Copper serves as a co-factor for a host of metalloenzymes that contribute to malignant progression. The orally bioavailable copper chelating agent tetrathiomolybdate (TM) has been associated with a ...significant survival benefit in high-risk triple negative breast cancer (TNBC) patients. Despite these promising data, the mechanisms by which copper depletion impacts metastasis are poorly understood and this remains a major barrier to advancing TM to a randomized phase II trial. Here, using two independent TNBC models, we report a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit elevated intracellular copper levels and a marked sensitivity to TM. Global proteomic and metabolomic profiling identifies TM-mediated inactivation of Complex IV as the primary metabolic defect in the SOX2/OCT4+ cell population. We also identify AMPK/mTORC1 energy sensor as an important downstream pathway and show that AMPK inhibition rescues TM-mediated loss of invasion. Furthermore, loss of the mitochondria-specific copper chaperone, COX17, restricts copper deficiency to mitochondria and phenocopies TM-mediated alterations. These findings identify a copper-metabolism-metastasis axis with potential to enrich patient populations in next-generation therapeutic trials.
mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key ...regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.
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•mTORC1-S6K1 and CK1 phosphorylate SRPK2 to induce its nuclear translocation•SRPK2 activates SR proteins and U1-70K to promote splicing of lipogenic transcripts•SRPK2 inhibition results in intron retention and mRNA instability of lipogenic genes•Inhibition of SRPK2 signaling suppresses lipid metabolism and cancer cell growth
An mTOR-dependent pathway is a key post-transcriptional regulator of lipogenic enzymes that are involved in tumor growth.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Aneuploidy causes severe developmental defects and is a near universal feature of tumor cells. Despite its profound effects, the cellular processes affected by aneuploidy are not well characterized. ...Here, we examined the consequences of aneuploidy on the proteome of aneuploid budding yeast strains. We show that although protein levels largely scale with gene copy number, subunits of multi-protein complexes are notable exceptions. Posttranslational mechanisms attenuate their expression when their encoding genes are in excess. Our proteomic analyses further revealed a novel aneuploidy-associated protein expression signature characteristic of altered metabolism and redox homeostasis. Indeed aneuploid cells harbor increased levels of reactive oxygen species (ROS). Interestingly, increased protein turnover attenuates ROS levels and this novel aneuploidy-associated signature and improves the fitness of most aneuploid strains. Our results show that aneuploidy causes alterations in metabolism and redox homeostasis. Cells respond to these alterations through both transcriptional and posttranscriptional mechanisms.
Many proteins that respond to DNA damage are recruited to DNA lesions. We used a proteomics approach that coupled isotopic labeling with chromatin fractionation and mass spectrometry to uncover ...proteins that associate with damaged DNA, many of which are involved in DNA repair or nucleolar function. We show that polycomb group members are recruited by poly(ADP ribose) polymerase (PARP) to DNA lesions following UV laser microirradiation. Loss of polycomb components results in IR sensitivity of mammalian cells and Caenorhabditis elegans. PARP also recruits two components of the repressive nucleosome remodeling and deacetylase (NuRD) complex, chromodomain helicase DNA-binding protein 4 (CHD4) and metastasis associated 1 (MTA1), to DNA lesions. PARP plays a role in removing nascent RNA and elongating RNA polymerase II from sites of DNA damage. We propose that PARP sets up a transient repressive chromatin structure at sites of DNA damage to block transcription and facilitate DNA repair.
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Neoantigens generated by somatic nonsynonymous mutations are key targets of tumor-specific T cells, but only a small number of mutations predicted to be immunogenic are presented by MHC molecules on ...cancer cells. Vaccination studies in mice and patients have shown that the majority of neoepitopes that elicit T cell responses fail to induce significant antitumor activity, for incompletely understood reasons. We report that radiotherapy upregulates the expression of genes containing immunogenic mutations in a poorly immunogenic mouse model of triple-negative breast cancer. Vaccination with neoepitopes encoded by these genes elicited CD8+ and CD4+ T cells that, whereas ineffective in preventing tumor growth, improved the therapeutic efficacy of radiotherapy. Mechanistically, neoantigen-specific CD8+ T cells preferentially killed irradiated tumor cells. Neoantigen-specific CD4+ T cells were required for the therapeutic efficacy of vaccination and acted by producing Th1 cytokines, killing irradiated tumor cells, and promoting epitope spread. Such a cytotoxic activity relied on the ability of radiation to upregulate class II MHC molecules as well as the death receptors FAS/CD95 and DR5 on the surface of tumor cells. These results provide proof-of-principle evidence that radiotherapy works in concert with neoantigen vaccination to improve tumor control.
Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent ...stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
A mechanistic understanding of signaling networks requires identification and analysis of phosphorylation sites. Mass spectrometry offers a rapid and highly sensitive approach to mapping ...phosphorylation sites. However, mass spectrometry has significant limitations that must be considered when planning to carry out phosphorylation-site mapping. Here we provide an overview of key information that should be taken into consideration before beginning phosphorylation-site analysis, as well as a step-by-step guide for carrying out successful experiments.
Execution of the DNA damage response (DDR) relies upon a dynamic array of protein modifications. Using quantitative proteomics, we have globally profiled ubiquitination, acetylation, and ...phosphorylation in response to UV and ionizing radiation. To improve acetylation site profiling, we developed the strategy FACET-IP. Our datasets of 33,500 ubiquitination and 16,740 acetylation sites provide valuable insight into DDR remodeling of the proteome. We find that K6- and K33-linked polyubiquitination undergo bulk increases in response to DNA damage, raising the possibility that these linkages are largely dedicated to DDR function. We also show that Cullin-RING ligases mediate 10% of DNA damage-induced ubiquitination events and that EXO1 is an SCF-Cyclin F substrate in the response to UV radiation. Our extensive datasets uncover additional regulated sites on known DDR players such as PCNA and identify previously unknown DDR targets such as CENPs, underscoring the broad impact of the DDR on cellular physiology.
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•Proteomic discovery of DNA damage-regulated ubiquitination and acetylation sites•FACET-IP approach improves acetylation site detection•Atypical K6- and K33-polyubiquitin linkages increase in response to UV radiation•SCF-Cyclin F mediates EXO1 ubiquitination in response to UV radiation
Elia et al. have globally profiled ubiquitination, acetylation, and phosphorylation in the DNA damage response (DDR). Their valuable datasets of 33,500 ubiquitination and 16,740 acetylation sites implicate K6- and K33-linked polyubiquitination in the DDR, demonstrate that CRLs mediate 10% of DDR ubiquitination events, and reveal that EXO1 is an SCF-Cyclin F substrate.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
To further our understanding of immunopeptidomics, improved tools are needed to identify peptides presented by major histocompatibility complex class I (MHC-I). Many existing tools are limited by ...their reliance upon chemical affinity data, which is less biologically relevant than sampling by mass spectrometry, and other tools are limited by incomplete exploration of machine learning approaches. Herein, we assemble publicly available data describing human peptides discovered by sampling the MHC-I immunopeptidome with mass spectrometry and use this database to train random forest classifiers (ForestMHC) to predict presentation by MHC-I.
As measured by precision in the top 1% of predictions, our method outperforms NetMHC and NetMHCpan on test sets, and it outperforms both these methods and MixMHCpred on new data from an ovarian carcinoma cell line. We also find that random forest scores correlate monotonically, but not linearly, with known chemical binding affinities, and an information-based analysis of classifier features shows the importance of anchor positions for our classification. The random-forest approach also outperforms a deep neural network and a convolutional neural network trained on identical data. Finally, we use our large database to confirm that gene expression partially determines peptide presentation.
ForestMHC is a promising method to identify peptides bound by MHC-I. We have demonstrated the utility of random forest-based approaches in predicting peptide presentation by MHC-I, assembled the largest known database of MS binding data, and mined this database to show the effect of gene expression on peptide presentation. ForestMHC has potential applicability to basic immunology, rational vaccine design, and neoantigen binding prediction for cancer immunotherapy. This method is publicly available for applications and further validation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Hippo pathway controls metazoan organ growth by regulating cell proliferation and apoptosis. Many components have been identified, but our knowledge of the composition and structure of this ...pathway is still incomplete. Using existing pathway components as baits, we generated by mass spectrometry a high-confidence Drosophila Hippo protein-protein interaction network (Hippo-PPIN) consisting of 153 proteins and 204 interactions. Depletion of 67% of the proteins by RNA interference regulated the transcriptional coactivator Yorkie (Yki) either positively or negatively. We selected for further characterization a new member of the alpha-arrestin family, Leash, and show that it promotes degradation of Yki through the lysosomal pathway. Given the importance of the Hippo pathway in tumor development, the Hippo-PPIN will contribute to our understanding of this network in both normal growth and cancer.
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