Dual role of the adhesion G-protein coupled receptor ADRGE5/CD97 in glioblastoma invasion and proliferation.

CD97, an adhesion G-protein coupled receptor highly expressed in glioblastoma (GBM), consists of two noncovalently bound domains: the N-terminal fragment (NTF) and C-terminal fragment. The C-terminal fragment contains a GPCR domain that couples to Gα12/13, while the NTF interacts with extracellular matrix components and other receptors. We investigated the effects of changing CD97 levels and its function on primary patient-derived GBM stem cells (pdGSCs) in vitro and in vivo. We created two functional mutants: a constitutively active ΔNTF and the noncleavable dominant-negative H436A mutant. The CD97 knockdown in pdGSCs decreased, while overexpression of CD97 increased tumor size. Unlike other constructs, the ΔNTF mutant promoted tumor cell proliferation, but the tumors were comparable in size to those with CD97 overexpression. As expected, the GBM tumors overexpressing CD97 were very invasive, but surprisingly, the knockdown did not inhibit invasiveness and even induced it in noninvasive U87 tumors. Importantly, our results indicate that NTF was present in the tumor core cells but absent in the pdGSCs invading the brain. Furthermore, the expression of noncleavable H436A mutant led to large tumors that invade by sending massive protrusions, but the invasion of individual tumor cells was substantially reduced. These data suggest that NTF association with CD97 GPCR domain inhibits individual cell dissemination but not overall tumor invasion. However, NTF dissociation facilitates pdGSCs brain infiltration and may promote tumor proliferation. Thus, the interplay between two functional domains regulates CD97 activity resulting in either enhanced cell adhesion or stimulation of tumor cell invasion and proliferation.

Genetic manipulation of adhesion GPCR CD97/ADGRE5 modulates invasion in patient-derived glioma stem cells.

INTRODUCTION: Effective glioblastoma (GBM) treatment is limited by high invasiveness and heterogeneity. Current therapies target proliferating Glioma Stem Cell (GSC) subpopulations while sparing invading GSCs, which eventually engender tumor recurrence after treatment. Surface receptor CD97/ADRGE5 is associated with invasion and metastasis regulation in non-CNS cancers. Although CD97 expression level positively correlates with poor GBM patient prognosis, its role in this tumor is unclear. METHODS: Here, we examined CD97 function in primary patient-derived GSCs (pdGSCs) obtained from five GBM tumors, belonging to three major genetic subtypes. We compared endogenous CD97 levels in pdGSCs to the corresponding patient MRI's radiographic invasion pattern aggressiveness. We manipulated CD97 levels in these pdGSCs by knockdown and overexpression and analyzed: (i) stem and subtype marker expression, (ii) in vitro invasive properties, and (iii) cell proliferation. RESULTS: Endogenous CD97 levels in pdGSCs positively correlated with radiographic invasion pattern aggressiveness on patient MRIs, and in vitro invasion rate. CD97 knockdown decreased pdGSC invasion rates in vitro, most markedly in mesenchymal subtype pdGSCs, as well as classical subtype pdGSCs. Invasion rates in vitro increased after CD97 overexpression predominately in proneural subtype pdGSCs. In the pdGSC line with the lowest endogenous CD97 level, CD97 overexpression increased the proliferation rate almost threefold. CONCLUSIONS: For the first time in pdGSCs, we have shown that CD97 knockdown decreases and overexpression increases invasion rate in vitro. The effect of CD97 on invasion is pdGSC subtype-dependent. Future in vivo and mechanistic studies are needed for validation. Pharmacologic CD97 inhibitors should be identified, as they may potentially therapeutically diminish GBM invasion.

Identification of ADGRE5 as discriminating MYC target between Burkitt lymphoma and diffuse large B-cell lymphoma.

