Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations.

Chromosomal translocations encode oncogenic fusion proteins that have been proven to be causally involved in tumorigenesis. Our understanding of whether such genomic alterations also affect non-coding RNAs is limited, and their impact on circular RNAs (circRNAs) has not been explored. Here, we show that well-established cancer-associated chromosomal translocations give rise to fusion circRNAs (f-circRNA) that are produced from transcribed exons of distinct genes affected by the translocations. F-circRNAs contribute to cellular transformation, promote cell viability and resistance upon therapy, and have tumor-promoting properties in in vivo models. Our work expands the current knowledge regarding molecular mechanisms involved in cancer onset and progression, with potential diagnostic and therapeutic implications.

The BRAF pseudogene functions as a competitive endogenous RNA and induces lymphoma in vivo.

Research over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo "CDS" or "3' UTR" develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer.

Accurate transcriptome-wide identification and quantification of alternative polyadenylation from RNA-seq data with APAIQ.

Alternative polyadenylation (APA) enables a gene to generate multiple transcripts with different 3' ends, which is dynamic across different cell types or conditions. Many computational methods have been developed to characterize sample-specific APA using the corresponding RNA-seq data, but suffered from high error rate on both polyadenylation site (PAS) identification and quantification of PAS usage (PAU), and bias toward 3' untranslated regions. Here we developed a tool for APA identification and quantification (APAIQ) from RNA-seq data, which can accurately identify PAS and quantify PAU in a transcriptome-wide manner. Using 3' end-seq data as the benchmark, we showed that APAIQ outperforms current methods on PAS identification and PAU quantification, including DaPars2, Aptardi, mountainClimber, SANPolyA, and QAPA. Finally, applying APAIQ on 421 RNA-seq samples from liver cancer patients, we identified >540 tumor-associated APA events and experimentally validated two intronic polyadenylation candidates, demonstrating its capacity to unveil cancer-related APA with a large-scale RNA-seq data set.

A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language?

Here, we present a unifying hypothesis about how messenger RNAs, transcribed pseudogenes, and long noncoding RNAs "talk" to each other using microRNA response elements (MREs) as letters of a new language. We propose that this "competing endogenous RNA" (ceRNA) activity forms a large-scale regulatory network across the transcriptome, greatly expanding the functional genetic information in the human genome and playing important roles in pathological conditions, such as cancer.

Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs.

Here, we demonstrate that protein-coding RNA transcripts can crosstalk by competing for common microRNAs, with microRNA response elements as the foundation of this interaction. We have termed such RNA transcripts as competing endogenous RNAs (ceRNAs). We tested this hypothesis in the context of PTEN, a key tumor suppressor whose abundance determines critical outcomes in tumorigenesis. By a combined computational and experimental approach, we identified and validated endogenous protein-coding transcripts that regulate PTEN, antagonize PI3K/AKT signaling, and possess growth- and tumor-suppressive properties. Notably, we also show that these genes display concordant expression patterns with PTEN and copy number loss in cancers. Our study presents a road map for the prediction and validation of ceRNA activity and networks and thus imparts a trans-regulatory function to protein-coding mRNAs.

In vivo identification of tumor- suppressive PTEN ceRNAs in an oncogenic BRAF-induced mouse model of melanoma.

We recently proposed that competitive endogenous RNAs (ceRNAs) sequester microRNAs to regulate mRNA transcripts containing common microRNA recognition elements (MREs). However, the functional role of ceRNAs in cancer remains unknown. Loss of PTEN, a tumor suppressor regulated by ceRNA activity, frequently occurs in melanoma. Here, we report the discovery of significant enrichment of putative PTEN ceRNAs among genes whose loss accelerates tumorigenesis following Sleeping Beauty insertional mutagenesis in a mouse model of melanoma. We validated several putative PTEN ceRNAs and further characterized one, the ZEB2 transcript. We show that ZEB2 modulates PTEN protein levels in a microRNA-dependent, protein coding-independent manner. Attenuation of ZEB2 expression activates the PI3K/AKT pathway, enhances cell transformation, and commonly occurs in human melanomas and other cancers expressing low PTEN levels. Our study genetically identifies multiple putative microRNA decoys for PTEN, validates ZEB2 mRNA as a bona fide PTEN ceRNA, and demonstrates that abrogated ZEB2 expression cooperates with BRAF(V600E) to promote melanomagenesis.

Spatially resolved niche and tumor microenvironmental alterations in gastric cancer peritoneal metastases.

