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Circular RNAs were once considered artifacts of transcriptome sequencing but have recently been identified as functionally relevant in multiple cancers. Although there is still no clear main function of circRNAs, several studies have revealed that circRNAs are expressed in a variety of eukaryotic organisms, demonstrate conservation across species, and are expressed in a regulated manner often independent of their parental linear isoforms. circNFATC3, an abundant and uncharacterized circular isoform of NFATC3 gene (which is formed form backsplicing of exon 2 and 3) in solid tumors was identified from transcriptomic data. Here we show that circNFATC3 gain of and loss of function experiments using RNAi mediated circRNA silencing and circular mini vector-mediated overexpression of circularized constructs in breast and ovarian cancer cell lines affects molecular phenotypes. Knockdown of circNFATC3 induces a reduction in cell proliferation, invasion, migration, and oxidative phosphorylation. Gain of function of circNFATc3 in MDA-MB -231 cells and SKOV3 cells shows a significant increase in cell proliferation, migration, and respiration. The above results suggest that circNFATC3 is a functionally relevant circular RNA in cancer

The immune system is a well-known vital regulator of tumor growth, and one of the main hallmarks of cancer is evading the immune system. Immune system deregulation can lead to immune surveillance evasion, sustained cancer growth, proliferation, and metastasis. Tumor-mediated disruption of the immune system is accomplished by different mechanisms that involve extensive crosstalk with the immediate microenvironment, which includes endothelial cells, immune cells, and stromal cells, to create a favorable tumor niche that facilitates the development of cancer. The essential role of non-coding RNAs such as microRNAs (miRNAs) in the mechanism of cancer cell immune evasion has been highlighted in recent studies. miRNAs are small non-coding RNAs that regulate a wide range of post-transcriptional gene expression in a cell. Recent studies have focused on the function that miRNAs play in controlling the expression of target proteins linked to immune modulation. Studies show that miRNAs modulate the immune response in cancers by regulating the expression of different immune-modulatory molecules associated with immune effector cells, such as macrophages, dendritic cells, B-cells, and natural killer cells, as well as those present in tumor cells and the tumor microenvironment. This review explores the relationship between miRNAs, their altered patterns of expression in tumors, immune modulation, and the functional control of a wide range of immune cells, thereby offering detailed insights on the crosstalk of tumor-immune cells and their use as prognostic markers or therapeutic agents.

Circular RNAs (circRNAs) are an evolutionarily conserved novel class of non-coding endogenous RNAs (ncRNAs) found in the eukaryotic transcriptome, originally believed to be aberrant RNA splicing by-products with decreased functionality. However, recent advances in high-throughput genomic technology have allowed circRNAs to be characterized in detail and revealed their role in controlling various biological and molecular processes, the most essential being gene regulation. Because of the structural stability, high expression, availability of microRNA (miRNA) binding sites and tissue-specific expression, circRNAs have become hot topic of research in RNA biology. Compared to the linear RNA, circRNAs are produced differentially by backsplicing exons or lariat introns from a pre-messenger RNA (mRNA) forming a covalently closed loop structure missing 3′ poly-(A) tail or 5′ cap, rendering them immune to exonuclease-mediated degradation. Emerging research has identified multifaceted roles of circRNAs as miRNA and RNA binding protein (RBP) sponges and transcription, translation, and splicing event regulators. CircRNAs have been involved in many human illnesses, including cancer and neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease, due to their aberrant expression in different pathological conditions. The functional versatility exhibited by circRNAs enables them to serve as potential diagnostic or predictive biomarkers for various diseases. This review discusses the properties, characterization, profiling, and the diverse molecular mechanisms of circRNAs and their use as potential therapeutic targets in different human malignancies.

