Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma.

LINE-1 (L1) retrotransposons are mobile genetic elements comprising ~17% of the human genome. New L1 insertions can profoundly alter gene function and cause disease, though their significance in cancer remains unclear. Here, we applied enhanced retrotransposon capture sequencing (RC-seq) to 19 hepatocellular carcinoma (HCC) genomes and elucidated two archetypal L1-mediated mechanisms enabling tumorigenesis. In the first example, 4/19 (21.1%) donors presented germline retrotransposition events in the tumor suppressor mutated in colorectal cancers (MCC). MCC expression was ablated in each case, enabling oncogenic ß-catenin/Wnt signaling. In the second example, suppression of tumorigenicity 18 (ST18) was activated by a tumor-specific L1 insertion. Experimental assays confirmed that the L1 interrupted a negative feedback loop by blocking ST18 repression of its enhancer. ST18 was also frequently amplified in HCC nodules from Mdr2(-/-) mice, supporting its assignment as a candidate liver oncogene. These proof-of-principle results substantiate L1-mediated retrotransposition as an important etiological factor in HCC.

Apoptosis in mesenchymal stromal cells activates an immunosuppressive secretome predicting clinical response in Crohn's disease.

In vivo apoptosis of human mesenchymal stromal cells (MSCs) plays a critical role in delivering immunomodulation. Yet, caspase activity not only mediates the dying process but also death-independent functions that may shape the immunogenicity of apoptotic cells. Therefore, a better characterization of the immunological profile of apoptotic MSCs (ApoMSCs) could shed light on their mechanistic action and therapeutic applications. We analyzed the transcriptomes of MSCs undergoing apoptosis and identified several immunomodulatory factors and chemokines dependent on caspase activation following Fas stimulation. The ApoMSC secretome inhibited human T cell proliferation and activation, and chemoattracted monocytes in vitro. Both immunomodulatory activities were dependent on the cyclooxygenase2 (COX2)/prostaglandin E2 (PGE2) axis. To assess the clinical relevance of ApoMSC signature, we used the peripheral blood mononuclear cells (PBMCs) from a cohort of fistulizing Crohn's disease (CD) patients who had undergone MSC treatment (ADMIRE-CD). Compared with healthy donors, MSCs exposed to patients' PBMCs underwent apoptosis and released PGE2 in a caspase-dependent manner. Both PGE2 and apoptosis were significantly associated with clinical responses to MSCs. Our findings identify a new mechanism whereby caspase activation delivers ApoMSC immunosuppression. Remarkably, such molecular signatures could implicate translational tools for predicting patients' clinical responses to MSC therapy in CD.

Immunogenomics of Colorectal Cancer Response to Checkpoint Blockade: Analysis of the KEYNOTE 177 Trial and Validation Cohorts.

BACKGROUND & AIMS: Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy. METHODS: We selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME. RESULTS: In hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages. CONCLUSIONS: Our study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.

O-linked mucin-type glycosylation regulates the transcriptional programme downstream of EGFR.

Aberrant mucin-type O-linked glycosylation is a common occurrence in cancer where the upregulation of sialyltransferases is often seen leading to the early termination of O-glycan chains. Mucin-type O-linked glycosylation is not limited to mucins and occurs on many cell surface glycoproteins including EGFR, where the number of sites can be limited. Upon EGF ligation, EGFR induces a signaling cascade and may also translocate to the nucleus where it directly regulates gene transcription, a process modulated by Galectin-3 and MUC1 in some cancers. Here, we show that upon EGF binding, breast cancer cells carrying different O-glycans respond by transcribing different gene expression signatures. MMP10, the principal gene upregulated when cells carrying sialylated core 1 glycans were stimulated with EGF, is also upregulated in ER-positive breast carcinoma reported to express high levels of ST3Gal1 and hence mainly core 1 sialylated O-glycans. In contrast, isogenic cells engineered to carry core 2 glycans upregulate CX3CL1 and FGFBP1 and these genes are upregulated in ER-negative breast carcinomas, also known to express longer core 2 O-glycans. Changes in O-glycosylation did not significantly alter signal transduction downstream of EGFR in core 1 or core 2 O-glycan expressing cells. However, striking changes were observed in the formation of an EGFR/galectin-3/MUC1/ß-catenin complex at the cell surface that is present in cells carrying short core 1-based O-glycans but absent in core 2 carrying cells.

Molecular Biology and Evolution of Cancer: From Discovery to Action.

Cancer progression is an evolutionary process. During this process, evolving cancer cell populations encounter restrictive ecological niches within the body, such as the primary tumor, circulatory system, and diverse metastatic sites. Efforts to prevent or delay cancer evolution-and progression-require a deep understanding of the underlying molecular evolutionary processes. Herein we discuss a suite of concepts and tools from evolutionary and ecological theory that can inform cancer biology in new and meaningful ways. We also highlight current challenges to applying these concepts, and propose ways in which incorporating these concepts could identify new therapeutic modes and vulnerabilities in cancer.