BACKGROUND AND AIMS: Peritoneal metastasis (PM) in gastric cancer (GC) is associated with poor prognosis and significant morbidity. We sought to understand the genomic, transcriptomic, and tumor microenvironment (TME) features that contribute to peritoneal organotropism in GC. METHODS: We conducted a comprehensive multi-omic analysis of 548 samples from 326 patients, including primary tumors, matched normal tissues, peritoneal metastases, and adjacent-normal peritoneal tissues. We employed whole exome sequencing, whole transcriptome sequencing, and digital spatial profiling to investigate molecular alterations, gene expression patterns, and TME characteristics associated with PM. RESULTS: Our analysis identified specific genomic alterations in primary tumors, including mutations in ELF3, CDH1, and PIGR, and TME signatures, such as stromal infiltration and M2 macrophage enrichment, associated with increased risk of PM. We observed distinct transcriptional programs and immune compositions in GCPM compared to liver metastases, highlighting the importance of the TME in transcoelomic metastasis. We found differential expression of therapeutic targets between primary tumors and PM, with lower CLDN18.2 and FGFR2b expression in PM. We unravel the roles of the TME in niche reprogramming within the peritoneum, and provide evidence of pre-metastatic niche conditioning even in early GC without clinical PM. These findings were further validated using a humanized mouse model, which demonstrated niche remodeling in the peritoneum during transcoelomic metastasis. CONCLUSION: Our study provides a comprehensive molecular characterization of GCPM and unveils key biological principles underlying transcoelomic metastasis. The identified predictive markers, therapeutic targets, and TME alterations offer potential avenues for targeted interventions and improved patient outcomes.

Global analysis of RNA-binding proteins identifies a positive feedback loop between LARP1 and MYC that promotes tumorigenesis.

In addition to genomic alterations, aberrant changes in post-transcriptional regulation can modify gene function and drive cancer development. RNA-binding proteins (RBPs) are a large class of post-transcriptional regulators that have been increasingly implicated in carcinogenesis. By integrating multi-omics data, we identify LARP1 as one of the most upregulated RBPs in colorectal cancer (CRC) and demonstrate its oncogenic properties. We perform LARP1:RNA interactome profiling and unveil a previously unexplored role for LARP1 in targeting the 3'UTR of oncogenes in CRC. Notably, we identify the proto-oncogenic transcription factor MYC as a key LARP1-regulated target. Our data show that LARP1 positively modulates MYC expression by associating with its 3'UTR. In addition, antisense oligonucleotide-mediated blocking of the interaction between LARP1 and the MYC 3'UTR reduces MYC expression and in vitro CRC growth. Furthermore, a systematic analysis of LARP1:protein interactions reveals IGF2BP3 and YBX1 as LARP1-interacting proteins that also regulate MYC expression and CRC development. Finally, we demonstrate that MYC reciprocally modulates LARP1 expression by targeting its enhancer. In summary, our data reveal a critical, previously uncharacterized role of LARP1 in promoting CRC tumorigenesis, validate its direct regulation of the proto-oncogene MYC and delineate a model of the positive feedback loop between MYC and LARP1 that promotes CRC growth and development.

Circular RNAs and Untranslated Regions in Acute Myeloid Leukemia.

Before the advent of next-generation sequencing, research on acute myeloid leukemia (AML) mostly centered on protein-coding genes. In recent years, breakthroughs in RNA sequencing technologies and whole transcriptome analysis have led to the discovery that approximately 97.5% of the human genome is transcribed into non-coding RNAs (ncRNAs). This paradigm shift has led to an explosion of research interest in different classes of non-coding RNAs, such as circular RNAs (circRNAs) as well as non-coding untranslated regions (UTRs) of protein-coding messenger RNAs. The critical roles of circRNAs and UTRs in AML pathogenesis have become increasingly apparent. In this review, we discuss the cellular mechanisms of circRNAs and summarize recent studies that reveal their biological roles in AML. Furthermore, we also review the contribution of 3'UTRs to disease progression. Finally, we discuss the potential of circRNAs and 3'UTRs as new biomarkers for disease stratification and/or the prediction of treatment response and targets for the development of RNA-directed therapeutic applications.

A comprehensive expression landscape of RNA-binding proteins (RBPs) across 16 human cancer types.

RNA-binding proteins (RBPs) are key regulators of posttranscriptional processes such as RNA maturation, localization, turnover and translation. Despite their dysregulation in various diseases including cancer, the landscape of RBP expression in human cancer has not been well elucidated. Here, we built a comprehensive expression landscape of 1504 RBPs across 16 human cancer types, which revealed that RBPs are predominantly upregulated in tumours and this phenomenon is affected by the tumour immune subtypes and microenvironment. Across different cancer types, 109 RBPs are consistently upregulated while 41 RBPs are consistently downregulated. These up-regulated and down-regulated RBPs show distinct molecular characteristics and prognostic effects, whereas their dysregulation is mediated by distinct cis/trans-regulatory mechanisms. Finally, we validated one candidate PABPC1L that might promote colon tumorigenesis by regulating mRNA splicing. In summary, we built a comprehensive expression landscape of RBPs across different cancer types and identified consistently dysregulated RBPs which could be novel targets for developing broad-spectrum anticancer agents.