Figure S2. Figure a, b, c Shows functional predicted networks from RNAseq data of circANKRD12 silenced MDA-MB-231 cells induces inflammatory immune responses and cancer cell invasion with activation Z score and p values. (PPTX 6928 kb) (PPTX 6949 kb)

S2. File represents additional figures for validating the circANKRD12 in cell lines. (PPTX 17563 kb) (PPTX 17572 kb)

Figure S1. (a) The abundance of circANKRD12 and ANKRD12 linear RNA. Semi qRT-PCR data indicating the abundance of circANKRD12 and ANKRD12 mRNA in the cytoplasmic and nuclear fractions of SKOV3 cells, nuclear and cytoplasmic purity markers were also assessed using nuclear specific marker 7SK and cytoplasmic specific marker CYTB. The gel picture represents PCR amplified products of circular RNAs with three different sets of primers. (b) Real-time data shows the abundance of circANKRD12 and ANKRD12 mRNA in the cytoplasm and nucleus. (c) The abundance of circANKRD12 and ANKRD12 mRNA in a panel of cancer cells. (PPTX 2728 kb) (PPTX 2750 kb)

Tables S1-S5. Tables representing primers used for different gene expression studies, siRNAs and pathways involved in circANKRD12 gene knockdown condition. (PPTX 3253 kb) (PPTX 3268 kb)

Circular RNAs (circRNAs) are abundant in eukaryotic transcriptomes and have been linked to various human disorders. However, understanding genetic control of circular RNA expression is in early stages. Here we present the first integrated analysis of circRNAs and genome sequence variation from lymphoblastoid cell lines of the 1000 genomes project. We identified thousands of circRNAs in the RNA-seq data and show their association with local single nucleotide polymorphic sites, referred to as circQTLs, which influence the circRNA transcript abundance. Strikingly, we found that circQTLs exist independently of eQTLs with most circQTLs having no effect on mRNA expression. Only a fraction of the polymorphic sites are shared and linked to both circRNA and mRNA expression with mostly similar effects on circular and linear RNA. A shared intronic QTL, rs55928920, of HMSD gene drives the circular and linear expression in opposite directions, potentially modulating circRNA levels at the expense of mRNA. Finally, circQTLs and eQTLs are largely independent and exist in separate linkage disequilibrium (LD) blocks with circQTLs highly enriched for functional genomic elements and regulatory regions. This study reveals a previously uncharacterized role of DNA sequence variation in human circular RNA regulation.

BackgroundThree-dimensional (3D) tumor cultures in vitro have recently regained attention as they have been acclaimed to have higher similarity to in vivo tumors than cells grown in conventional monolayers (2D). Cancer cells in 2D monolayers fail to mimic the exact tumor microenvironment existing in the real clinical samples. The 3D anchorage-independent cancer models can better explain the cell adhesion properties in ovarian cancer cells.ResultsTranscriptome profiling of 3D models compared to 2D models in various cancer cell lines shows differential expression of TGF-β-mediated and cell adhesion pathways. Presence of TGF-β in these cell lines shows an increased invasion potential which is specific to cell type. We decided to choose two ovarian cancer cell lines as model systems to study Epithelial to Mesenchymal Transition (EMT) and cancer invasion based on their invasion potential and ability to switch the cadherins expression. TGF-β modulation in EMT and cancer invasion was successfully depicted in both 2D and 3D models of SKOV3 and CAOV3 cell lines. Functional evaluations in 3D and 2D models demonstrate that the addition of the exogenous TGF-β can induce EMT and invasion in cancer cells by turning them into aggressive phenotypes. TGF-β receptor kinase I inhibitor (LY364947) can revert TGF-β effect in these cells.ConclusionsIn a nutshell, TGF-β can induce EMT and migration, increase aggressiveness, increase cell survival, alter cell characteristics, remodel the Extracellular Matrix (ECM) and increase cell metabolism favorable for tumor invasion and metastasis.

Background Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. Methods We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells. Results In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression. Conclusions Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker. Electronic supplementary material The online version of this article (10.1186/s12885-019-5723-0) contains supplementary material, which is available to authorized users.

Breast cancer is regarded as a heterogeneous and complicated disease that remains the prime focus in the domain of public health concern. Next-generation sequencing technologies provided a new perspective dimension to non-coding RNAs, which were initially considered to be transcriptional noise or a product generated from erroneous transcription. Even though understanding of biological and molecular functions of noncoding RNA remains enigmatic, researchers have established the pivotal role of these RNAs in governing a plethora of biological phenomena that includes cancer-associated cellular processes such as proliferation, invasion, migration, apoptosis, and stemness. In addition to this, the transmission of microRNAs and long non-coding RNAs was identified as a source of communication to breast cancer cells either locally or systemically. The present review provides in-depth information with an aim at discovering the fundamental potential of non-coding RNAs, by providing knowledge of biogenesis and functional roles of micro RNA and long non-coding RNAs in breast cancer and breast cancer stem cells, as either oncogenic drivers or tumor suppressors. Furthermore, non-coding RNAs and their potential role as diagnostic and therapeutic moieties have also been summarized.