MEGA-V: detection of variant gene sets in patient cohorts.

Summary: : Detecting significant associations between genetic variants and disease may prove particularly challenging when the variants are rare in the population and/or act together with other variants to cause the disease. We have developed a statistical framework named Mutation Enrichment Gene set Analysis of Variants (MEGA-V) that specifically detects the enrichments of genetic alterations within a process in a cohort of interest. By focusing on the mutations of several genes contributing to the same function rather than on those affecting a single gene, MEGA-V increases the power to detect statistically significant associations. Availability and Implementation: MEGA-V is available at https://github.com/ciccalab/MEGA. Contact: francesca.ciccarelli@kcl.ac.uk. Supplementary information: Supplementary data are available at Bioinformatics online.

NCG 5.0: updates of a manually curated repository of cancer genes and associated properties from cancer mutational screenings.

The Network of Cancer Genes (NCG, http://ncg.kcl.ac.uk/) is a manually curated repository of cancer genes derived from the scientific literature. Due to the increasing amount of cancer genomic data, we have introduced a more robust procedure to extract cancer genes from published cancer mutational screenings and two curators independently reviewed each publication. NCG release 5.0 (August 2015) collects 1571 cancer genes from 175 published studies that describe 188 mutational screenings of 13 315 cancer samples from 49 cancer types and 24 primary sites. In addition to collecting cancer genes, NCG also provides information on the experimental validation that supports the role of these genes in cancer and annotates their properties (duplicability, evolutionary origin, expression profile, function and interactions with proteins and miRNAs).

Network of Cancer Genes (NCG 3.0): integration and analysis of genetic and network properties of cancer genes.

The identification of a constantly increasing number of genes whose mutations are causally implicated in tumor initiation and progression (cancer genes) requires the development of tools to store and analyze them. The Network of Cancer Genes (NCG 3.0) collects information on 1494 cancer genes that have been found mutated in 16 different cancer types. These genes were collected from the Cancer Gene Census as well as from 18 whole exome and 11 whole-genome screenings of cancer samples. For each cancer gene, NCG 3.0 provides a summary of the gene features and the cross-reference to other databases. In addition, it describes duplicability, evolutionary origin, orthology, network properties, interaction partners, microRNA regulation and functional roles of cancer genes and of all genes that are related to them. This integrated network of information can be used to better characterize cancer genes in the context of the system in which they act. The data can also be used to identify novel candidates that share the same properties of known cancer genes and may therefore play a similar role in cancer. NCG 3.0 is freely available at http://bio.ifom-ieo-campus.it/ncg.

Double-negative B cells and DNASE1L3 colocalise with microbiota in gut-associated lymphoid tissue.

Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.

Ultradeep sequencing of a human ultraconserved region reveals somatic and constitutional genomic instability.

Early detection of cancer-associated genomic instability is crucial, particularly in tumour types in which this instability represents the essential underlying mechanism of tumourigenesis. Currently used methods require the presence of already established neoplastic cells because they only detect clonal mutations. In principle, parallel sequencing of single DNA filaments could reveal the early phases of tumour initiation by detecting low-frequency mutations, provided an adequate depth of coverage and an effective control of the experimental error. We applied ultradeep sequencing to estimate the genomic instability of individuals with hereditary non-polyposis colorectal cancer (HNPCC). To overcome the experimental error, we used an ultraconserved region (UCR) of the human genome as an internal control. By comparing the mutability outside and inside the UCR, we observed a tendency of the ultraconserved element to accumulate significantly fewer mutations than the flanking segments in both neoplastic and nonneoplastic HNPCC samples. No difference between the two regions was detectable in cells from healthy donors, indicating that all three HNPCC samples have mutation rates higher than the healthy genome. This is the first, to our knowledge, direct evidence of an intrinsic genomic instability of individuals with heterozygous mutations in mismatch repair genes, and constitutes the proof of principle for the development of a more sensitive molecular assay of genomic instability.

SPG20 is mutated in Troyer syndrome, an hereditary spastic paraplegia.

Troyer syndrome (TRS) is an autosomal recessive complicated hereditary spastic paraplegia (HSP) that occurs with high frequency in the Old Order Amish. We report mapping of the TRS locus to chromosome 13q12.3 and identify a frameshift mutation in SPG20, encoding spartin. Comparative sequence analysis indicates that spartin shares similarity with molecules involved in endosomal trafficking and with spastin, a molecule implicated in microtubule interaction that is commonly mutated in HSP.

Mechanistic insights into the interactions between cancer drivers and the tumour immune microenvironment.

