SnapShot: Unconventional miRNA Functions.

MicroRNAs (miRNAs) are synonymous with post-transcriptional repression of target genes. A number of studies, however, have reported miRNAs functioning outside this paradigm, and this SnapShot outlines these unconventional ways in which miRNAs can exert regulatory functions. To view this SnapShot, open or download the PDF.

Non-coding RNAs in disease: from mechanisms to therapeutics.

Non-coding RNAs (ncRNAs) are a heterogeneous group of transcripts that, by definition, are not translated into proteins. Since their discovery, ncRNAs have emerged as important regulators of multiple biological functions across a range of cell types and tissues, and their dysregulation has been implicated in disease. Notably, much research has focused on the link between microRNAs (miRNAs) and human cancers, although other ncRNAs, such as long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), are also emerging as relevant contributors to human disease. In this Review, we summarize our current understanding of the roles of miRNAs, lncRNAs and circRNAs in cancer and other major human diseases, notably cardiovascular, neurological and infectious diseases. Further, we discuss the potential use of ncRNAs as biomarkers of disease and as therapeutic targets.

APPLE and translation: When a small peptideproduced from a "non-coding RNA" matters!

Sun et al. (2021) identified a novel translation initiation mechanism mediated through a new type of regulator named APPLE, a small peptide produced from a non-coding RNA transcript in acute myeloid leukemia, providing unforeseen opportunities for targeting the translation machinery in cancer cells.

Loss of p53 drives neuron reprogramming in head and neck cancer.

The solid tumour microenvironment includes nerve fibres that arise from the peripheral nervous system1,2. Recent work indicates that newly formed adrenergic nerve fibres promote tumour growth, but the origin of these nerves and the mechanism of their inception are unknown1,3. Here, by comparing the transcriptomes of cancer-associated trigeminal sensory neurons with those of endogenous neurons in mouse models of oral cancer, we identified an adrenergic differentiation signature. We show that loss of TP53 leads to adrenergic transdifferentiation of tumour-associated sensory nerves through loss of the microRNA miR-34a. Tumour growth was inhibited by sensory denervation or pharmacological blockade of adrenergic receptors, but not by chemical sympathectomy of pre-existing adrenergic nerves. A retrospective analysis of samples from oral cancer revealed that p53 status was associated with nerve density, which was in turn associated with poor clinical outcomes. This crosstalk between cancer cells and neurons represents mechanism by which tumour-associated neurons are reprogrammed towards an adrenergic phenotype that can stimulate tumour progression, and is a potential target for anticancer therapy.

17ß-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17ß-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17ß-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17ß-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

Classical and noncanonical functions of miRNAs in cancers.

Alterations in microRNAs (miRNAs) expression are causative in the initiation and progression of human cancers. The molecular events responsible for the widespread differential expression of miRNAs in malignancy are exemplified by their location in cancer-associated genomic regions, epigenetic mechanisms, transcriptional dysregulation, chemical modifications and editing, and alterations in miRNA biogenesis proteins. The classical miRNA function is synonymous with post-transcriptional repression of target protein genes. However, several studies have reported miRNAs functioning outside this paradigm and some of these novel modes of regulation of gene expression have been implicated in cancers. Here, we summarize key aspects of miRNA involvement in cancer, with a special focus on these lesser-studied mechanisms of action.

Atlas of metastatic gastric cancer links ferroptosis to disease progression and immunotherapy response.

