Proteomic profiling of oncogene-induced HCC in mouse models
Ontology highlight
ABSTRACT: Oncogene activation drives changes in pathways related to cell proliferation, metabolism, and immune evasion, offering insights into molecular events underpinning hepatocarcinogenesis
Project description:Enhancers play key roles in gene regulation. However, comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Through gene regulatory network modeling, we identified that dynamic cell state transitions, a critical missing component in prevalent enhancer discovery strategies, can be utilized to improve the cells’ sensitivity to enhancer perturbation. Guided by the modeling results, we performed a mid-transition CRISPRi-based enhancer screen utilizing human embryonic stem cell definitive endoderm differentiation as a dynamic transition system. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core lineage-specifying transcription factors (TFs), including many enhancers with weak to moderate effects. Integrating the screening results with enhancer activity measurements (ATAC-seq, H3K27ac ChIP-seq) and three-dimensional enhancer-promoter interaction information (CTCF looping, Hi-C), we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Together, our dynamic network-guided enhancer screen and the CIA enhancer prediction model provide generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.
Project description:Emergomyces africanus is a thermally dimorphic fungus and one of the leading causes of emergomycosis, a neglected infection primarily affecting immunocompromised individuals. Despite its clinical relevance, how E. africanus adapts to the host environment remains unknown. Recent studies suggest that extracellular vesicles (EVs) may play a key role during host adaptation by modulating immune responses and transporting virulence factors. It remains unknown whether E. africanus produces EVs. Here, we demonstrated EV production in E. africanus and investigated their size and composition after cultivation in nutrient-rich and nutrient-limited media, mimicking environmental and host-like conditions. In addition, we evaluated the effect of E. africanus EVs released in nutrient-limited media on bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMDMs). Under nutrient limitation, E. africanus released EVs enriched in proteins that, in other models, are virulence-associated, including catalase, HSP60, and chitinase, whereas EVs from rich media carried proteins linked to anabolic pathways. The presence of -1,3-glucans and a higher content of chitin-like structures were also detected in EVs released in nutrient-limited conditions. EVs from nutrient-limited conditions activated BMDCs, increasing MHC-II and CD40 expression and promoting a pro-inflammatory cytokine profile (IL-6 and TNF-α). In contrast, BMDMs exhibited elevated IL-10 levels, suggesting an anti-inflammatory shift. Remarkably, EV pre-treatment enhanced BMDM antifungal activity, significantly reducing E. africanus viability post-infection. These findings show that E. africanus dynamically adjust their EVs cargo in response to environmental cues, directly influencing immune modulation and fungal survival. Indeed, pre-treatment of the insect Galleria mellonella with these EVs induced a protective response against a lethal inoculum of Histoplasma capsulatum. This study reinforces the potential of EVs as therapeutic targets and offers new insights into how E. africanus adapts to the host environment.
Project description:Bone marrow mesenchymal stromal cells (MSCs) are a major source of secreted factors that control hematopoietic stem and progenitor cell (HSPC) function. We previously reported the generation of revitalized MSCs (rMSCs), which support functional HSCs in culture more effectively than control MSCs. In a secretomic screen using rMSCs, we identified semaphorin 3A (SEM3A) as a secreted factor upregulated as part of a pro-inflammatory signature that may underly HSPC expansion by rMSCs. Similarly, SEM3A expression is upregulated by BM-MSCs in vivo in response to hematopoietic stress. Recombinant SEM3A directly promotes HSPC quiescence ex vivo. Analysis of a SEM3A loss of function mutation in vivo revealed hematopoietic progenitor expansion and accelerated recovery after myeloablation, consistent with a role for SEM3A in regulating the HSPC stress response. This work highlights proteomic screening using rMSCs as a method to identify novel secreted niche factors and uncovers a novel role for SEM3A in promoting HSPC quiescence in stress hematopoiesis.
Project description:Chromatin architecture plays a key role in development and cancer, yet most studies lack mechanistic depth due to widespread epigenomic remodeling. To address this, we tracked chromatin structure dynamics during the progression of endocrine resistance in ER+ breast cancer using Hi-C, chromatin accessibility, epigenomic, and transcriptomic pro_iling. We uncovered a critical role for H3K9 methylation and the demethylase KDM4C association with SWI/SNF in driving proliferation of cells fated to become resistant through a non-genomic estrogen-mediated mechanism. These _indings highlight the mechanistic contribution of chromatin regulation in therapy resistance and offer a blueprint for studying similar processes in cancer, development, and cell fate decisions.
Project description:Aging somatic cells are characterized by specific chromosome aneuploidy. This work examines the role of DNA replication within the centromeres of chromosomes Y (ChrY) and 21 (Chr21). The aneuploidy of these two chromosomes is associated with Alzheimer's Disease (AD) pathology. Using human neural progenitor cells engineered to overexpress wild-type (wt) and pseudo-hyper-phosphorylated (php) Tau proteins, we developed a novel method to analyze replication dynamics in centromeric regions. Our findings reveal overexpression reduces the replication initiation events that are active within alpha satellite sequences in control condition. Mass spectrometry analysis on immunoprecipitated Tau identified nuclear interactors of Tau, particularly in its php form, which might directly influence chromatin architecture and gene expression. This research provides critical insights into the molecular mechanisms of aneuploidy in tauopathies.