BACKGROUND: The crosstalk between cancer and the tumour immune microenvironment (TIME) has attracted significant interest in the latest years because of its impact on cancer evolution and response to treatment. Despite this, cancer-specific tumour-TIME interactions and their mechanistic insights are still poorly understood. METHODS: Here, we compute the significant interactions occurring between cancer-specific genetic drivers and five anti- and pro-tumour TIME features in 32 cancer types using Lasso regularised ordinal regression. Focusing on head and neck squamous cancer (HNSC), we rebuild the functional networks linking specific TIME driver alterations to the TIME state they associate with. RESULTS: The 477 TIME drivers that we identify are multifunctional genes whose alterations are selected early in cancer evolution and recur across and within cancer types. Tumour suppressors and oncogenes have an opposite effect on the TIME and the overall anti-tumour TIME driver burden is predictive of response to immunotherapy. TIME driver alterations predict the immune profiles of HNSC molecular subtypes, and perturbations in keratinization, apoptosis and interferon signalling underpin specific driver-TIME interactions. CONCLUSIONS: Overall, our study delivers a comprehensive resource of TIME drivers, gives mechanistic insights into their immune-regulatory role, and provides an additional framework for patient prioritisation to immunotherapy. The full list of TIME drivers and associated properties are available at http://www.network-cancer-genes.org .

The transcriptional landscape of endogenous retroelements delineates esophageal adenocarcinoma subtypes.

Most cancer types exhibit aberrant transcriptional activity, including derepression of retrotransposable elements (RTEs). However, the degree, specificity and potential consequences of RTE transcriptional activation may differ substantially among cancer types and subtypes. Representing one extreme of the spectrum, we characterize the transcriptional activity of RTEs in cohorts of esophageal adenocarcinoma (EAC) and its precursor Barrett's esophagus (BE) from the OCCAMS (Oesophageal Cancer Clinical and Molecular Stratification) consortium, and from TCGA (The Cancer Genome Atlas). We found exceptionally high RTE inclusion in the EAC transcriptome, driven primarily by transcription of genes incorporating intronic or adjacent RTEs, rather than by autonomous RTE transcription. Nevertheless, numerous chimeric transcripts straddling RTEs and genes, and transcripts from stand-alone RTEs, particularly KLF5- and SOX9-controlled HERVH proviruses, were overexpressed specifically in EAC. Notably, incomplete mRNA splicing and EAC-characteristic intronic RTE inclusion was mirrored by relative loss of the respective fully-spliced, functional mRNA isoforms, consistent with compromised cellular fitness. Defective RNA splicing was linked with strong transcriptional activation of a HERVH provirus on Chr Xp22.32 and defined EAC subtypes with distinct molecular features and prognosis. Our study defines distinguishable RTE transcriptional profiles of EAC, reflecting distinct underlying processes and prognosis, thus providing a framework for targeted studies.

Modification of gene duplicability during the evolution of protein interaction network.

Duplications of genes encoding highly connected and essential proteins are selected against in several species but not in human, where duplicated genes encode highly connected proteins. To understand when and how gene duplicability changed in evolution, we compare gene and network properties in four species (Escherichia coli, yeast, fly, and human) that are representative of the increase in evolutionary complexity, defined as progressive growth in the number of genes, cells, and cell types. We find that the origin and conservation of a gene significantly correlates with the properties of the encoded protein in the protein-protein interaction network. All four species preserve a core of singleton and central hubs that originated early in evolution, are highly conserved, and accomplish basic biological functions. Another group of hubs appeared in metazoans and duplicated in vertebrates, mostly through vertebrate-specific whole genome duplication. Such recent and duplicated hubs are frequently targets of microRNAs and show tissue-selective expression, suggesting that these are alternative mechanisms to control their dosage. Our study shows how networks modified during evolution and contributes to explaining the occurrence of somatic genetic diseases, such as cancer, in terms of network perturbations.

Network of Cancer Genes: a web resource to analyze duplicability, orthology and network properties of cancer genes.

The Network of Cancer Genes (NCG) collects and integrates data on 736 human genes that are mutated in various types of cancer. For each gene, NCG provides information on duplicability, orthology, evolutionary appearance and topological properties of the encoded protein in a comprehensive version of the human protein-protein interaction network. NCG also stores information on all primary interactors of cancer proteins, thus providing a complete overview of 5357 proteins that constitute direct and indirect determinants of human cancer. With the constant delivery of results from the mutational screenings of cancer genomes, NCG represents a versatile resource for retrieving detailed information on particular cancer genes, as well as for identifying common properties of precompiled lists of cancer genes. NCG is freely available at: http://bio.ifom-ieo-campus.it/ncg.

Reflected stemness as a potential driver of the tumour microenvironment.

