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Background Despite advances in early detection and therapies, cancer is still one of the most common causes of death worldwide. Since each tumor is unique, there is a need to implement personalized care and develop robust tools for monitoring treatment response to assess drug efficacy and prevent disease relapse. Main body Recent developments in liquid biopsies have enabled real-time noninvasive monitoring of tumor burden through the detection of molecules shed by tumors in the blood. These molecules include circulating tumor nucleic acids (ctNAs), comprising cell-free DNA or RNA molecules passively and/or actively released from tumor cells. Often highlighted for their diagnostic, predictive, and prognostic potential, these biomarkers possess valuable information about tumor characteristics and evolution. While circulating tumor DNA (ctDNA) has been in the spotlight for the last decade, less is known about circulating tumor RNA (ctRNA). There are unanswered questions about why some tumors shed high amounts of ctNAs while others have undetectable levels. Also, there are gaps in our understanding of associations between tumor evolution and ctNA characteristics and shedding kinetics. In this review, we summarize current knowledge about ctNA biology and release mechanisms and put this information into the context of tumor evolution and clinical utility. Conclusions A deeper understanding of the biology of ctDNA and ctRNA may inform the use of liquid biopsies in personalized medicine to improve cancer patient outcomes.

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.Leidal et al. show that the LC3-conjugation pathway, which is part of the autophagy machinery, controls extracellular vesicle cargo loading and secretion of RNA-binding proteins.

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m 1 and m 2 , reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m 1 isoform with a single-methylated adenosine (2′- O -methyladenosine, Am), which is then converted to a dimethylated m 2 isoform ( N 6 ,2′- O -dimethyladenosine, m 6 Am). The relative m 1 and m 2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m 2 isoform. We show FTO is inhibited by the oncometabolite d -2-hydroxyglutarate, resulting in increased m 2 -snRNA levels. Furthermore, cells that exhibit high m 2 -snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing. Two different methylation states of the adenosine adjacent to the snRNA cap are found in the biogenesis process of snRNAs, Am and m6Am, whose levels are regulated by FTO and are related to alternative pre-mRNA splicing.

The abundance of mRNA is mainly determined by the rates of RNA transcription and decay. Here, we present a method for unbiased estimation of differential mRNA decay rate from RNA-sequencing data by modeling the kinetics of mRNA metabolism. We show that in all primary human tissues tested, and particularly in the central nervous system, many pathways are regulated at the mRNA stability level. We present a parsimonious regulatory model consisting of two RNA-binding proteins and four microRNAs that modulate the mRNA stability landscape of the brain, which suggests a new link between RBFOX proteins and Alzheimer’s disease. We show that downregulation of RBFOX1 leads to destabilization of mRNAs encoding for synaptic transmission proteins, which may contribute to the loss of synaptic function in Alzheimer’s disease. RBFOX1 downregulation is more likely to occur in older and female individuals, consistent with the association of Alzheimer’s disease with age and gender. “mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer’s disease.”

Individual cells within a tumour can exhibit distinct genetic and molecular features. The impact of such diversification on metastatic potential is unknown. Here we identify clonal human breast cancer subpopulations that display different levels of morphological and molecular diversity. Highly variable subpopulations are more proficient at metastatic colonization and chemotherapeutic survival. Through single-cell RNA-sequencing, inter-cell transcript expression variability is identified as a defining feature of the highly variable subpopulations that leads to protein-level variation. Furthermore, we identify high variability in the spliceosomal machinery gene set. Engineered variable expression of the spliceosomal gene SNRNP40 promotes metastasis, attributable to cells with low expression. Clinically, low SNRNP40 expression is associated with metastatic relapse. Our findings reveal transcriptomic variability generation as a mechanism by which cancer subpopulations can diversify gene expression states, which may allow for enhanced fitness under changing environmental pressures encountered during cancer progression. Phenotypic and genetic intra-tumor heterogeneity have an important role in cancer progression and therapeutic resistance. Here, the authors show that phenotypically variable tumor subpopulations exhibit higher metastatic potential and display enhanced intra-clonal transcriptomic variability, likely promoted by deregulated spliceosome activity.

Epidemiological studies have established a positive association between obesity and the incidence of postmenopausal breast cancer. Moreover, it is known that obesity promotes stem cell-like properties of breast cancer cells. However, the cancer cell-autonomous mechanisms underlying this correlation are not well defined. Here we demonstrate that obesity-associated tumor formation is driven by cellular adaptation rather than expansion of pre-existing clones within the cancer cell population. While there is no correlation with specific mutations, cellular adaptation to obesity is governed by palmitic acid (PA) and leads to enhanced tumor formation capacity of breast cancer cells. This process is governed epigenetically through increased chromatin occupancy of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPB). Obesity-induced epigenetic activation of C/EBPB regulates cancer stem-like properties by modulating the expression of key downstream regulators including CLDN1 and LCN2 . Collectively, our findings demonstrate that obesity drives cellular adaptation to PA drives tumor initiation in the obese setting through activation of a C/EBPB dependent transcriptional network. Obesity is linked to cancer risk in post-menopausal breast cancer. At the molecular level this is governed by cellular adaption to palmitic acid through epigenetic activation of a C/EBPB-dependent transcriptional network that drives tumor formation.