Project description:Epigenetic mechanisms tightly regulate gene expression at the chromatin level and have recently been found to colocalize with the splicing machinery during active transcription; however, the precise functional consequences of these interactions are uncertain. Here, we identify unique interactions of the CoREST repressor complex with the RNA splicing machinery and their functional consequences. Using mass spectrometry, in vivo binding assays, and Cryo-EM we find that CoREST-splicing factor interactions are direct and perturbed by the bivalent inhibitor, corin, leading to robust changes in RNA splicing in melanoma and other malignancies. Moreover, these corin-associated splicing changes are shown to promote global effects on oncogenic and survival-associated splice variants leading to a tumor-suppressive phenotype. Using predictive machine learning models, MHC IP-MS, and ELISpot assays we identify thousands of neopeptides derived from unannotated splice sites which generate corin-induced splice neoantigens that are demonstrated to be immunogenic in vitro. Corin is further shown to inhibit tumor growth and enhance checkpoint blockade in a manner dependent on host T cells that trigger an anti-tumor T cell response in vivo and promote dramatic expansion of cytotoxic T cells in an immune cold melanoma tumor model, effectively sensitizing them to anti-PD1 immunotherapy. These data position CoREST inhibition as a unique therapeutic opportunity in cancer which reverses oncogenic splicing programs across broad tumor types while also creating tumor-associated neoantigens which may enhance the immunogenicity of current therapeutics and may be readily translated to the clinic.
Project description:To identify the subproteome degraded by lysosomes in human cancer cells, we compared whole cell proteomes of wild-type and autophagy-deficient non-small cell lung cancer cells in the presence or absence of a lysosomal degradation inhibitor (N/L). Further, we compared whole cell proteomes of cells treated with a selective autophagy inhibitor in the presence or absence of N/L to identify lysosome substrate proteins that fail to be degraded upon treatment with this small molecule.
Project description:Intrauterine growth restriction (IUGR) is associated with increased risk of cardiometabolic disease later in life and have been shown to affects female and male offspring differently, but the mechanisms remain unclear. The purpose of this study was to identify proteomic differences and metabolic risk markers in IUGR from male and female neonates when compare with appropriate for gestational age (AGA) that will provide a better understanding of IUGR pathogenesis and its associated risks. Our result revealed alterations in IUGR cord plasma proteomes with most of the enriched proteins implicated in peroxisome pathways. This effect was evident in females but not in males. Furthermore, we observed that catalase activity, peroxisome enzyme, was significantly increased in females (P<0.05) but unchanged in males. Finally, we identified risk proteins associated with obesity, type-2 diabetes, and glucose intolerance such as EGF containing fibulin extracellular matrix protein 1, proprotein convertase subtilisin/kexin type 9 (PCSK9) and transforming growth factor beta receptor 3 (TGFBR3) proteins unique to females while coagulation factor IX (C9) and retinol binding protein 4 (RBP4) are for males. Our data reveal that IUGR may display sexual dimorphism which may be associated with differences in lifelong risk for cardiometabolic disease between males and females.
Project description:The strongest risk factors for Alzheimer’s disease (AD) include the 4 allele of apolipoprotein E (APOE), the R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), and female sex. Here, we combine APOE4 and TREM2R47H (R47H) in female P301S tauopathy mice to identify the pathways activated when AD risk is the strongest, thereby highlighting detrimental disease mechanisms. We find that the R47H variant induces neurodegeneration in 9- to- 10-month-old female APOE4 tauopathy mice. The combination of APOE4 and R47H (APOE4-R47H) worsened hyperphosphorylated tau pathology in the frontal cortex and amplified tauopathy-induced cell-autonomous microglial cGAS-STING signaling and downstream interferon response. APOE4-R47H microglia displayed cGAS- and BAX-dependent upregulation of senescence, showing association between neurotoxic signatures and implicating mitochondrial permeabilization in pathogenesis. By uncovering pathways enhanced by the strongest AD risk factors, our study points to cGAS-STING signaling and associated microglial senescence as potential drivers of AD risk.
Project description:Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase responsible for ubiquitination and proteasome degradation of substrate p27, thus driving cellular proliferation. We have shown previously that knockout of Skp2 in an immunocompetent transgenic mouse model of OS improved survival, drove apoptosis, and induced anti-tumor immunity. Here, we applied single-cell RNA-sequencing (scRNA-seq) to study primary OS tumors derived from Osx-Cre driven conditional knockout of Rb1 and Trp53 in mice. We further compared this model with OS models with functional disruption of Skp2: one with Skp2 knockout and the other with the Skp2-p27 interaction disrupted (resulting in p27 overexpression). We report that murine OS models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. Skp2 disruption led to reduction of T cell exhaustion and upregulation of interferon signaling, as well as induction of cell-type specific replicative and endoplasmic reticulum stress, which we corroborated via mass spectrometry proteomics. Further, we observed that interferon induction was correlated with improved survival in OS patients. Additionally, our scRNA-seq analysis uncovered decreased expression of metastasis-related gene signatures in Skp2-disrupted OS, which we validated by observation of a strong reduction in lung metastasis in the Skp2 knockout mice. Finally, we report several potential mechanisms of escape from targeting Skp2 in OS, including upregulation of Myc targets, induction of genomic instability, overexpression of alternative E3 ligases, and lineage plasticity. These mechanistic insights into OS tumor biology and Skp2 function suggest novel targets for new, synergistic therapies, while the data and our comprehensive analysis may serve as a public resource for further big data-driven OS research.