberrantly activated in a variety of tumors. Recently, a new gene was identified, CT-GABRA3, the transcription of which initiates inside the segmental duplication but extends far outside. According to the latest genome annotation, CT- GABRA3 starts near MAGEA3, with which it shares a bidirectional promoter. In an earlier annotation, however, the duplicated segment was positioned in the opposite orientation, and CT-GABRA3 was instead coupled with MAGEA6. To resolve this discrepancy, and based on the contention that genes connected by a bidirectional promoter are almost always co-expressed, we decided to compare the expression profiles of CT-GABRA3, MAGEA3, and MAGEA6. We found that in tumor tissues and cell lines of different origins, the expression of CT-GABRA3 was better correlated with that of MAGEA6. Moreover, in a cellular model of experimental induction with a DNA demethylation agent, activation CT-GABRA3 was associated with that of MAGEA6, but not with that of MAGEA3. Together these results support a connection between CT-GABRA3 and MAGEA6 and illustrate how promoter-sharing genes can be exploited to resolve genome assembly uncertainties.

Dynamics and predicted drug response of a gene network linking dedifferentiation with beta-catenin dysfunction in hepatocellular carcinoma.

BACKGROUND & AIMS: Alterations of individual genes variably affect the development of hepatocellular carcinoma (HCC). Thus, we aimed to characterize the function of tumor-promoting genes in the context of gene regulatory networks (GRNs). METHODS: Using data from The Cancer Genome Atlas, from the LIRI-JP (Liver Cancer - RIKEN, JP project), and from our transcriptomic, transfection and mouse transgenic experiments, we identify a GRN which functionally links LIN28B-dependent dedifferentiation with dysfunction of ß-catenin (CTNNB1). We further generated and validated a quantitative mathematical model of the GRN using human cell lines and in vivo expression data. RESULTS: We found that LIN28B and CTNNB1 form a GRN with SMARCA4, Let-7b (MIRLET7B), SOX9, TP53 and MYC. GRN functionality is detected in HCC and gastrointestinal cancers, but not in other cancer types. GRN status negatively correlates with HCC prognosis, and positively correlates with hyperproliferation, dedifferentiation and HGF/MET pathway activation, suggesting that it contributes to a transcriptomic profile typical of the proliferative class of HCC. The mathematical model predicts how the expression of GRN components changes when the expression of another GRN member varies or is inhibited by a pharmacological drug. The dynamics of GRN component expression reveal distinct cell states that can switch reversibly in normal conditions, and irreversibly in HCC. The mathematical model is available via a web-based tool which can evaluate the GRN status of HCC samples and predict the impact of therapeutic agents on the GRN. CONCLUSIONS: We conclude that identification and modelling of the GRN provide insights into the prognosis of HCC and the mechanisms by which tumor-promoting genes impact on HCC development. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a heterogeneous disease driven by the concomitant deregulation of several genes functionally organized as networks. Here, we identified a gene regulatory network involved in a subset of HCCs. This subset is characterized by increased proliferation and poor prognosis. We developed a mathematical model which uncovers the dynamics of the network and allows us to predict the impact of a therapeutic agent, not only on its specific target but on all the genes belonging to the network.

Oncogenic roles of DNA hypomethylation through the activation of cancer-germline genes.

Global loss of DNA methylation is frequently observed in the genome of human tumors. Although this epigenetic alteration is clearly associated with cancer progression, the way it exerts its pro-tumoral effect remains incompletely understood. A remarkable consequence of DNA hypomethylation in tumors is the aberrant activation of "cancer-germline" genes (also known as "cancer-testis" genes), which comprise a diverse group of germline-specific genes that use DNA methylation as a primary mechanism for repression in normal somatic tissues. Here we review the evidence that such cancer-germline genes contribute to key processes of tumor development. Notably, several cancer-germline genes were found to stimulate oncogenic pathways involved in cell proliferation (SSX, DDX43, MAEL, PIWIL1), angiogenesis (DDX53), immortality (BORIS/CTCFL), and metastasis (CT-GABRA3). Others appear to inhibit tumor suppressor pathways, including those controlling growth inhibition signals (MAGEA11, MAGEB2), apoptosis (MAGEA2, MAGEC2), and genome integrity (HORMAD1, NXF2). Cancer-germline genes were also implicated in the regulation of tumor metabolism (MAGEA3/MAGEA6). Together, our survey substantiates the concept that DNA hypomethylation promotes tumorigenesis via transcriptional activation of oncogenes. Importantly, considering their highly restricted pattern of expression, cancer-germline genes may represent valuable targets for the development of anti-cancer therapies with limited side effects.

A gene expression signature identifying transient DNMT1 depletion as a causal factor of cancer-germline gene activation in melanoma.

BACKGROUND: Many human tumors show aberrant activation of a group of germline-specific genes, termed cancer-germline (CG) genes, several of which appear to exert oncogenic functions. Although activation of CG genes in tumors has been linked to promoter DNA demethylation, the mechanisms underlying this epigenetic alteration remain unclear. Two main processes have been proposed: awaking of a gametogenic program directing demethylation of target DNA sequences via specific regulators, or general deficiency of DNA methylation activities resulting from mis-targeting or down-regulation of the DNMT1 methyltransferase. RESULTS: By the analysis of transcriptomic data, we searched to identify gene expression changes associated with CG gene activation in melanoma cells. We found no evidence linking CG gene activation with differential expression of gametogenic regulators. Instead, CG gene activation correlated with decreased expression of a set of mitosis/division-related genes (ICCG genes). Interestingly, a similar gene expression signature was previously associated with depletion of DNMT1. Consistently, analysis of a large set of melanoma tissues revealed that DNMT1 expression levels were often lower in samples showing activation of multiple CG genes. Moreover, by using immortalized melanocytes and fibroblasts carrying an inducible anti-DNMT1 small hairpin RNA (shRNA), we demonstrate that transient depletion of DNMT1 can lead to long-term activation of CG genes and repression of ICCG genes at the same time. For one of the ICCG genes (CDCA7L), we found that its down-regulation in melanoma cells was associated with deposition of repressive chromatin marks, including H3K27me3. CONCLUSIONS: Together, our observations point towards transient DNMT1 depletion as a causal factor of CG gene activation in vivo in melanoma.