The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

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Minimal information for studies of extracellular vesicles 2018 (MISEV2018) : a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

CA : A Cancer Journal for Clinicians

The role of non-coding RNAs (ncRNAs) in disease is best understood for microRNAs in cancer. However, there is increasing interest in the disease-related roles of other ncRNAs — including piRNAs, snoRNAs, T-UCRs and lncRNAs — and in using this knowledge for therapy.

Non-coding RNAs in human disease

The study of extracellular vesicles (EVs) has the potential to identify unknown cellular and molecular mechanisms in intercellular communication and in organ homeostasis and disease. Exosomes, with an average diameter of ~100 nanometers, are a subset of EVs. The biogenesis of exosomes involves their origin in endosomes, and subsequent interactions with other intracellular vesicles and organelles generate the final content of the exosomes. Their diverse constituents include nucleic acids, proteins, lipids, amino acids, and metabolites, which can reflect their cell of origin. In various diseases, exosomes offer a window into altered cellular or tissue states, and their detection in biological fluids potentially offers a multicomponent diagnostic readout. The efficient exchange of cellular components through exosomes can inform their applied use in designing exosome-based therapeutics.

The biology, function, and biomedical applications of exosomes

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

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Biological properties of extracellular vesicles and their physiological functions

Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1; also known as deleted in oral cancer or DOC1) is a tumor suppressor gene known to play functional roles in both cell cycle regulation and in the epigenetic control of embryonic stem cell differentiation, the latter as a core subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex. In the vast majority of oral squamous cell carcinomas (OSCC), expression of the CDK2AP1 protein is reduced or lost. Notwithstanding the latter (and the DOC1 acronym), mutations or deletions in its coding sequence are extremely rare. Accordingly, CDK2AP1 protein-deficient oral cancer cell lines express as much CDK2AP1 mRNA as proficient cell lines. Here, by combining in silico and in vitro approaches, and by taking advantage of patient-derived data and tumor material in the analysis of loss of CDK2AP1 expression, we identified a set of microRNAs, namely miR-21-5p, miR-23b-3p, miR-26b-5p, miR-93-5p, and miR-155-5p, which inhibit its translation in both cell lines and patient-derived OSCCs. Of note, no synergistic effects were observed of the different miRs on the CDK2AP1-3-UTR common target. We also developed a novel approach to the combined ISH/IF tissue microarray analysis to study the expression patterns of miRs and their target genes in the context of tumor architecture. Last, we show that CDK2AP1 loss, as the result of miRNA expression, correlates with overall survival, thus highlighting the clinical relevance of these processes for carcinomas of the oral cavity. 

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The deleted in oral cancer (DOC1 aka CDK2AP1) tumor suppressor gene is downregulated in oral squamous cell carcinoma by multiple microRNAs


 Introduction: Long non-coding RNAs (lncRNAs) are important regulators of gene expression and are involved in cancer progression and metastasis. Although studies have shown that aberrant expression of lncRNAs mediates disease progression in ovarian cancer (OC), the role of lncRNA ADAMTS9-AS2 has not been well studied.
 Objective: To determine the role of ADAMTS9-AS2 on OC pathogenesis.
 Methods: We examined ADAMTS9-AS2 expression in human OC cells and tumor tissues using in situ hybridization (ISH). ADAMTS9-AS2 was overexpressed in A2780cp20 and IGROV1cp20 cells and evaluated its effects on cell proliferation, viability and apoptosis. To evaluate the role of ADAMTS9-AS2 in tumor suppression, nude mice were injected with ADAMTS9-AS2 over expressing cells. Reverse phase protein array (RPPA) was performed to identify the downstream regulators. Luciferase reporter assay was performed to confirm the interaction of targeted protein with ADAMTS9-AS2. In addition, epigenetic regulation of ADAMTS9-AS2 was studied using 5-Azacytidine.
 Results: A2780cp20 and IGROV1cp20 cells expressed lower amounts of ADAMTS9-AS2, while SKOV3 and PEO4 cells showed higher expression levels. ISH analysis showed dense nuclear staining of ADAMTS9-AS2 in normal fallopian tube sections, whereas OC sections showed reduced expression. Kaplan-Meier survival analysis showed that patients with high expression of ADAMTS9-AS2 had significantly better overall survival (p=0.0079) and progression-free survival (p=0.00018) than those with low ADAMTS9-AS2 expression. Overexpression of ADAMTS9-AS2 in A2780cp20 cells resulted in decreased proliferation (p<0.01) and increased apoptosis (p<0.001). ADAMTS9-AS2 overexpressing A2780cp20 and IGROV1cp20 tumor bearing mice showed significant reduction in tumor growth (p=0.002; A2780cp20, p=0.01; IGROV1cp20) when compared to control A2780cp20 and IGROV1 tumor bearing mice. RPPA analysis showed that ANNEXIN A1 was the most upregulated protein upon ADAMTS9-AS2 overexpression. We confirmed that ANNEXIN A1 expression is regulated by ADAMTS9-AS2 by silencing ADAMTS9-AS2. Further, luciferase reporter assay results confirmed the interaction between ADAMTS9-AS2 and ANNEXIN A1 promoter and silencing ADAMTS9-AS2 with siRNA abolished this effect. Consistently with our previous in vitro findings, we observed a significant increase of ANNEXIN A1 expression in the ADAMTS9-AS2 overexpressing mice, compared with the A2780cp20 control. Further, azacytidine treated A2780cp20 cells showed an increase in ADAMTS9-AS2 expression.
 Conclusion: ADAMTS9-AS2 acts as a tumor suppressor, by enhancing the expression of ANNEXIN A1, and thereby regulating tumor growth. Thus, ADAMTS9-AS2-ANNEXIN A1 axis may represent a novel therapeutic target in OC.
 Citation Format: Emine Bayraktar, Guy Katz, Dahai Jiang, Cristian Rodriguez-Aguayo, Lingegowda S. Mangala, Santosh K. Dasari, Elaine Stur, Cristina Ivan, Gabriel Lopez-Berestein, Anil K. Sood. Elucidating the role of ADAMTS9AS2 lncRNA in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1550.

Cristina Ivan

Papers

The deleted in oral cancer (DOC1 aka CDK2AP1) tumor suppressor gene is downregulated in oral squamous cell carcinoma by multiple microRNAs

Abstract 1550: Elucidating the role of ADAMTS9AS2 lncRNA in ovarian cancer