// Ikhlak Ahmed 1 , Thasni Karedath 1 , Simeon S. Andrews 1 , Iman K. Al-Azwani 2 , Yasmin Ali Mohamoud 2 , Denis Querleu 3 , Arash Rafii 4 , Joel A. Malek 1, 2 1 Department of Genetic medicine, Weill Cornell Medicine-Qatar, Education City, Ar-Rayyan, Qatar 2 Genomics Core, Weill Cornell Medicine-Qatar, Education City, Ar-Rayyan, Qatar 3 Department of Gynecologic Oncology, Universite Montepllier 1, Montpellier, France 4 Stem Cell and Microenvironment Laboratory, Weill Cornell Medicine-Qatar, Education City, Ar-Rayyan, Qatar Correspondence to: Joel A. Malek, e-mail: jom2042@qatar-med.cornell.edu Keywords: circular RNA, ovarian cancer, peritoneal metastasis, lymph node metastasis, miRNA Received: December 07, 2015 Accepted: April 02, 2016 Published: April 22, 2016 ABSTRACT Recently, a class of endogenous species of RNA called circular RNA (circRNA) has been shown to regulate gene expression in mammals and their role in cellular function is just beginning to be understood. To investigate the role of circRNAs in ovarian cancer, we performed paired-end RNA sequencing of primary sites, peritoneal and lymph node metastases from three patients with stage IIIC ovarian cancer. We developed an in-house computational pipeline to identify and characterize the circRNA expression from paired-end RNA-Seq libraries. This pipeline revealed thousands of circular isoforms in Epithelial Ovarian Carcinoma (EOC). These circRNAs are enriched for potentially effective miRNA seed matches. A significantly larger number of circRNAs are differentially expressed between tumor sites than mRNAs. Circular and linear expression exhibits an inverse trend for many cancer related pathways and signaling pathways like NFkB, PI3k/AKT and TGF-β typically activated for mRNA in metastases are inhibited for circRNA expression. Further, circRNAs show a more robust expression pattern across patients than mRNA forms indicating their suitability as biomarkers in highly heterogeneous cancer transcriptomes. The consistency of circular RNA expression may offer new candidates for cancer treatment and prognosis.

The present study evaluated the ability of methanolic extract of Centella asiatica (Linn) Urban (Umbelliferae) to induce apoptosis in different cancer cell lines. MCF-7 cells emerged as the most sensitive cell line for in vitro growth inhibitory activity. C. asiatica extract induced apoptosis in MCF-7 cells as indicated by nuclear condensation, increased annexin staining, loss of mitochondrial membrane potential and induction of DNA breaks identified by TUNEL reactivity. It is possible that the use of C. asiatica extract as a component in herbal medicines could be justifiable.

The present study evaluated the ability of methanolic extract of Centella asiatica (Linn) Urban (Umbelliferae) to induce apoptosis in different cancer cell lines. MCF-7 cells emerged as the most sensitive cell line for in vitro growth inhibitory activity. C. asiatica extract induced apoptosis in MCF-7 cells as indicated by nuclear condensation, increased annexin staining, loss of mitochondrial membrane potential and induction of DNA breaks identified by TUNEL reactivity. It is possible that the use of C. asiatica extract as a component in herbal medicines could be justifiable.