The multilayered complexity of ceRNA crosstalk and competition.

Recent reports have described an intricate interplay among diverse RNA species, including protein-coding messenger RNAs and non-coding RNAs such as long non-coding RNAs, pseudogenes and circular RNAs. These RNA transcripts act as competing endogenous RNAs (ceRNAs) or natural microRNA sponges - they communicate with and co-regulate each other by competing for binding to shared microRNAs, a family of small non-coding RNAs that are important post-transcriptional regulators of gene expression. Understanding this novel RNA crosstalk will lead to significant insight into gene regulatory networks and have implications in human development and disease.

A FTH1 gene:pseudogene:microRNA network regulates tumorigenesis in prostate cancer.

Non-coding RNAs play a vital role in diverse cellular processes. Pseudogenes, which are non-coding homologs of protein-coding genes, were once considered non-functional evolutional relics. However, recent studies have shown that pseudogene transcripts can regulate their parental transcripts by sequestering shared microRNAs (miRNAs), thus acting as competing endogenous RNAs (ceRNAs). In this study, we utilize an unbiased screen to identify the ferritin heavy chain 1 (FTH1) transcript and multiple FTH1 pseudogenes as targets of several oncogenic miRNAs in prostate cancer (PCa). We characterize the critical role of this FTH1 gene:pseudogene:miRNA network in regulating tumorigenesis in PCa, whereby oncogenic miRNAs downregulate the expression of FTH1 and its pseudogenes to drive oncogenesis. We further show that impairing miRNA binding and subsequent ceRNA crosstalk completely rescues the slow growth phenotype in vitro and in vivo. Our results also demonstrate the reciprocal regulation between the pseudogenes and intracellular iron levels, which are crucial for multiple physiological and pathophysiological processes. In summary, we describe an extensive gene:pseudogene network comprising multiple miRNAs and multiple pseudogenes derived from a single parental gene. The network could be regulated through multiple mechanisms to modulate iron storage in various signaling pathways, the deregulation of which results in PCa development and progression.

A novel SOCS5/miR-18/miR-25 axis promotes tumorigenesis in liver cancer.

The role of miRNAs with tumor suppressive activity in liver cancer has been well studied. However, little is known about potential oncomiRs in HCC. In our study, we conducted a systematic evaluation of candidate oncomiRs and found that upregulation of miR-18a and miR-25 in HCC was associated with poor patient survival and promoted proliferation in HCC cell lines. These two miRNAs belong to the polycistronic paralogous miR-17-92 and miR-25-106b clusters respectively. Although the members of both clusters are often upregulated in HCC, the contribution of individual miRNAs in these clusters to HCC tumorigenesis is not fully understood. We validated SOCS5 as a bona fide target of both miRNAs, and established, for the first time, the tumor suppressive role of SOCS5 in liver cancer. We further investigated the mechanism by which SOCS5 contributes to tumorigenesis, demonstrated that this SOCS5/miR-18a/miR-25 axis regulates the tumor suppressor TSC1 and downstream mTOR signaling, and highlighted the potential therapeutic use of miR-18a and miR-25 inhibition in restoring SOCS5 levels in HCC.

The Yin-Yang Regulation of Reactive Oxygen Species and MicroRNAs in Cancer.

Reactive oxygen species (ROS) are highly reactive oxygen-containing chemical species formed as a by-product of normal aerobic respiration and also from a number of other cellular enzymatic reactions. ROS function as key mediators of cellular signaling pathways involved in proliferation, survival, apoptosis, and immune response. However, elevated and sustained ROS production promotes tumor initiation by inducing DNA damage or mutation and activates oncogenic signaling pathways to promote cancer progression. Recent studies have shown that ROS can facilitate carcinogenesis by controlling microRNA (miRNA) expression through regulating miRNA biogenesis, transcription, and epigenetic modifications. Likewise, miRNAs have been shown to control cellular ROS homeostasis by regulating the expression of proteins involved in ROS production and elimination. In this review, we summarized the significance of ROS in cancer initiation, progression, and the regulatory crosstalk between ROS and miRNAs in cancer.

A non-canonical tumor suppressive role for the long non-coding RNA MALAT1 in colon and breast cancers.

