miRNAs as novel immunoregulators 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.

Identification of human genetic variants controlling circular RNA expression.

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 the 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.

Silencing of ANKRD12 circRNA induces molecular and functional changes associated with invasive phenotypes.

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.

Cell Type-Specific TGF-ß Mediated EMT in 3D and 2D Models and Its Reversal by TGF-ß Receptor Kinase Inhibitor in Ovarian Cancer Cell Lines.

Transcriptome profiling of 3D models compared to 2D models in various cancer cell lines shows differential expression of TGF-ß-mediated and cell adhesion pathways. Presence of TGF-ß in these cell lines shows an increased invasion potential which is specific to cell type. In the present study, we identified exogenous addition of TGF-ß can induce Epithelial to Mesenchymal Transition (EMT) in a few cancer cell lines. RNA sequencing and real time PCR were carried out in different ovarian cancer cell lines to identify molecular profiling and metabolic profiling. Since EMT induction by TGF-ß is cell-type specific, we decided to select two promising ovarian cancer cell lines as model systems to study EMT. TGF-ß modulation in EMT and cancer invasion were successfully depicted in both 2D and 3D models of SKOV3 and CAOV3 cell lines. Functional evaluation in 3D and 2D models demonstrates that the addition of the exogenous TGF-ß can induce EMT and invasion in cancer cells by turning them into aggressive phenotypes. TGF-ß receptor kinase I inhibitor (LY364947) can revert the TGF-ß effect in these cells. In a nutshell, TGF-ß 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. We concluded that transcriptomic and phenotypic effect of TGF-ß and its inhibitor is cell-type specific and not cancer specific.

Metabolomics for mitochondrial and cancer studies.

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.

Cytokine- and chemokine-induced inflammatory colorectal tumor microenvironment: Emerging avenue for targeted therapy.

Colorectal cancer (CRC) is a predominant life-threatening cancer, with liver and peritoneal metastases as the primary causes of death. Intestinal inflammation, a known CRC risk factor, nurtures a local inflammatory environment enriched with tumor cells, endothelial cells, immune cells, cancer-associated fibroblasts, immunosuppressive cells, and secretory growth factors. The complex interactions of aberrantly expressed cytokines, chemokines, growth factors, and matrix-remodeling enzymes promote CRC pathogenesis and evoke systemic responses that affect disease outcomes. Mounting evidence suggests that these cytokines and chemokines play a role in the progression of CRC through immunosuppression and modulation of the tumor microenvironment, which is partly achieved by the recruitment of immunosuppressive cells. These cells impart features such as cancer stem cell-like properties, drug resistance, invasion, and formation of the premetastatic niche in distant organs, promoting metastasis and aggressive CRC growth. A deeper understanding of the cytokine- and chemokine-mediated signaling networks that link tumor progression and metastasis will provide insights into the mechanistic details of disease aggressiveness and facilitate the development of novel therapeutics for CRC. Here, we summarized the current knowledge of cytokine- and chemokine-mediated crosstalk in the inflammatory tumor microenvironment, which drives immunosuppression, resistance to therapeutics, and metastasis during CRC progression. We also outlined the potential of this crosstalk as a novel therapeutic target for CRC. The major cytokine/chemokine pathways involved in cancer immunotherapy are also discussed in this review.

Regulation of Circular RNA CircNFATC3 in Cancer Cells Alters Proliferation, Migration, and Oxidative Phosphorylation.

Circular RNAs were once considered artifacts of transcriptome sequencing but have recently been identified as functionally relevant in different types of cancer. Although there is still no clear main function of circRNAs, several studies have revealed that circRNAs are expressed in various eukaryotic organisms in a regulated manner often independent of their parental linear isoforms demonstrating conservation across species. circNFATC3, an abundant and uncharacterized circular RNA of exon 2 and 3 from NFATC3, was identified in transcriptomic data of solid tumors. Here we show that circNFATC3 gain- 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 affect molecular phenotypes. The knockdown of circNFATC3 induces a reduction in cell proliferation, invasion, migration, and oxidative phosphorylation. Gain-of-function of circNFATC3 in MDA-MB-231 and SK-OV-3 cells show a significant increase in cell proliferation, migration, and respiration. The above results suggest that circNFATC3 is a functionally relevant circular RNA in breast and ovarian cancer.

Insights Into the Role of CircRNAs: Biogenesis, Characterization, Functional, and Clinical Impact in Human Malignancies.

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.

Non-Coding RNAs as Regulators and Markers for Targeting of Breast Cancer and Cancer Stem Cells.

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.

Altered expression pattern of circular RNAs in primary and metastatic sites of epithelial ovarian carcinoma.

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-ß 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.