Embryonic stem cells have the ability to give rise to different cell types and also the self reniewal capacity. Those features are essentially controlled by pluripotency factors which maintain cells into an undifferentiated state. Cancer cells are distinguished from normal cells by a dedifferentiation process in which the expression of pluripotency factors can be increased. c-Myc, Oct4 and KLF4 are pluripotency transcription factors. Oct-1, homologue of Oct-4, have been recently revealed to be pro-tumorigenic and his expression, such as c-Myc, is increased in various cancers. In this study, we investigated the role of this transcription factors into the cell metabolism. The role of the oncogene MYC on tumor cell metabolism has been largely studied. In cancer cells, c-Myc induces the expression of genes involved in glycolysis and glutaminolysis. However, the entire metabolic changes induced by c-Myc have not yet been elucidated. Furthermore, little is known about the role of Oct-1 and KLF4 in cancer metabolism. We have used the metabolic footprinting approach in order to characterize the Oct-1, c-Myc and KLF4 deficient cells and to identify the induced consecutive metabolic remodeling. Because of the variability of mutations observed in cancer cells, we investigated the effects induced by Oct-1, KLF4 and c-Myc deficiencies in mouse and rat embryonic fibroblasts by using UPLC (Ultra performance liquid chromatography). The uptake and release of metabolites was quantified by providing different substrates (glutamine or fatty acid). The glucose consumption and the lactate production which defined as the Warburg effect was measured. The mitochondrial function was investigated by oxygen consumption rate measurement. We found that the metabolites produced by Oct-1, KLF4 and c-Myc deficient cells seems to present a signature which can explain there over-expression in tumors. Our results suggest that embryonic stem cells related pluripotency transcription factors are implicated into the metabolic remodeling of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1272. doi:10.1158/1538-7445.AM2011-1272

Cancer cells have recently been shown to be resistant to anoikis, a form of cell death due to detachment, thus decreasing responsiveness to therapy. Catecholamines, antioxidants, and oncogenes have been shown to rescue metabolic defects caused during cell detachment. Thus, there is a strong motivation to study metabolic alterations associated with anoikis resistance in cancer. Despite efforts to understand the link between anoikis and cancer metabolism, extensive metabolic analysis of cancer cells is lacking. Tissue engineering of cancer cells to create three-dimensional cell environments can also be used as a model system to study cancer metabolism. We postulate that anoikis can be regulated by metabolism in tissue engineered cultures of ovarian cancer (OC) cells. Our findings using quantification of metabolites by conducting Ultra Performance Liquid Chromatography and metabolic assays pertaining to central carbon and nitrogen metabolism provided clues as to which pathways relevant to glycolysis, the pentose phosphate pathway, the TCA and urea cycles, lipid cycle, and amino acid and nucleotide metabolisms, may be altered by anoxia in cancer cells, resulting in identification of dominant factors which regulate OC metabolism. Amino acid supplementation significantly increased the TCA cycle flux at the level of citrate synthase and the fatty acid-derived acetyl-CoA to CO2. The information we have gained elucidates the critical role of metabolism in anoikis resistance in OCC in tissue engineered cultures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 981. doi:10.1158/1538-7445.AM2011-981

Endothelial, fibroblast cells, and extracellular matrix play a major role in progression, growth and spread of cancers. The extent to which each plays a role in metabolic adaptations in cancer metabolism is unknown. Because of the unique signature of metabolites in endothelial and fibroblasts when compared with epithelial cancer cell metabolism, metabolic profiling is an ideal approach to study induced metabolic changes in cancer cells during cocultures with fibroblasts and endothelia cells. Additionally, interaction of extracellular matrix with cancer cells has been shown to play a key role in cancer invasion and dissemination. In current work, we study the role of microenvironment (various cell types and extracellular matrix proteins) on alterations and adaptations of cancer metabolism. Metabolic profiling of various cancer cells (lung, prostate, breast, and ovarian) in coculture with fibroblasts and endothelial cells was done using metabolic flux analysis in various extracellular matrix environments (collagen coating, collagen gel, and matrigel). Metabolic flux analysis (MFA) refers to a methodology whereby intracellular fluxes (i.e. conversion rates of metabolites through individual reactions) are calculated using a stoichiometric model for the major intracellular reactions and applying mass balances around intracellular metabolites. MFA is a powerful approach for understanding and comparing different metabolic states, and offer insights into the functional capabilities of a metabolic network. The power of this approach is that it takes into account a large set of measurements as well as the complex inter-dependence among the various pathways due to the sharing of a common pool of co-factors such as NADH, NADPH, thus providing a more complete and integrated picture of the metabolic state than isolated measurements relevant to a few pathways. Our findings using quantification of 82 metabolites pertaining to central carbon and nitrogen metabolism provided clues as to which pathways relevant to glycolysis, the pentose phosphate pathway, the TCA and urea cycles, lipid cycle, and amino acid and nucleotide metabolisms, may be altered by endothelial and fibroblasts in cancer cells, resulting in identification of dominant factors which regulate cancer cell metabolism. During extracellular matrix cultures, amino acid supplementation, irrespective of extracellular matrix type, induced dramatic changes. Amino acid supplementation also significantly increased the TCA cycle flux at the level of citrate synthase and oxidized 96% to 97% of the fatty acid-derived acetyl-CoA to CO2. The information we gain elucidates the critical role of microenvironment and nutritional supplementation in cancer metabolism and allows development of therapeutics for modulating the communication between cancer and its microenvironment to prevent cancer invasion and dissemination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 45.

