During metastatic progression, circulating cancer cells become lodged within the microvasculature of end organs, where most die from mechanical deformation. Although this phenomenon was first ...described over a half-century ago, the mechanisms enabling certain cells to survive this metastasis-suppressive barrier remain unknown. By applying whole-transcriptome RNA-sequencing technology to isogenic cancer cells of differing metastatic capacities, we identified a mutation encoding a truncated form of the pannexin-1 (PANX1) channel, PANX1(1-89), as recurrently enriched in highly metastatic breast cancer cells. PANX1(1-89) functions to permit metastatic cell survival during traumatic deformation in the microvasculature by augmenting ATP release from mechanosensitive PANX1 channels activated by membrane stretch. PANX1-mediated ATP release acts as an autocrine suppressor of deformation-induced apoptosis through P2Y-purinergic receptors. Finally, small-molecule therapeutic inhibition of PANX1 channels is found to reduce the efficiency of breast cancer metastasis. These data suggest a molecular basis for metastatic cell survival on microvasculature-induced biomechanical trauma.
Metastatic colonization is the primary cause of death from colorectal cancer (CRC). We employed genome-scale in vivo short hairpin RNA (shRNA) screening and validation to identify 26 promoters of CRC ...liver colonization. Among these genes, we identified a cluster that contains multiple targetable genes, including ITPR3, which promoted liver-metastatic colonization and elicited similar downstream gene expression programs. ITPR3 is a caffeine-sensitive inositol 1,4,5-triphosphate (IP3) receptor that releases calcium from the endoplasmic reticulum and enhanced metastatic colonization by inducing expression of RELB, a transcription factor that is associated with non-canonical NF-κB signaling. Genetic, cell biological, pharmacologic, and clinical association studies revealed that ITPR3 and RELB drive CRC colony formation by promoting cell survival upon substratum detachment or hypoxic exposure. RELB was sufficient to drive colonization downstream of ITPR3. Our findings implicate the ITPR3/calcium/RELB axis in CRC metastatic colony formation and uncover multiple clinico-pathologically associated targetable proteins as drivers of CRC metastatic colonization.
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•Loss of ITPR3 in colorectal cancer cells inhibits metastatic liver colonization•RELB acts downstream of ITPR3/calcium to promote colorectal cancer liver metastasis•ITPR3 and RELB regulate survival in response to hypoxia and substratum detachment•Exposure of colorectal cancer cells to ITPR3 inhibitor caffeine reduces liver metastasis
Moy et al. define a clinically relevant pathway that promotes colorectal cancer cell survival and liver metastasis via the type 3 inositol 1,4,5-triphosphate receptor ITPR3, a calcium channel that controls calcium release from the endoplasmic reticulum, and RELB, a transcription factor that is associated with non-canonical NF-κB signaling.
Colorectal cancer (CRC) is a leading cause of cancer mortality. Creatine metabolism was previously shown to critically regulate colon cancer progression. We report that RGX-202, an oral ...small-molecule SLC6A8 transporter inhibitor, robustly inhibits creatine import in vitro and in vivo, reduces intracellular phosphocreatine and ATP levels, and induces tumor apoptosis. RGX-202 suppressed CRC growth across KRAS wild-type and KRAS mutant xenograft, syngeneic, and patient-derived xenograft (PDX) tumors. Antitumor efficacy correlated with tumoral expression of creatine kinase B. Combining RGX-202 with 5-fluorouracil or the DHODH inhibitor leflunomide caused regressions of multiple colorectal xenograft and PDX tumors of distinct mutational backgrounds. RGX-202 also perturbed creatine metabolism in patients with metastatic CRC in a phase 1 trial, mirroring pharmacodynamic effects on creatine metabolism observed in mice. This is, to our knowledge, the first demonstration of preclinical and human pharmacodynamic activity for creatine metabolism targeting in oncology, thus revealing a critical therapeutic target.
Tumourigenesis and cancer progression require enhanced global protein translation
. Such enhanced translation is caused by oncogenic and tumour-suppressive events that drive the synthesis and ...activity of translational machinery
. 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.
Colorectal cancer metastasis to the liver is a major cause of cancer-related death; however, the genes and pathways that govern this metastatic colonization event remain poorly characterized. Here, ...using a large-scale in vivo RNAi screen, we identified liver and red blood cell pyruvate kinase (PKLR) as a driver of metastatic liver colonization. PKLR expression was increased in liver metastases as well as in primary colorectal tumors of patients with metastatic disease. Evaluation of a murine liver colonization model revealed that PKLR promotes cell survival in the tumor core during conditions of high cell density and oxygen deprivation by increasing glutathione, the primary endogenous antioxidant. PKLR negatively regulated the glycolytic activity of PKM2, the major pyruvate kinase isoenzyme known to regulate cellular glutathione levels. Glutathione is critical for metastasis, and we determined that the rate-limiting enzyme of glutathione synthesis, GCLC, becomes overexpressed in patient liver metastases, promotes cell survival under hypoxic and cell-dense conditions, and mediates metastatic liver colonization. RNAi-mediated inhibition of glutathione synthesis impaired survival of multiple colon cancer cell lines, and pharmacological targeting of this metabolic pathway reduced colonization in a primary patient-derived xenograft model. Our findings highlight the impact of metabolic reprogramming within the niche as metastases progress and suggest clinical potential for targeting this pathway in colorectal cancer.