BACKGROUND & AIMS: Metastases from gastric adenocarcinoma (GAC) lead to high morbidity and mortality. Developing innovative and effective therapies requires a comprehensive understanding of the tumor and immune biology of advanced GAC. Yet, collecting matched specimens from advanced, treatment-naïve GAC patients poses a significant challenge, limiting the scope of current research, which has predominantly focused on localized tumors. This gap hinders a deeper insight into the metastatic dynamics of GAC. METHODS: We performed in-depth single-cell transcriptome and immune profiling on 68 paired, treatment-naïve, primary-metastatic tumors to delineate alterations in cancer cells and their tumor microenvironment (TME) during metastatic progression. To validate our observations, we conducted comprehensive functional studies both in vitro and in vivo, employing cell lines, multiple PDX and novel mouse models of GAC. RESULTS: Liver and peritoneal metastases exhibited distinct properties in cancer cells and dynamics of TME phenotypes, supporting the notion that cancer cells and their local TMEs co-evolve at metastatic sites. Our study also revealed differential activation of cancer meta-programs across metastases. We observed evasion of cancer cell ferroptosis via GPX4 upregulation during GAC progression. Conditional depletion of Gpx4 or pharmacological inhibition of ferroptosis resistance significantly attenuated tumor growth and metastatic progression. Additionally, ferroptosis-resensitizing treatments augmented the efficacy of CAR T-cell therapy. CONCLUSIONS: This study represents the largest single-cell dataset of metastatic GACs to date. High-resolution mapping of the molecular and cellular dynamics of GAC metastasis has revealed a rationale for targeting ferroptosis defense in combination with CAR T-cell therapy as a novel therapeutic strategy with potential immense clinical implications.

miR-328 functions as an RNA decoy to modulate hnRNP E2 regulation of mRNA translation in leukemic blasts.

MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.

MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

Metastatic melanoma poses significant challenges as a highly lethal disease. Despite the success of molecular targeting using BRAFV600E inhibitors (BRAFis) and immunotherapy, the emergence of early recurrence remains an issue and there is the need for novel therapeutic approaches. This study aimed at creating a targeted delivery system for the oncosuppressor microRNA 126 (miR126) and testing its effectiveness in combination with a phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) inhibitor for treating metastatic melanoma resistant to BRAFis. To achieve this, we synthesized chitosan nanoparticles containing a chemically modified miR126 sequence. These nanoparticles were further functionalized with an antibody specific to the chondroitin sulfate proteoglycan 4 (CSPG4) melanoma marker. After evaluation in vitro, the efficacy of this treatment was evaluated through an in vivo experiment using mice bearing resistant human melanoma. The co-administration of miR126 and the PI3K/AKT inhibitor in these experiments significantly reduced tumor growth and inhibited the formation of liver and lung metastases. These results provide evidence for a strategy to target an oncosuppressive nucleic acid sequence to tumor cells while simultaneously protecting it from plasma degradation. The system described in this study exhibits encouraging potential for the effective treatment of therapy-resistant metastatic melanoma while also presenting a prospective approach for other forms of cancer.

Synergic activity of FGFR2 and MEK inhibitors in the treatment of FGFR2-amplified cancers of unknown primary.

Patients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96) and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. The tissue-of-origin prediction was obtained from the tumor microRNA expression profile and confirmed by single-cell transcriptomics. Genomic testing and fluorescence in situ hybridization analysis identified FGFR2 gene amplification in both models, in the form of homogeneously staining region (HSR) in CUP#55 and double minutes in CUP#96. FGFR2 was recognized as the main oncogenic driver and therapeutic target. FGFR2-targeting drug BGJ398 (infigratinib) in combination with the MEK inhibitor trametinib proved to be synergic and exceptionally active, both in vitro and in vivo. The effects of the combined treatment by single-cell gene expression analysis revealed a remarkable plasticity of tumor cells and the greater sensitivity of cells with epithelial phenotype. This study brings personalized therapy closer to CUP patients and provides the rationale for FGFR2 and MEK targeting in metastatic tumors with FGFR2 pathway activation.

Translational modeling-based evidence for enhanced efficacy of standard-of-care drugs in combination with anti-microRNA-155 in non-small-cell lung cancer.