A fundamental requirement for cancer initiation is the activation of developmental programmes by mutant cells. Oncogenic signals often confer an undifferentiated, stem cell-like phenotype that supports the long-term proliferative potential of cancer cells. Although cancer is a genetically driven disease, mutations in cancer-driver genes alone are insufficient for tumour formation, and the proliferation of cells harbouring oncogenic mutations depends on their microenvironment. In this Opinion article we discuss how the reprogrammed status of cancer cells not only represents the essence of their tumorigenicity but triggers 'reflected stemness' in their surrounding normal counterparts. We propose that this reciprocal interaction underpins the establishment of the tumour microenvironment (TME).

A SIMPLI (Single-cell Identification from MultiPLexed Images) approach for spatially-resolved tissue phenotyping at single-cell resolution.

Multiplexed imaging technologies enable the study of biological tissues at single-cell resolution while preserving spatial information. Currently, high-dimension imaging data analysis is technology-specific and requires multiple tools, restricting analytical scalability and result reproducibility. Here we present SIMPLI (Single-cell Identification from MultiPLexed Images), a flexible and technology-agnostic software that unifies all steps of multiplexed imaging data analysis. After raw image processing, SIMPLI performs a spatially resolved, single-cell analysis of the tissue slide as well as cell-independent quantifications of marker expression to investigate features undetectable at the cell level. SIMPLI is highly customisable and can run on desktop computers as well as high-performance computing environments, enabling workflow parallelisation for large datasets. SIMPLI produces multiple tabular and graphical outputs at each step of the analysis. Its containerised implementation and minimum configuration requirements make SIMPLI a portable and reproducible solution for multiplexed imaging data analysis. Software is available at "SIMPLI [ https://github.com/ciccalab/SIMPLI ]".

Comparative assessment of genes driving cancer and somatic evolution in non-cancer tissues: an update of the Network of Cancer Genes (NCG) resource.

BACKGROUND: Genetic alterations of somatic cells can drive non-malignant clone formation and promote cancer initiation. However, the link between these processes remains unclear and hampers our understanding of tissue homeostasis and cancer development. RESULTS: Here, we collect a literature-based repertoire of 3355 well-known or predicted drivers of cancer and non-cancer somatic evolution in 122 cancer types and 12 non-cancer tissues. Mapping the alterations of these genes in 7953 pan-cancer samples reveals that, despite the large size, the known compendium of drivers is still incomplete and biased towards frequently occurring coding mutations. High overlap exists between drivers of cancer and non-cancer somatic evolution, although significant differences emerge in their recurrence. We confirm and expand the unique properties of drivers and identify a core of evolutionarily conserved and essential genes whose germline variation is strongly counter-selected. Somatic alteration in even one of these genes is sufficient to drive clonal expansion but not malignant transformation. CONCLUSIONS: Our study offers a comprehensive overview of our current understanding of the genetic events initiating clone expansion and cancer revealing significant gaps and biases that still need to be addressed. The compendium of cancer and non-cancer somatic drivers, their literature support, and properties are accessible in the Network of Cancer Genes and Healthy Drivers resource at http://www.network-cancer-genes.org/ .

Exome sequencing of hepatocellular carcinoma in lemurs identifies potential cancer drivers: A pilot study.

Background and objectives: Hepatocellular carcinoma occurs frequently in prosimians, but the cause of these liver cancers in this group is unknown. Characterizing the genetic changes associated with hepatocellular carcinoma in prosimians may point to possible causes, treatments and methods of prevention, aiding conservation efforts that are particularly crucial to the survival of endangered lemurs. Although genomic studies of cancer in non-human primates have been hampered by a lack of tools, recent studies have demonstrated the efficacy of using human exome capture reagents across primates. Methodology: In this proof-of-principle study, we applied human exome capture reagents to tumor-normal pairs from five lemurs with hepatocellular carcinoma to characterize the mutational landscape of this disease in lemurs. Results: Several genes implicated in human hepatocellular carcinoma, including ARID1A, TP53 and CTNNB1, were mutated in multiple lemurs, and analysis of cancer driver genes mutated in these samples identified enrichment of genes involved with TP53 degradation and regulation. In addition to these similarities with human hepatocellular carcinoma, we also noted unique features, including six genes that contain mutations in all five lemurs. Interestingly, these genes are infrequently mutated in human hepatocellular carcinoma, suggesting potential differences in the etiology and/or progression of this cancer in lemurs and humans. Conclusions and implications: Collectively, this pilot study suggests that human exome capture reagents are a promising tool for genomic studies of cancer in lemurs and other non-human primates. Lay Summary: Hepatocellular carcinoma occurs frequently in prosimians, but the cause of these liver cancers is unknown. In this proof-of-principle study, we applied human DNA sequencing tools to tumor-normal pairs from five lemurs with hepatocellular carcinoma and compared the lemur mutation profiles to those of human hepatocellular carcinomas.