Functional test of a naturally occurred tumor modifier gene provides insights to melanoma development

: Occurrence of degenerative interactions is thought to serve as a mechanism underlying hybrid unfitness in most animal systems. However, the molecular mechanisms underpinning the genetic interaction and how they contribute to overall hybrid incompatibilities are limited to only a handful of examples. A vertebrate model organism, Xiphophorus, is used to study hybrid dysfunction, and it has been shown from this model that diseases, such as melanoma, can occur in certain interspecies hybrids. Melanoma development is due to hybrid inheritance of an oncogene, xmrk, and loss of a co-evolved tumor modifier. It was recently found that adgre5, a G protein-coupled receptor involved in cell adhesion, is a tumor regulator gene in naturally hybridizing Xiphophorus species Xiphophorus birchmanni (X. birchmanni) and Xiphophorus malinche (X. malinche). We hypothesized that 1 of the 2 parental alleles of adgre5 is involved in regulation of cell growth, migration, and melanomagenesis. Accordingly, we assessed the function of adgre5 alleles from each parental species of the melanoma-bearing hybrids using in vitro cell growth and migration assays. In addition, we expressed each adgre5 allele with the xmrk oncogene in transgenic medaka. We found that cells transfected with the X. birchmanni adgre5 exhibited decreased growth and migration compared to those with the X. malinche allele. Moreover, X. birchmanni allele of adgre5 completely inhibited melanoma development in xmrk-transgenic medaka, while X. malinche adgre5 expression did not exhibit melanoma suppressive activity in medaka. These findings provide evidence that adgre5 is a natural melanoma suppressor and provide new insight in melanoma etiology

To Detach, Migrate, Adhere, and Metastasize: CD97/ADGRE5 in Cancer

Tumorigenesis is a multistep process, during which cells acquire a series of mutations that lead to unrestrained cell growth and proliferation, inhibition of cell differentiation, and evasion of cell death. Growing tumors stimulate angiogenesis, providing them with nutrients and oxygen. Ultimately, tumor cells invade the surrounding tissue and metastasize; a process responsible for about 90% of cancer-related deaths. Adhesion G protein-coupled receptors (aGPCRs) modulate the cellular processes closely related to tumor cell biology, such as adhesion and detachment, migration, polarity, and guidance. Soon after first being described, individual human aGPCRs were found to be involved in tumorigenesis. Twenty-five years ago, CD97/ADGRE5 was discovered to be induced in one of the most severe tumors, dedifferentiated anaplastic thyroid carcinoma. After decades of research, the time has come to review our knowledge of the presence and function of CD97 in cancer. In summary, CD97 is obviously induced or altered in many tumor entities; this has been shown consistently in nearly one hundred published studies. However, its high expression at circulating and tumor-infiltrating immune cells renders the systemic targeting of CD97 in tumors difficult

Genetic analysis in a familial case with high bone mineral density suggests additive effects at two loci

Osteoporosis is the most common bone disease, characterized by a low bone mineral density (BMD) and increased risk of fracture. At the other end of the BMD spectrum, some individuals present strong, fracture-resistant, bones. Both osteoporosis and high BMD are heritable and their genetic architecture encompasses polygenic inheritance of common variants and some cases of monogenic highly penetrant variants in causal genes. We have investigated the genetics of high BMD in a family segregating this trait in an apparently Mendelian dominant pattern. We searched for rare causal variants by whole-exome sequencing in three affected and three nonaffected family members. Using this approach, we have identified 38 rare coding variants present in the proband and absent in the three individuals with normal BMD. Although we have found four variants shared by the three affected members of the family, we have not been able to relate any of these to the high-BMD phenotype. In contrast, we have identified missense variants in two genes, VAV3 and ADGRE5, each shared by two of out of three affected members, whose loss of function fits with the phenotype of the family. In particular, the proband, a woman displaying the highest BMD (sum Z-score = 7), carries both variants, whereas the other two affected members carry one each. VAV3 encodes a guanine-nucleotide-exchange factor with an important role in osteoclast activation and function. Although no previous cases of VAV3 mutations have been reported in humans, Vav3 knockout (KO) mice display dense bones, similarly to the high-BMD phenotype present in our family. The ADGRE5 gene encodes an adhesion G protein-coupled receptor expressed in osteoclasts whose KO mouse displays increased trabecular bone volume. Combined, these mouse and human data highlight VAV3 and ADGRE5 as novel putative high-BMD genes with additive effects, and potential therapeutic targets for osteoporosis. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research

The expanding functional roles and signaling mechanisms of adhesion G protein–coupled receptors

The adhesion class of G protein–coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N‐terminal region that is linked to a C‐terminal seven transmembrane (7TM) domain via a GPCR‐autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N‐terminal fragment (NTF) bound to the 7TM of the C‐terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell–cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs

The Evolutionary History of Vertebrate Adhesion GPCRs and Its Implication on Their Classification

Adhesion G protein-coupled receptors (aGPCRs) form a structurally separate class of GPCRs with an unresolved evolutionary history and classification. Based on phylogenetic relations of human aGPCRs, nine families (A–G, L, V) were distinguished. Taking advantage of available genome data, we determined the aGPCR repertoires in all vertebrate classes. Although most aGPCR families show a high numerical stability in vertebrate genomes, the full repertoire of family E, F, and G members appeared only after the fish–tetrapod split. We did not find any evidence for new aGPCR families in vertebrates which are not present in the human genome. Based on ortholog sequence alignments, selection analysis clearly indicated two types of tetrapod aGPCRs: (i) aGPCR under strong purifying selection in tetrapod evolution (families A, B, D, L, V); and (ii) aGPCR with signatures of positive selection in some tetrapod linages (families C, E, G, F). The alignments of aGPCRs also allowed for a revised definition of reference positions within the seven-transmembranehelix domain (relative position numbering scheme). Based on our phylogenetic cluster analysis, we suggest a revised nomenclature of aGPCRs including their transcript variants. Herein, the former families E and L are combined to one family (L) and GPR128/ADGRG7 forms a separate family (E). Furthermore, our analyses provide valuable information about the (patho)physiological relevance of individual aGPCR members

Emerging roles of adhesion G protein-coupled receptors.

Adhesion G protein-coupled receptors (aGPCRs) form a sub-group within the GPCR superfamily. Their distinctive structure contains an abnormally large N-terminal, extracellular region with a GPCR autoproteolysis-inducing (GAIN) domain. In most aGPCRs, the GAIN domain constitutively cleaves the receptor into two fragments. This process is often required for aGPCR signalling. Over the last two decades, much research has focussed on aGPCR-ligand interactions, in an attempt to deorphanize the family. Most ligands have been found to bind to regions N-terminal to the GAIN domain. These receptors may bind a variety of ligands, ranging across membrane-bound proteins and extracellular matrix components. Recent advancements have revealed a conserved method of aGPCR activation involving a tethered ligand within the GAIN domain. Evidence for this comes from increased activity in receptor mutants exposing the tethered ligand. As a result, G protein-coupling partners of aGPCRs have been more extensively characterised, making use of their tethered ligand to create constitutively active mutants. This has led to demonstrations of aGPCR function in, for example, neurodevelopment and tumour growth. However, questions remain around the ligands that may bind many aGPCRs, how this binding is translated into changes in the GAIN domain, and the exact mechanism of aGPCR activation following GAIN domain conformational changes. This review aims to examine the current knowledge around aGPCR activation, including ligand binding sites, the mechanism of GAIN domain-mediated receptor activation and how aGPCR transmembrane domains may relate to activation. Other aspects of aGPCR signalling will be touched upon, such as downstream effectors and physiological roles

Activation of Adhesion GPCR EMR2/ADGRE2 Induces Macrophage Differentiation and Inflammatory Responses via Gα16/Akt/MAPK/NF-κB Signaling Pathways

EMR2/ADGRE2 is a human myeloid-restricted adhesion G protein-coupled receptor critically implicated in vibratory urticaria, a rare type of allergy caused by vibration-induced mast cell activation. In addition, EMR2 is also highly expressed by monocyte/macrophages and has been linked to neutrophil migration and activation. Despite these findings, little is known of EMR2-mediated signaling and its role in myeloid biology. In this report, we show that activation of EMR2 via a receptor-specific monoclonal antibody promotes the differentiation of human THP-1 monocytic cell line and induces the expression of pro-inflammatory mediators, including IL-8, TNF-α, and MMP-9. Using specific signaling inhibitors and siRNA knockdowns, biochemical and functional analyses reveal that the EMR2-mediated signaling is initiated by Gα16, followed by the subsequent activation of Akt, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and nuclear factor kappa-light-chain-enhancer of activated B cells. Our results demonstrate a functional role for EMR2 in the differentiation and inflammatory activation of human monocytic cells and provide potential targets for myeloid cell-mediated inflammatory disorders

