Pushing the Frontiers of Cancer Research: Highlights from the Frontiers in Cancer Science Conference 2023.

The 15th annual Frontiers in Cancer Science (FCS) conference gathered scientific experts who shared the latest research converging upon several themes of cancer biology. These themes included the dysregulation of metabolism, cell death, and other signaling processes in cancer cells; using patient "omics" datasets and single-cell and spatial approaches to investigate heterogeneity, understand therapy resistance, and identify targets; innovative strategies for inhibiting tumors, including rational drug combinations and improved drug delivery mechanisms; and advances in models that can facilitate screening for cancer vulnerabilities and drug testing. We hope the insights from this meeting will stimulate further progress in the field.

Dengue and Zika RNA-RNA interactomes reveal pro- and anti-viral RNA in human cells.

BACKGROUND: Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication. RESULTS: Here, we use proximity ligation sequencing to identify virus-host RNA interactions for four strains of Zika virus (ZIKV) and one strain of dengue virus (DENV-1) in human cells. We find hundreds of coding and non-coding RNAs that bind to DENV and ZIKV viruses. Host RNAs tend to bind to single-stranded regions along the virus genomes according to hybridization energetics. Compared to SARS-CoV-2 interactors, ZIKV-interacting host RNAs tend to be downregulated upon virus infection. Knockdown of several short non-coding RNAs, including miR19a-3p, and 7SK RNA results in a decrease in viral replication, suggesting that they act as virus-permissive factors. In addition, the 3'UTR of DYNLT1 mRNA acts as a virus-restrictive factor by binding to the conserved dumbbell region on DENV and ZIKV 3'UTR to decrease virus replication. We also identify a conserved set of host RNAs that interacts with DENV, ZIKV, and SARS-CoV-2, suggesting that these RNAs are broadly important for RNA virus infection. CONCLUSIONS: This study demonstrates that host RNAs can impact virus replication in permissive and restrictive ways, expanding our understanding of host factors and RNA-based gene regulation during viral pathogenesis.

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.

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.

3'UTR heterogeneity and cancer progression.

The majority of human mRNAs generate alternative 3' untranslated regions (UTRs) through various processes, including RNA modifications such as RNA editing, m6A methylation, and alternative polyadenylation (APA), with 3'UTR splicing as an emerging mechanism. Multiple factors, ranging from the genome to transcriptome level, regulate these processes and contribute to 3'UTR heterogeneity. Genomic variants in 3'UTR regions as well as aberrant 3'UTR processing alter the transcriptomic landscape and are associated with cancer. Increasing evidence, aided by high-resolution sequencing technologies and large-scale computational analyses, points towards potential crosstalk between these processes, whose deregulation may further contribute to cancer pathogenesis. In-depth characterization of these events will increase our appreciation of their significance and help to drive therapeutic development in this field.

Pan-cancer pervasive upregulation of 3' UTR splicing drives tumourigenesis.

Most mammalian genes generate messenger RNAs with variable untranslated regions (UTRs) that are important post-transcriptional regulators. In cancer, shortening at 3' UTR ends via alternative polyadenylation can activate oncogenes. However, internal 3' UTR splicing remains poorly understood as splicing studies have traditionally focused on protein-coding alterations. Here we systematically map the pan-cancer landscape of 3' UTR splicing and present this in SpUR ( http://www.cbrc.kaust.edu.sa/spur/home/ ). 3' UTR splicing is widespread, upregulated in cancers, correlated with poor prognosis and more prevalent in oncogenes. We show that antisense oligonucleotide-mediated inhibition of 3' UTR splicing efficiently reduces oncogene expression and impedes tumour progression. Notably, CTNNB1 3' UTR splicing is the most consistently dysregulated event across cancers. We validate its upregulation in hepatocellular carcinoma and colon adenocarcinoma, and show that the spliced 3' UTR variant is the predominant contributor to its oncogenic functions. Overall, our study highlights the importance of 3' UTR splicing in cancer and may launch new avenues for RNA-based anti-cancer therapeutics.

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.

MiR-138 is a potent regulator of the heterogenous MYC transcript population in cancers.

3'UTR shortening in cancer has been shown to activate oncogenes, partly through the loss of microRNA-mediated repression. This suggests that many reported microRNA-oncogene target interactions may not be present in cancer cells. One of the most well-studied oncogenes is the transcription factor MYC, which is overexpressed in more than half of all cancers. MYC overexpression is not always accompanied by underlying genetic aberrations. In this study, we demonstrate that the MYC 3'UTR is shortened in colorectal cancer (CRC). Using unbiased computational and experimental approaches, we identify and validate microRNAs that target the MYC coding region. In particular, we show that miR-138 inhibits MYC expression and suppresses tumor growth of CRC and hepatocellular carcinoma (HCC) cell lines. Critically, the intravenous administration of miR-138 significantly impedes MYC-driven tumor growth in vivo. Taken together, our results highlight the previously uncharacterized shortening of the MYC 3'UTR in cancer, and identify miR-138 as a potent regulator of the heterogenous MYC transcript population.

Pseudogene-mediated DNA demethylation leads to oncogene activation.