Long noncoding RNAs (lncRNAs) constitute one of the largest classes of transcripts and have been widely implicated in various diseases such as cancer. Increasing evidence suggests that several lncRNAs are dysregulated and play critical roles in tumorigenesis. LncRNAs can be regulated by key oncogenes and tumor suppressors, adding complexity to the intricate crosstalk between protein coding genes and the noncoding transcriptome. In our study, we investigated the effect that dysregulation of the key tumor suppressor PTEN has on the noncoding transcriptome. We identified the lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) as a target of PTEN and find that this regulation is conserved in both human and mouse as well as with both chronic and acute PTEN dysregulation. We show that this regulation is at least in part microRNA (miRNA)-dependent, and characterize the miRNAs that may be mediating this crosstalk. In summary, we establish and characterize a non-canonical PTEN-microRNA-MALAT1 axis that regulates tumorigenesis and describe for the first time that the MALAT1 lncRNA possesses novel tumor suppressive properties in colon and breast cancers.

Noncoding RNA:RNA Regulatory Networks in Cancer.

Noncoding RNAs (ncRNAs) constitute the majority of the human transcribed genome. This largest class of RNA transcripts plays diverse roles in a multitude of cellular processes, and has been implicated in many pathological conditions, especially cancer. The different subclasses of ncRNAs include microRNAs, a class of short ncRNAs; and a variety of long ncRNAs (lncRNAs), such as lincRNAs, antisense RNAs, pseudogenes, and circular RNAs. Many studies have demonstrated the involvement of these ncRNAs in competitive regulatory interactions, known as competing endogenous RNA (ceRNA) networks, whereby lncRNAs can act as microRNA decoys to modulate gene expression. These interactions are often interconnected, thus aberrant expression of any network component could derail the complex regulatory circuitry, culminating in cancer development and progression. Recent integrative analyses have provided evidence that new computational platforms and experimental approaches can be harnessed together to distinguish key ceRNA interactions in specific cancers, which could facilitate the identification of robust biomarkers and therapeutic targets, and hence, more effective cancer therapies and better patient outcome and survival.

Long noncoding RNAs: lincs between human health and disease.

Long noncoding RNAs (lncRNAs) represent one of the largest classes of transcripts and are highly diverse in terms of characteristics and functions. Advances in high-throughput sequencing platforms have enabled the rapid discovery and identification of lncRNAs as key regulatory molecules involved in various cellular processes and their dysregulation in various human diseases. Here, we summarize the current knowledge of the functions and underlying mechanisms of lncRNA activity with a particular focus on cancer biology. We also discuss the potential of lncRNAs as diagnostic and therapeutic targets for clinical applications.

Integrated transcriptional and competitive endogenous RNA networks are cross-regulated in permissive molecular environments.

Competitive endogenous (ce)RNAs cross-regulate each other through sequestration of shared microRNAs and form complex regulatory networks based on their microRNA signature. However, the molecular requirements for ceRNA cross-regulation and the extent of ceRNA networks remain unknown. Here, we present a mathematical mass-action model to determine the optimal conditions for ceRNA activity in silico. This model was validated using phosphatase and tensin homolog (PTEN) and its ceRNA VAMP (vesicle-associated membrane protein)-associated protein A (VAPA) as paradigmatic examples. A computational assessment of the complexity of ceRNA networks revealed that transcription factor and ceRNA networks are intimately intertwined. Notably, we found that ceRNA networks are responsive to transcription factor up-regulation or their aberrant expression in cancer. Thus, given optimal molecular conditions, alterations of one ceRNA can have striking effects on integrated ceRNA and transcriptional networks.

MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation.

MicroRNAs (miRNAs) are short RNAs that direct messenger RNA degradation or disrupt mRNA translation in a sequence-dependent manner. For more than a decade, attempts to study the interaction of miRNAs with their targets were confined to the 3' untranslated regions of mRNAs, fuelling an underlying assumption that these regions are the principal recipients of miRNA activity. Here we focus on the mouse Nanog, Oct4 (also known as Pou5f1) and Sox2 genes and demonstrate the existence of many naturally occurring miRNA targets in their amino acid coding sequence (CDS). Some of the mouse targets analysed do not contain the miRNA seed, whereas others span exon-exon junctions or are not conserved in the human and rhesus genomes. miR-134, miR-296 and miR-470, upregulated on retinoic-acid-induced differentiation of mouse embryonic stem cells, target the CDS of each transcription factor in various combinations, leading to transcriptional and morphological changes characteristic of differentiating mouse embryonic stem cells, and resulting in a new phenotype. Silent mutations at the predicted targets abolish miRNA activity, prevent the downregulation of the corresponding genes and delay the induced phenotype. Our findings demonstrate the abundance of CDS-located miRNA targets, some of which can be species-specific, and support an augmented model whereby animal miRNAs exercise their control on mRNAs through targets that can reside beyond the 3' untranslated region.