BACKGROUND: Elevated microRNA-155 (miR-155) expression in non-small-cell lung cancer (NSCLC) promotes cisplatin resistance and negatively impacts treatment outcomes. However, miR-155 can also boost anti-tumor immunity by suppressing PD-L1 expression. Therapeutic targeting of miR-155 through its antagonist, anti-miR-155, has proven challenging due to its dual molecular effects. METHODS: We developed a multiscale mechanistic model, calibrated with in vivo data and then extrapolated to humans, to investigate the therapeutic effects of nanoparticle-delivered anti-miR-155 in NSCLC, alone or in combination with standard-of-care drugs. RESULTS: Model simulations and analyses of the clinical scenario revealed that monotherapy with anti-miR-155 at a dose of 2.5 mg/kg administered once every three weeks has substantial anti-cancer activity. It led to a median progression-free survival (PFS) of 6.7 months, which compared favorably to cisplatin and immune checkpoint inhibitors. Further, we explored the combinations of anti-miR-155 with standard-of-care drugs, and found strongly synergistic two- and three-drug combinations. A three-drug combination of anti-miR-155, cisplatin, and pembrolizumab resulted in a median PFS of 13.1 months, while a two-drug combination of anti-miR-155 and cisplatin resulted in a median PFS of 11.3 months, which emerged as a more practical option due to its simple design and cost-effectiveness. Our analyses also provided valuable insights into unfavorable dose ratios for drug combinations, highlighting the need for optimizing dose regimens to prevent antagonistic effects. CONCLUSIONS: This work bridges the gap between preclinical development and clinical translation of anti-miR-155 and unravels the potential of anti-miR-155 combination therapies in NSCLC.

TGF-ß1 and TGF-ßR1 variants are associated with clinical outcomes in smoking-related head and neck cancer patients treated with chemoradiation through modulating microRNA-mediated regulation.

TGF-ß1 and TGF-ßR1 play important roles in immune and inflammatory responses. Genetic variants of TGF-ß1 rs1800470 and TGF-ßR1 rs334348 have emerged as potentially prognostic biomarkers for HPV-related head and neck cancer, while their prognostic effect on survival of smoking-related head and neck cancer remains unknown. This study included 1403 patients with smoking-related head and neck cancer, and all these patients were genotyped for TGF-ß1 rs1800470 and TGF-ßR1 rs334348. Both univariate and multivariate analyses were performed to evaluate associations between the two functional genetic variants in microRNA binding sites of TGF-ß1 and TGF-ßR1 and survivals. Patients with TGF-ß1 rs1800470 CT or CC genotype had 30-35% risk reductions for OS, DSS, and DFS compared to patients with TT genotype among overall patients, ever smokers, and patients administered chemoradiation. Furthermore, patients with TGF-ßR1 rs334348 GA or GG genotype had significant 50-60% risk reductions for OS, DSS, and DFS compared to patients with AA genotype among overall patients and patients administered chemoradiation; among ever smokers, the risk reductions even reached 60-70%. The TCGA dataset was used for validation. These findings suggest that TGF-ß1 rs1800470 and TGF-ßR1 rs334348 significantly affect survival outcomes in patients with smoking-related head and neck cancer, especially in the subgroups of ever smokers and patients treated with chemoradiation. These genetic variants may serve as prognostic indicators for patients with smoking-related head and neck cancer and could play a role in advancing the field of personalized chemoradiation, thereby improving patient survival and quality of life.

Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.

Epithelial SOX9 drives progression and metastases of gastric adenocarcinoma by promoting immunosuppressive tumour microenvironment.

OBJECTIVE: Many cancers engage embryonic genes for rapid growth and evading the immune system. SOX9 has been upregulated in many tumours, yet the role of SOX9 in mediating immunosuppressive tumour microenvironment is unclear. Here, we aim to dissect the role of SOX9-mediated cancer stemness attributes and immunosuppressive microenvironment in advanced gastric adenocarcinoma (GAC) for novel therapeutic discoveries. METHODS: Bulk RNAseq/scRNA-seq, patient-derived cells/models and extensive functional studies were used to identify the expression and functions of SOX9 and its target genes in vitro and in vivo. Immune responses were studied in PBMCs or CD45+ immune cells cocultured with tumour cells with SOX9high or knockout and the KP-Luc2 syngeneic models were used for efficacy of combinations. RESULTS: SOX9 is one of the most upregulated SOX genes in GAC and highly expressed in primary and metastatic tissues and associated with poor prognosis. Depletion of SOX9 in patient-derived GAC cells significantly decreased cancer stemness attributes, tumour formation and metastases and consistently increased CD8+ T cell responses when cocultured with PBMCs/CD45+ cells from GAC patients. RNA sequencing identified the leukaemia inhibitory factor (LIF) as the top secreted molecule regulated by SOX9 in tumour cells and was enriched in malignant ascites and mediated SOX9-induced M2 macrophage repolarisation and inhibited T cell function. CONCLUSION: Epithelial SOX9 is critical in suppressing CD8+ T cell responses and modified macrophage function in GAC through the paracrine LIF factor. Cotargeting LIF/LIFR and CSF1R has great potential in targeting SOX9-mediated cancer stemness, T cell immunosuppression and metastases suggesting the novel combination therapy against advanced GAC.