Pseudogenes, noncoding homologs of protein-coding genes, once considered nonfunctional evolutionary relics, have recently been linked to patient prognoses and cancer subtypes. Despite this potential clinical importance, only a handful of >12,000 pseudogenes in humans have been characterized in cancers to date. Here, we describe a previously unrecognized role for pseudogenes as potent epigenetic regulators that can demethylate and activate oncogenes. We focused on SALL4, a known oncogene in hepatocellular carcinoma (HCC) with eight pseudogenes. Using a locus-specific demethylating technology, we identified the critical CpG region for SALL4 expression. We demonstrated that SALL4 pseudogene 5 hypomethylates this region through interaction with DNMT1, resulting in SALL4 up-regulation. Intriguingly, pseudogene 5 is significantly up-regulated in a hepatitis B virus model before SALL4 induction, and both are increased in patients with HBV-HCC. Our results suggest that pseudogene-mediated demethylation represents a novel mechanism of oncogene activation in cancer.

Systematic Analysis of Intronic miRNAs Reveals Cooperativity within the Multicomponent FTX Locus to Promote Colon Cancer Development.

Approximately half of all miRNA reside within intronic regions and are often cotranscribed with their host genes. However, most studies of intronic miRNA focus on individual miRNA, while conversely most studies of protein-coding and noncoding genes frequently ignore any intron-derived miRNA. We hypothesize that the individual components of such multigenic loci may play cooperative or competing roles in driving disease progression and that examining the combinatorial effect of these components would uncover deeper insights into their functional importance. To address this, we performed systematic analyses of intronic miRNA:host loci in colon cancer. The FTX locus, comprising of a long noncoding RNA FTX and multiple intronic miRNA, was highly upregulated in cancer, and cooperativity within this multicomponent locus promoted cancer growth. FTX interacted with DHX9 and DICER and regulated A-to-I RNA editing and miRNA expression. These results show for the first time that a long noncoding RNA can regulate A-to-I RNA editing, further expanding the functional repertoire of long noncoding RNA. Intronic miR-374b and miR-545 inhibited tumor suppressors PTEN and RIG-I to enhance proto-oncogenic PI3K-AKT signaling. Furthermore, intronic miR-421 may exert an autoregulatory effect on miR-374b and miR-545. Taken together, our data unveil the intricate interplay between intronic miRNA and their host transcripts in the modulation of key signaling pathways and disease progression, adding new perspectives to the functional landscape of multigenic loci. SIGNIFICANCE: This study illustrates the functional relationships between individual components of multigenic loci in regulating cancer progression.See related commentary by Calin, p. 1212.

Non-Coding RNAs: Uncharted Mediators of Thyroid Cancer Pathogenesis.

Thyroid cancer is the most prevalent malignancy of the endocrine system and the ninth most common cancer globally. Despite the advances in the management of thyroid cancer, there are critical issues with the diagnosis and treatment of thyroid cancer that result in the poor overall survival of undifferentiated and metastatic thyroid cancer patients. Recent studies have revealed the role of different non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) that are dysregulated during thyroid cancer development or the acquisition of resistance to therapeutics, and may play key roles in treatment failure and poor prognosis of the thyroid cancer patients. Here, we systematically review the emerging roles and molecular mechanisms of ncRNAs that regulate thyroid tumorigenesis and drug response. We then propose the potential clinical implications of ncRNAs as novel diagnostic and prognostic biomarkers for thyroid cancer.

Therapeutic RNA Strategies for Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation with persistent respiratory symptoms. Current therapeutics for COPD are largely borrowed from the drug armamentarium for the treatment of asthma, which has different pathophysiological mechanisms from COPD. COPD has been linked to dysregulated expression of mRNAs and noncoding (nc)RNAs including miRNAs, PIWI-interacting (pi)RNAs, long noncoding (lnc)RNAs, and circular (circ)RNAs. This review highlights and discusses some recent advances towards development of RNA therapeutics for COPD.

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 Butterfly Effect of RNA Alterations on Transcriptomic Equilibrium.

: Post-transcriptional regulation plays a key role in modulating gene expression, and the perturbation of transcriptomic equilibrium has been shown to drive the development of multiple diseases including cancer. Recent studies have revealed the existence of multiple post-transcriptional processes that coordinatively regulate the expression and function of each RNA transcript. In this review, we summarize the latest research describing various mechanisms by which small alterations in RNA processing or function can potentially reshape the transcriptomic landscape, and the impact that this may have on cancer development.

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 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.

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.

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.

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.

Identification of competing endogenous RNAs of the tumor suppressor gene PTEN: A probabilistic approach.

Regulation by microRNAs (miRNAs) and modulation of miRNA activity are critical components of diverse cellular processes. Recent research has shown that miRNA-based regulation of the tumor suppressor gene PTEN can be modulated by the expression of other miRNA targets acting as competing endogenous RNAs (ceRNAs). However, the key sequence-based features enabling a transcript to act as an effective ceRNA are not well understood and a quantitative model associating statistical significance to such features is currently lacking. To identify and assess features characterizing target recognition by PTEN-regulating miRNAs, we analyze multiple datasets from PAR-CLIP experiments in conjunction with RNA-Seq data. We consider a set of miRNAs known to regulate PTEN and identify high-confidence binding sites for these miRNAs on the 3' UTR of protein coding genes. Based on the number and spatial distribution of these binding sites, we calculate a set of probabilistic features that are used to make predictions for novel ceRNAs of PTEN. Using a series of experiments in human prostate cancer cell lines, we validate the highest ranking prediction (TNRC6B) as a ceRNA of PTEN. The approach developed can be applied to map ceRNA networks of critical cellular regulators and to develop novel insights into crosstalk between different pathways involved in cancer.