Blood vessels support tumours by providing nutrients and oxygen, while also acting as conduits for the dissemination of cancer 1 . Here we use mouse models of breast and lung cancer to investigate whether endothelial cells also have active ‘instructive’ roles in the dissemination of cancer. We purified genetically tagged endothelial ribosomes and their associated transcripts from highly and poorly metastatic tumours. Deep sequencing revealed that metastatic tumours induced expression of the axon-guidance gene Slit2 in endothelium, establishing differential expression between the endothelial (high Slit2 expression) and tumoural (low Slit2 expression) compartments. Endothelial-derived SLIT2 protein and its receptor ROBO1 promoted the migration of cancer cells towards endothelial cells and intravasation. Deleting endothelial Slit2 suppressed metastatic dissemination in mouse models of breast and lung cancer. Conversely, deletion of tumoural Slit2 enhanced metastatic progression. We identified double-stranded RNA derived from tumour cells as an upstream signal that induces expression of endothelial SLIT2 by acting on the RNA-sensing receptor TLR3. Accordingly, a set of endogenous retroviral element RNAs were upregulated in metastatic cells and detected extracellularly. Thus, cancer cells co-opt innate RNA sensing to induce a chemotactic signalling pathway in endothelium that drives intravasation and metastasis. These findings reveal that endothelial cells have a direct instructive role in driving metastatic dissemination, and demonstrate that a single gene ( Slit2 ) can promote or suppress cancer progression depending on its cellular source. Expression of the axon-guidance gene Slit2 in endothelium, induced by endothelial sensing of tumour-derived double-stranded RNA, promotes metastatic dissemination in mouse models of breast and lung cancer.

The commonly mutated human KRAS oncogene encodes two distinct KRAS4A and KRAS4B proteins generated by differential splicing. We demonstrate here that coordinated regulation of both isoforms through control of splicing is essential for development of Kras mutant tumors. The minor KRAS4A isoform is enriched in cancer stem-like cells, where it responds to hypoxia, while the major KRAS4B is induced by ER stress. KRAS4A splicing is controlled by the DCAF15/RBM39 pathway, and deletion of KRAS4A or pharmacological inhibition of RBM39 using Indisulam leads to inhibition of cancer stem cells. Our data identify existing clinical drugs that target KRAS4A splicing, and suggest that levels of the minor KRAS4A isoform in human tumors can be a biomarker of sensitivity to some existing cancer therapeutics. Kras is frequently mutated in lung cancer and two isoforms are generated via alternative splicing. Here, the authors show that the two isoforms have divergent roles in cancer stem cells and the main tumour cell population, which are regulated by hypoxia and endoplasmic reticulum stress.

Colorectal cancer (CRC) is a major cause of human death. Mortality is primarily due to metastatic organ colonization, with the liver being the main organ affected. We modeled metastatic CRC (mCRC) liver colonization using patient-derived primary and metastatic tumor xenografts (PDX). Such PDX modeling predicted patient survival outcomes. In vivo selection of multiple PDXs for enhanced metastatic colonization capacity upregulated the gluconeogenic enzyme PCK1, which enhanced liver metastatic growth by driving pyrimidine nucleotide biosynthesis under hypoxia. Consistently, highly metastatic tumors upregulated multiple pyrimidine biosynthesis intermediary metabolites. Therapeutic inhibition of the pyrimidine biosynthetic enzyme DHODH with leflunomide substantially impaired CRC liver metastatic colonization and hypoxic growth. Our findings provide a potential mechanistic basis for the epidemiologic association of anti-gluconeogenic drugs with improved CRC metastasis outcomes, reveal the exploitation of a gluconeogenesis enzyme for pyrimidine biosynthesis under hypoxia, and implicate DHODH and PCK1 as metabolic therapeutic targets in CRC metastatic progression. Colorectal cancer, also known as bowel cancer, is the second most deadly cancer in the United States, where it affects over 140,000 people each year. This cancer often spreads to the liver, in a process known as metastasis. To do this, the colorectal cancer cells must survive the low oxygen levels found in the blood that carries them from the gut to the liver, and in the liver itself. The majority of colorectal cancer cells that arrive in the liver die, but some survive leading to secondary tumors. To investigate how the colorectal cancer cells that survive and metastasize to the liver accomplish this feat, Yamaguchi, Weinberg, Nguyen et al. took colorectal tumors from patients and introduced them into mice. This showed that tumors from patients with the worst outcomes tended to metastasize more efficiently in mice. Next, Yamaguchi et al. looked to see which genes were active in the colorectal cancer cells that were able to metastasize and compared them to those that were active in the cells that could not. This analysis revealed that the gene coding for a protein called PCK1 was more active in the cells that could metastasize. In healthy cells, PCK1 promotes the generation of the sugar glucose, and Yamaguchi et al. observed that, in a low oxygen environment, higher levels of PCK1 allowed colorectal cancer cells to proliferate faster. Unexpectedly, this was due to PCK1 increasing the production of a molecule needed to make nucleotides, which are the building blocks for DNA and RNA. Consistent with this, metastasizing colorectal cancer cells generated more nucleotide precursors, and inhibiting the enzyme involved in nucleotide synthesis (DHODH) with an arthritis drug called leflunomide stopped colorectal cancer cells from spreading. Metastasizing colorectal cancer cells depend on PCK1 and the nucleotide-synthesizing enzyme to grow, so therapies that target these proteins may help more patients to survive this kind of cancer. The findings also suggest that the arthritis drug leflunomide should be explored further as a potential drug for the treatment of colorectal cancer.

Tumourigenesis and cancer progression require enhanced global protein translation 1 – 3 . Such enhanced translation is caused by oncogenic and tumour-suppressive events that drive the synthesis and activity of translational machinery 4 , 5 . Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes, including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour-suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase—along with its downstream cognate tRNAs—elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth. Passarelli et al. identify leucyl-tRNA synthetase (LARS) as a critical tumour suppressor that upregulates translation of growth-inhibitory genes, including EMP3 and GGT5 , thereby impairing breast cancer development.