Epigenetic deregulation in cancer: Enzyme players and non-coding RNAs.

Data obtained from cutting-edge research have shown that deregulated epigenetic marks are critical hallmarks of cancer. Rapidly emerging scientific evidence has helped in developing a proper understanding of the mechanisms leading to control of cellular functions, from changes in chromatin accessibility, transcription and translation, and in post-translational modifications. Firstly, mechanisms of DNA methylation and demethylation are introduced, as well as modifications of DNA and RNA, with particular focus on N6-methyladenosine (m6A), discussing the effects of these modifications in normal cells and in malignancies. Then, chromatin modifying proteins and remodelling complexes are discussed. Many enzymes and accessory proteins in these complexes have been found mutated or have undergone differential splicing, leading to defective protein complexes. Epigenetic mechanisms acting on nucleosomes by polycomb repressive complexes and on chromatin by SWI/SNF complexes on nucleosome assembly/disassembly, as well as main mutated genes linked to cancers, are reviewed. Among enzymes acting on histones and other proteins erasing the reversible modifications are histone deacetylases (HDACs). Sirtuins are of interest since most of these enzymes not only deacylate histones and other proteins, but also post-translationally modify proteins adding a Mono-ADP-ribose (MAR) moiety. MAR can be read by MACRO-domain containing proteins such as histone MacroH2A1, with specific function in chromatin assembly. Finally, recent advances are presented on non-coding RNAs with a scaffold function, prospecting their role in assembly of chromatin modifying complexes, recruiting enzyme players to chromatin regions. Lastly, the imbalance in metabolites production due to mitochondrial dysfunction is presented, with the potential of these metabolites to inhibit enzymes, either writers, readers or erasers of epitranscriptome marks. In the perspectives, studies are overwied on drugs under development aiming to limit excessive enzyme activities and to reactivate chromatin modifying complexes, for therapeutic application. This knowledge may lead to novel drugs and personalised medicine for cancer patients.

Emerging role of oncogenic long noncoding RNA as cancer biomarkers.

The view of long noncoding RNAs as nonfunctional "garbage" has been definitely outdated by the large body of evidence indicating this class of ncRNAs as "golden junk", especially in precision oncology. Indeed, in light of their oncogenic role and the higher expression in multiple cancer types compared with paired adjacent tissues, the clinical interest for lncRNAs as diagnostic and/or prognostic biomarkers has been rapidly increasing. The emergence of large-scale sequencing technologies, their subsequent diffusion even in small research and clinical centers, the technological advances for the detection of low-copy lncRNAs in body fluids, coupled to the huge reduction of operating costs, have nowadays made possible to rapidly and comprehensively profile them in multiple tumors and large cohorts. In this review, we first summarize some relevant data about the oncogenic role of well-studied lncRNAs having a clinical relevance. Then, we focus on the description of their potential use as diagnostic/prognostic biomarkers, including an updated overview about licensed patents or clinical trials on lncRNAs in oncology.

The Role of Non-Coding RNAs in Chromosomal Instability in Cancer.