Effect of Dietary Restriction and Subsequent Re-Alimentation on the Transcriptional Profile of Bovine Skeletal Muscle

peer-reviewedCompensatory growth (CG), an accelerated growth phenomenon which occurs following a period of dietary restriction is exploited worldwide in animal production systems as a method to lower feed costs. However the molecular mechanisms regulated CG expression remain to be elucidated fully. This study aimed to uncover the underlying biology regulating CG in cattle, through an examination of skeletal muscle transcriptional profiles utilising next generation mRNA sequencing technology. Twenty Holstein Friesian bulls were fed either a restricted diet for 125 days, with a target growth rate of 0.6 kg/day (Period 1), following which they were allowed feed ad libitum for a further 55 days (Period 2) or fed ad libitum for the entirety of the trial. M. longissimus dorsi biopsies were harvested from all bulls on days 120 and 15 of periods 1 and 2 respectively and RNAseq analysis was performed. During realimentation in Period 2, previously restricted animals displayed CG, growing at 1.8 times the rate of the ad libitum control animals. Compensating animals were also more feed efficient during re-alimentation and compensated for 48% of their previous dietary restriction. 1,430 and 940 genes were identified as significantly differentially expressed (Benjamini Hochberg adjusted P < 0.1) in periods 1 and 2 respectively. Additionally, 2,237 genes were differentially expressed in animals undergoing CG relative to dietary restriction. Dietary restriction in Period 1 was associated with altered expression of genes involved in lipid metabolism and energy production. CG expression in Period 2 occurred in association with greater expression of genes involved in cellular function and organisation. This study highlights some of the molecular mechanisms regulating CG in cattle. Differentially expressed genes identified are potential candidate genes for the identification of biomarkers for CG and feed efficiency, which may be incorporated into future breeding programme

Die Rolle des Transkriptionsfaktors MYC in Lymphomen

The transcription factor MYC, encoded by the MYC gene, plays a central role in many cellular processes, such as cell growth, apoptosis and cell communication. However, as an oncogene, MYC also plays a central role in initiation and progression of many different types of cancers, including malignant lymphomas, and is therefore the focus of many oncological studies. Malignant lymphomas do not refer to a single disease entity, but describe a broad range of lymphatic neoplasias that derive from mature lymphoid cells. They can be subclassified into over 60 subtypes based on their differentiation, morphology and/or clinical course. Based on histology, malignant lymphomas can be generally distinguished into Hodgkin's lymphoma (HL) and Non-Hodgkin's lymphoma (NHL). NHL can be further sub-grouped according to their cell of origin into B- and T-cell NHL. Further subclassification exists based on additional histological, clinical and molecular criteria including chromosomal alterations and gene expression profiles. The present work deals with Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), which both belong to aggressive high-grade B-cell lymphoma. Here, we explore the role of the transcription factor MYC in the pathogenesis and clinical course of these lymphoma types. One hallmark of BL is the t(8;14) chromosomal translocation, leading to an overexpression of MYC protein. MYC translocations are however, not restricted to BL, but can also occur in DLBCL, although at a much lower frequency. MYC break-positive BL differ significantly from the MYC break-positive DLBCL in their clinical course, with BL associated with a complete cure of the majority of cases. To investigate the molecular differences between these two lymphoma subtypes, we performed a metabolic and proteomic study, identifying pyruvate as one of the discriminatory metabolites. This metabolic phenotype was further confirmed by proteomic studies of pyruvate metabolism-associated proteins (Schwarzfischer, et al., 2017). In a second study, the genome-wide MYC binding pattern of BL, MYC breakpositive and -negative DLBCL was analyzed by chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) and RNA-based next generation sequencing (RNA-Seq). Significant differences in the MYC DNA-binding patterns were identified, which were also mirrored in the different gene expression patterns. One of these differentially expressed genes code for the cell surface receptor CD97 (ADGRE5), which is significantly over-expressed in BL, but absent in MYC breakpositive and -negative DLBCL. This finding was confirmed by independent validation experiments, including immunohistological staining of cell lines and primary patient samples (Kleo et al 2018 – submitted). This doctoral thesis was complemented by an investigation of long noncoding RNAs (lncRNAs) and their role in modulating the MYC-driven cellular transcriptome. Using NGS, we identified 13 lncRNAs, which were differentially expressed between BL and DLBCLs, one of which was strongly regulated by MYC. This IncRNA was able to modulate MYC-induced cell cycle genes with a strong impact on cell cycle progression. We therefore called this lncRNA MINCR (MYC-induced non-coding RNA) (Doose, et al., 2015). Taken together, this thesis provides additional evidence that MYC is not merely an on/off amplifier of gene activity but exerts specific actions on the gene expression program and – as a consequence – on cellular functions, a finding also true for aggressive lymphoma. Based on the MYC differences between BL and DLBCL, the identification of biomarkers for their distinction appears to be possible. Therefore it is justified to conclude that MYC plays an essential but diverse role in the pathogenesis of various lymphoma types, a finding which might be important for future treatment modalities. The analyses of this work were partly conducted in cooperation with other research groups and led to three publications, which provide the scientific basis for this cumulative thesis