Oct-1 and C-myc have recently been shown to be pro-tumorigenic and their expression is increased in various cancers. Despite recent surge in understanding the role of pluripotency related transcription factors of stem cells in tumorigenicity, little is understood of the role of pluripotency factors oct-1, c-myc, and oct-1 in cancer metabolism. We have used metabolic profiling of oct-1 and c-myc deficient and overexpressed cells to characterize intermediary metabolism and identify the critical nutrients in cancer metabolism. Cancer cell metabolism was investigated using systems biology tools such as metabolic flux analysis (MFA) to reveal intracellular ES cell metabolic flux distribution. Metabolic flux analysis (MFA) refers to a methodology whereby intracellular fluxes (i.e. conversion rates of metabolites through individual reactions) are calculated using a stoichiometric model for the major intracellular reactions and applying mass balances around intracellular metabolites. MFA is a powerful approach for understanding and comparing different metabolic states, and offer insights into the functional capabilities of a metabolic network. The power of this approach is that it takes into account a large set of measurements as well as the complex inter-dependence among the various pathways due to the sharing of a common pool of co-factors such as NADH, NADPH, thus providing a more complete and integrated picture of the metabolic state than isolated measurements relevant to a few pathways. Given that the turnover rate of intracellular metabolites is very fast compared to the time scale of perturbations imparted on the cells, we can assume a “pseudo-steady-state” whereby intracellular levels of metabolites do not change significantly. Under these conditions, the intracellular fluxes are linearly related to measured rates of uptake and release of extracellular metabolites. Since, the metabolites produced by oct-1 and c-myc deficient cells present a unique signature which is different from the metabolic profile of oct-1 and c-myc over expressed cells, we hypothesized that metabolic profiling based approach could lead to understanding of the role of these factors in cancer metabolism. The developed strategy first revealed pathways that were down regulated in the c-myc and oct-1 deficient cells. Second, the obtained optimal flux solutions were used to guide the up-regulation of various pathways and hence, determining the appropriate nutrient and hormonal supplements necessary for loss of oncogenic transformations. Combined biological and clinical studies using metabolic profiling of oct-1 and c-myc deficient and overexpressed cells is likely to have a major impact on the development of therapeutics targeting metabolism in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5092.

Metabolomics, a high-throughput global metabolite analysis, is a burgeoning field, and in recent times has shown substantial evidence to support its emerging role in cancer diagnosis, cancer recurrence, and prognosis, as well as its impact in identifying novel cancer biomarkers and developing cancer therapeutics. Newly evolving advances in disease diagnostics and therapy will further facilitate future growth in the field of metabolomics, especially in cancer, where there is a dire need for sensitive and more affordable diagnostic tools and an urgency to develop effective therapies and identify reliable biomarkers to predict accurately the response to a therapy. Here, we review the application of metabolomics in cancer and mitochondrial studies and its role in enabling the understanding of altered metabolism and malignant transformation during cancer growth and metastasis. The recent developments in the area of metabolic flux analysis may help to close the gap between clinical metabolomics research and the development of cancer metabolome. In the era of personalized medicine with more and more patient specific targeted therapies being used, we need reliable, dynamic, faster, and yet sensitive biomarkers both to track the disease and to develop and evolve therapies during the course of treatment. Recent advances in metabolomics along with the novel strategies to analyze, understand, and construct the metabolic pathways opens this window of opportunity in a very cost-effective manner. This article is part of a Special Issue entitled: Bioenergetics of Cancer.