Eukaryotic transfer RNAs can become selectively fragmented upon various stresses, generating tRNA‐derived small RNA fragments. Such fragmentation has been reported to impact a small fraction of the ...tRNA pool and thus presumed to not directly impact translation. We report that oxidative stress can rapidly generate tyrosine‐tRNAGUA fragments in human cells—causing significant depletion of the precursor tRNA. Tyrosine‐tRNAGUA depletion impaired translation of growth and metabolic genes enriched in cognate tyrosine codons. Depletion of tyrosine tRNAGUA or its translationally regulated targets USP3 and SCD repressed proliferation—revealing a dedicated tRNA‐regulated growth‐suppressive pathway for oxidative stress response. Tyrosine fragments are generated in a DIS3L2 exoribonuclease‐dependent manner and inhibit hnRNPA1‐mediated transcript destabilization. Moreover, tyrosine fragmentation is conserved in C. elegans. Thus, tRNA fragmentation can coordinately generate trans‐acting small RNAs and functionally deplete a tRNA. Our findings reveal the existence of an underlying adaptive codon‐based regulatory response inherent to the genetic code.
Synopsis
Whether tRNAs affect gene regulation beyond their central role as adaptors in mRNA translation remains poorly understood. This study uncovers a non‐canonical function for fragments of tyrosine‐coding tRNAGUA in controlling expression of growth promoting genes upon stress.
Oxidative stress‐induced tRNA fragmentation depletes both precursor and mature tRNATyrGUA in human cell lines.
tRNATyrGUA depletion impairs translation of growth genes that are enriched for its cognate tyrosine codons.
Tyrosine‐tRNA fragments are generated in a DIS3L2 ribonuclease‐dependent manner and inhibit RNA‐binding protein hnRNPA1.
Precursor tRNAGUA fragmentation response is conserved in C. elegans.
Ribonuclease‐generated fragments of one particular tRNA control expression of growth‐promoting genes upon stress.
The human genome contains 61 codons encoding 20 amino acids. Synonymous codons representing a given amino acid are decoded by a set of transfer RNAs (tRNAs) called isoacceptors. We report the ...surprising observation that two isoacceptor tRNAs that decode synonymous codons become modulated in opposing directions during breast cancer progression. Specifically, tRNA
became upregulated, whereas tRNA
became repressed as breast cancer cells attained enhanced metastatic capacity. Functionally, tRNA
promoted and tRNA
suppressed metastatic colonization in mouse xenograft models. These tRNAs mediated opposing effects on codon-dependent translation of growth-promoting genes, consistent with genomic enrichment or depletion of their cognate codons in mitotic genes. Our findings uncover a specific isoacceptor tRNA pair that act in opposition, divergently impacting growth-regulating genes and a disease phenotype. Degeneracy of the genetic code can thus be biologically exploited by human cancer cells via tRNA isoacceptor shifts that causally facilitate the transition toward a growth-promoting state.
N6-methyladenosine (m6A) is the most abundant internal modification of messenger RNA. While the presence of m6A on transcripts can impact nuclear RNA fates, a reader of this mark that mediates ...processing of nuclear transcripts has not been identified. We find that the RNA-binding protein HNRNPA2B1 binds m6A-bearing RNAs in vivo and in vitro and its biochemical footprint matches the m6A consensus motif. HNRNPA2B1 directly binds a set of nuclear transcripts and elicits similar alternative splicing effects as the m6A writer METTL3. Moreover, HNRNPA2B1 binds to m6A marks in a subset of primary miRNA transcripts, interacts with the microRNA Microprocessor complex protein DGCR8, and promotes primary miRNA processing. Also, HNRNPA2B1 loss and METTL3 depletion cause similar processing defects for these pri-miRNA precursors. We propose HNRNPA2B1 to be a nuclear reader of the m6A mark and to mediate, in part, this mark’s effects on primary microRNA processing and alternative splicing.
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•HNRNPA2B1 binds m6A-containing sites and the RGAC motif in nuclear transcripts•HNRNPA2B1 mediates m6A-dependent primary microRNA processing events•Modulation of HNRNPA2B1 and METTL3 causes similar changes to alternative splicing
The RNA-binding protein HNRNPA2B1 is a nuclear “reader” of the m6A mark, acting as an adaptor that recruits the Microprocessor complex to a subset of precursor miRNAs, facilitating their processing into mature miRNAs.
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