BACKGROUND: MYC is a heterogeneously expressed transcription factor that plays a multifunctional role in many biological processes such as cell proliferation and differentiation. It is also associated with many types of cancer including the malignant lymphomas. There are two types of aggressive B-cell lymphoma, namely Burkitt lymphoma (BL) and a subgroup of diffuse large cell lymphoma (DLBCL), which both carry MYC translocations and overexpress MYC but both differ significantly in their clinical outcome. In DLBCL, MYC translocations are associated with an aggressive behavior and poor outcome, whereas MYC-positive BL show a superior outcome. METHODS: To shed light on this phenomenon, we investigated the different modes of actions of MYC in aggressive B-cell lymphoma cell lines subdivided into three groups: (i) MYC-positive BL, (ii) DLBCL with MYC translocation (DLBCLpos) and (iii) DLBCL without MYC translocation (DLBCLneg) for control. In order to identify genome-wide MYC-DNA binding sites a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed. In addition, ChIP-Seq for H3K4me3 was used for determination of genomic regions accessible for transcriptional activity. These data were supplemented with gene expression data derived from RNA-Seq. RESULTS: Bioinformatics integration of all data sets revealed different MYC-binding patterns and transcriptional profiles in MYC-positive BL and DLBCL cell lines indicating different functional roles of MYC for gene regulation in aggressive B-cell lymphomas. Based on this multi-omics analysis we identified ADGRE5 (alias CD97) - a member of the EGF-TM7 subfamily of adhesion G protein-coupled receptors - as a MYC target gene, which is specifically expressed in BL but not in DLBCL regardless of MYC translocation. CONCLUSION: Our study describes a diverse genome-wide MYC-DNA binding pattern in BL and DLBCL cell lines with and without MYC translocations. Furthermore, we identified ADREG5 as a MYC target gene able to discriminate between BL and DLBCL irrespectively of the presence of MYC breaks in DLBCL. Since ADGRE5 plays an important role in tumor cell formation, metastasis and invasion, it might also be instrumental to better understand the different pathobiology of BL and DLBCL and help to explain discrepant clinical characteristics of BL and DLBCL.

Comprehensive analysis of ADGRE5 gene in human tumors: Clinical relevance, prognostic implications, and potential for personalized immunotherapy.

Purpose: The Adhesion G protein receptor E5 (ADGRE5) gene is involved in a wide range of biological functions in human tumors; however, its specific molecular mechanism and significance in the analysis of human tumors have not yet been determined. Here, we provide a comprehensive genomic architecture of ADGRE5 in the tumor immune microenvironment and its clinical relevance across a broad range of solid tumors. Methods: In this study, we used publicly available bioinformatics databases, with a primary focus on The Cancer Genome Atlas (TCGA) database and GTEx data, to conduct a comprehensive analysis of the impact on patient prognosis associated with ADGRE5. Results: Statistics of more than 30 solid tumors from TCGA and Cancer Cell Line Encyclopedia (CCLE) were examined. ADGRE5 was differentially expressed in several cancers and was significantly associated with survival outcomes. Higher ADGRE5 levels were associated with worse prognosis in adrenocortical carcinoma, low grade glioma of the brain (LGG), lung squamous cell carcinoma, liver hepatocellular carcinoma, and uveal melanoma (UVM). Additionally, ADGRE5 was found to be an independent risk factor for LGG and UVM. The clinical relevance of ADGRE5 in tumor immunogenicity was further investigated. The expression level of ADGRE5 was not only strongly associated with tumor infiltration, such as tumor-infiltrating immune cells and immune subtypes, but also with tumor mutation burden, pyroptosis, and epithelial-mesenchymal transition in various types of cancer (P < 0.05). Furthermore, we noted that ADGRE5 exhibited a positive association with targeted drug sensitivity and conversely, a negative association with traditional chemotherapeutic drug sensitivity. Thus, ADGRE5 is expected to be a guiding marker gene for clinical prognosis and personalized tumor immunotherapy.

The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability.

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of ß-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.

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.

The Interaction of CD97/ADGRE5 With ß-Catenin in Adherens Junctions Is Lost During Colorectal Carcinogenesis.

The adhesion G-protein-coupled receptor CD97/ADGRE5 is present in adherens junctions of human normal intestinal cells and upregulated in colorectal carcinomas. Here, we examined whether CD97 directly interacts with junctional proteins in normal and malignant colorectal tissue. We identified an association of CD97 with ß-catenin using a proximity ligation assay and confirmed the interaction between both endogenous proteins at the biochemical level by co-immunoprecipitation in human and mouse tissues and cell lines. Glutathione S-transferase-pulldown revealed that CD97 binds ß-catenin through its seven-span transmembrane/intracellular domain(s). To study tumor-associated changes in the interaction of CD97 and ß-catenin in situ, we quantified and correlated both proteins at the membrane, and in the cytoplasm and nuclei of colorectal carcinomas and their corresponding normal tissues (n = 111). In normal colon, membranous levels of CD97 and ß-catenin correlated strongly (p < 0.0001). To some degree both molecules disappeared in carcinomas simultaneously from the membrane of tumor cells (p = 0.017). CD97 accumulated in the cytoplasm, whereas ß-catenin emerged in the cytoplasm and nuclei. CD97 and ß-catenin levels in the cytoplasm correlated well (p < 0.0001). Irrespective of their subcellular localization, interaction of CD97 with ß-catenin in tumor cells was also restricted to the cell contacts. Accordingly, CD97 did not regulate ß-catenin-dependent TCF-mediated transcriptional activity. In summary, while CD97 and ß-catenin interact in adherens junctions, their interaction is lost and both molecules follow different functional paths inside tumor cells.