Chromosomal instability (CIN) is characterized by an increased frequency of changes in chromosome structure or number and is regarded as a hallmark of cancer. CIN plays a prevalent role in tumorigenesis and cancer progression by assisting the cancer cells' phenotypic adaptation to stress, which have been tightly linked to therapy resistance and metastasis. Both CIN-inducing and CIN-repressing agents are being clinically tested for the treatment of cancer to increase CIN levels to unsustainable levels leading to cell death or to decrease CIN levels to limit the development of drug resistance, respectively. Non-coding RNAs (ncRNAs) including microRNAs and long ncRNAs (lncRNAs) have been fundamentally implicated in CIN. The miR-22, miR-26a, miR-28, and miR-186 target important checkpoint proteins involved in mediating chromosomal stability and their expression modulation has been directly related to CIN occurrence. lncRNAs derived from telomeric, centrosomal, and enhancer regions play an important role in mediating genome stability, while specific lncRNA transcripts including genomic instability inducing RNA called Ginir, P53-responsive lncRNA termed as GUARDIN, colon cancer-associated transcript 2, PCAT2, and ncRNA activated by DNA damage called NORAD have been shown to act within CIN-associated pathways. In this review, we discuss how these ncRNAs either maintain or disrupt the stability of chromosomes and how these mechanisms could be exploited for novel therapeutic approaches targeting CIN in cancer patients. SIGNIFICANCE STATEMENT: Chromosomal instability increases tumor heterogeneity and thereby assists the phenotypic adaptation of cancer cells, causing therapy resistance and metastasis. Several microRNAs and long non-coding RNAs that have been causally linked to chromosomal instability could represent novel therapeutic targets. Understanding the role of non-coding RNAs in regulating different genes involved in driving chromosomal instability will give insights into how non-coding RNAs can be utilized toward modifying chemotherapeutic regimens in different cancers.

ALYREF, a novel factor involved in breast carcinogenesis, acts through transcriptional and post-transcriptional mechanisms selectively regulating the short NEAT1 isoform.

The RNA-binding protein ALYREF (THOC4) is involved in transcriptional regulation and nuclear mRNA export, though its role and molecular mode of action in breast carcinogenesis are completely unknown. Here, we identified high ALYREF expression as a factor for poor survival in breast cancer patients. ALYREF significantly influenced cellular growth, apoptosis and mitochondrial energy metabolism in breast cancer cells as well as breast tumorigenesis in orthotopic mouse models. Transcriptional profiling, phenocopy and rescue experiments identified the short isoform of the lncRNA NEAT1 as a molecular trigger for ALYREF effects in breast cancer. Mechanistically, we found that ALYREF binds to the NEAT1 promoter region to enhance the global NEAT1 transcriptional activity. Importantly, by stabilizing CPSF6, a protein that selectively activates the post-transcriptional generation of the short isoform of NEAT1, as well as by direct binding and stabilization of the short isoform of NEAT1, ALYREF selectively fine-tunes the expression of the short NEAT1 isoform. Overall, our study describes ALYREF as a novel factor contributing to breast carcinogenesis and identifies novel molecular mechanisms of regulation the two isoforms of NEAT1.

Standardisation of protocols can be crucial in long non-coding RNA research.

In this issue, Traversa et al. [1] reviewed our current knowledge about the role of circular and linear forms of PVT1 non-coding RNA in cancer and human diseases. They highlighted the technical challenges of these studies and raised a potential bias in the publications, which require more attention from researchers.

Decrypting noncoding RNA interactions, structures, and functional networks.

The world of noncoding RNAs (ncRNAs) is composed of an enormous and growing number of transcripts, ranging in length from tens of bases to tens of kilobases, involved in all biological processes and altered in expression and/or function in many types of human disorders. The premise of this review is the concept that ncRNAs, like many large proteins, have a multidomain architecture that organizes them spatially and functionally. As ncRNAs are beginning to be imprecisely classified into functional families, we review here how their structural properties might inform their functions with focus on structural architecture-function relationships. We will describe the properties of "interactor elements" (IEs) involved in direct physical interaction with nucleic acids, proteins, or lipids and of "structural elements" (SEs) directing their wiring within the "ncRNA interactor networks" through the emergence of secondary and/or tertiary structures. We suggest that spectrums of "letters" (ncRNA elements) are assembled into "words" (ncRNA domains) that are further organized into "phrases" (complete ncRNA structures) with functional meaning (signaling output) through complex "sentences" (the ncRNA interactor networks). This semiotic analogy can guide the exploitation of ncRNAs as new therapeutic targets through the development of IE-blockers and/or SE-lockers that will change the interactor partners' spectrum of proteins, RNAs, DNAs, or lipids and consequently influence disease phenotypes.