The RNF43_p.G659fs mutation occurs frequently in colorectal cancer, but its function remains poorly understood and there are no specific therapies directed against this alteration. In this study, we ...find that RNF43_p.G659fs promotes cell growth independent of Wnt signaling. We perform a drug repurposing library screen and discover that cells with RNF43_p.G659 mutations are selectively killed by inhibition of PI3K signaling. PI3K/mTOR inhibitors yield promising antitumor activity in RNF43
isogenic cell lines and xenograft models, as well as in patient-derived organoids harboring RNF43_p.G659fs mutations. We find that RNF43
binds p85 leading to increased PI3K signaling through p85 ubiquitination and degradation. Additionally, RNA-sequencing of RNF43
isogenic cells reveals decreased interferon response gene expression, that is reversed by PI3K/mTOR inhibition, suggesting that RNF43
may alter tumor immunity. Our findings suggest a therapeutic application for PI3K/mTOR inhibitors in treating RNF43_p.G659fs mutant cancers.
Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these ...lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis.
Abstract
DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express ...elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.
The angiogenic switch, the time at which a tumor becomes vascularized, is a critical step in tumor progression. Indeed, without blood supply, tumors will fail to grow beyond 1 mm
and are unlikely to ...disseminate. The extracellular matrix (ECM), a major component of the tumor microenvironment, is known to undergo significant changes during angiogenesis and tumor progression. However the extent of these changes remains unknown. In this study, we used quantitative proteomics to profile the composition of the ECM of pancreatic islets in a mouse model of insulinoma characterized by a precisely timed angiogenic switch. Out of the 120 ECM proteins quantified, 35 were detected in significantly different abundance as pancreatic islets progressed from being hyperplastic to angiogenic to insulinomas. Among these, the core ECM proteins, EFEMP1, fibrillin 1, and periostin were found in higher abundance, and decorin, Dmbt1, hemicentin, and Vwa5 in lower abundance. The angiogenic switch being a common feature of solid tumors, we propose that some of the proteins identified represent potential novel anti-angiogenic targets. In addition, we report the characterization of the ECM composition of normal pancreatic islets and propose that this could be of interest for the design of tissue-engineering strategies for treatment of diabetes.
Abstract
RNA-protein interactions determine the cellular fate of RNA and are central to regulating gene expression outcomes in health and disease. To date, no method exists that is able to identify ...proteins that interact with specific regions within endogenous RNAs in live cells. Here, we develop SHIFTR (Selective RNase H-mediated interactome framing for target RNA regions), an efficient and scalable approach to identify proteins bound to selected regions within endogenous RNAs using mass spectrometry. Compared to state-of-the-art techniques, SHIFTR is superior in accuracy, captures minimal background interactions and requires orders of magnitude lower input material. We establish SHIFTR workflows for targeting RNA classes of different length and abundance, including short and long non-coding RNAs, as well as mRNAs and demonstrate that SHIFTR is compatible with sequentially mapping interactomes for multiple target RNAs in a single experiment. Using SHIFTR, we comprehensively identify interactions of cis-regulatory elements located at the 5′ and 3′-terminal regions of authentic SARS-CoV-2 RNAs in infected cells and accurately recover known and novel interactions linked to the function of these viral RNA elements. SHIFTR enables the systematic mapping of region-resolved RNA interactomes for any RNA in any cell type and has the potential to revolutionize our understanding of transcriptomes and their regulation.
Graphical Abstract
Graphical Abstract
The extracellular matrix (ECM) is a major component of tumors and a significant contributor to cancer progression. In this study, we use proteomics to investigate the ECM of human mammary carcinoma ...xenografts and show that primary tumors of differing metastatic potential differ in ECM composition. Both tumor cells and stromal cells contribute to the tumor matrix and tumors of differing metastatic ability differ in both tumor- and stroma-derived ECM components. We define ECM signatures of poorly and highly metastatic mammary carcinomas and these signatures reveal up-regulation of signaling pathways including TGFβ and VEGF. We further demonstrate that several proteins characteristic of highly metastatic tumors (LTBP3, SNED1, EGLN1, and S100A2) play causal roles in metastasis, albeit at different steps. Finally we show that high expression of LTBP3 and SNED1 correlates with poor outcome for ER(-)/PR(-)breast cancer patients. This study thus identifies novel biomarkers that may serve as prognostic and diagnostic tools. DOI: http://dx.doi.org/10.7554/eLife.01308.001.
Pancreatic ductal adenocarcinoma (PDAC) has a collagen-rich dense extracellular matrix (ECM) that promotes malignancy of cancer cells and presents a barrier for drug delivery. Data analysis of our ...published mass spectrometry (MS)-based studies on enriched ECM from samples of progressive PDAC stages reveal that the C-terminal prodomains of fibrillar collagens are partially uncleaved in PDAC ECM, suggesting reduced procollagen C-proteinase activity. We further show that the enzyme responsible for procollagen C-proteinase activity, bone morphogenetic protein1 (BMP1), selectively suppresses tumor growth and metastasis in cells expressing high levels of COL1A1. Although BMP1, as a secreted proteinase, promotes fibrillar collagen deposition from both cancer cells and stromal cells, only cancer-cell-derived procollagen cleavage and deposition suppresses tumor malignancy. These studies reveal a role for cancer-cell-derived fibrillar collagen in selectively restraining tumor growth and suggest stratification of patients based on their tumor epithelial collagen I expression when considering treatments related to perturbation of fibrillar collagens.
It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations ...affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.
The metazoan mitochondrial translation machinery is unusual in having a single tRNAMet that fulfills the dual role of the initiator and elongator tRNAMet. A portion of the Met-tRNAMet pool is ...formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNAMet (fMet-tRNAmet), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNAMet levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of Met-tRNAMet formylation.
► Mutations in MTFMT cause Leigh syndrome and combined OXPHOS deficiency ► Fibroblasts from patients with mutations in MTFMT have abnormal tRNAMet pools ► Met-tRNAMet formylation is critical for efficient human mitochondrial translation
Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses
; however, the relationship between phenotypic characteristics and TCR ...properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8
T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.
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