73 research outputs found
Mesenchymal stromal cell activation by breast cancer secretomes in bioengineered 3D microenvironments
Mesenchymal stromal cells (MSCs) are key contributors of the tumour microenvironment and are known to promote cancer progression through reciprocal communication with cancer cells, but how they become activated is not fully understood. Here, we investigate how breast cancer cells from different stages of the metastatic cascade convert MSCs into tumour-associated MSCs (TA-MSCs) using unbiased, global approaches. Using mass spectrometry, we compared the secretomes of MCF-7 cells, invasive MDA-MB-231 cells, and sublines isolated from bone, lung, and brain metastases and identified ECM and exosome components associated with invasion and organ-specific metastasis. Next, we used synthetic hydrogels to investigate how these different secretomes activate MSCs in bioengineered 3D microenvironments. Using kinase activity profiling and RNA sequencing, we found that only MDA-MB-231 breast cancer secretomes convert MSCs into TA-MSCs, resulting in an immunomodulatory phenotype that was particularly prominent in response to bone-tropic cancer cells. We have investigated paracrine signalling from breast cancer cells to TA-MSCs in 3D, which may highlight new potential targets for anticancer therapy approaches aimed at targeting tumour stroma
CoreFlow: A computational platform for integration, analysis and modeling of complex biological data
Initial recommendations for performing, benchmarking, and reporting single-cell proteomics experiments
Analyzing proteins from single cells by tandem mass spectrometry (MS) has
become technically feasible. While such analysis has the potential to
accurately quantify thousands of proteins across thousands of single cells, the
accuracy and reproducibility of the results may be undermined by numerous
factors affecting experimental design, sample preparation, data acquisition,
and data analysis. Broadly accepted community guidelines and standardized
metrics will enhance rigor, data quality, and alignment between laboratories.
Here we propose best practices, quality controls, and data reporting
recommendations to assist in the broad adoption of reliable quantitative
workflows for single-cell proteomics.Comment: Supporting website: https://single-cell.net/guideline
Unravelling the GSK3β-related genotypic interaction network influencing hippocampal volume in recurrent major depressive disorder
Objective
Glycogen synthase kinase 3β (GSK3β) has been implicated in mood disorders. We previously reported associations between a GSK3β polymorphism and hippocampal volume in major depressive disorder (MDD). We then reported similar associations for a subset of GSK3β-regulated genes. We now investigate a comprehensive list of genes encoding proteins that directly interact with GSK3β to identify a genotypic network influencing hippocampal volume in MDD.
Participants and methods
We used discovery (N=141) and replication (N=77) recurrent MDD samples. Our gene list was generated from the NetworKIN database. Hippocampal measures were derived using an optimized Freesurfer protocol. We identified interacting single nucleotide polymorphisms using the machine learning algorithm Random Forest and verified interactions using likelihood ratio tests between nested linear regression models.
Results
The discovery sample showed multiple two-single nucleotide polymorphism interactions with hippocampal volume. The replication sample showed a replicable interaction (likelihood ratio test: P=0.0088, replication sample; P=0.017, discovery sample; Stoufferâs combined P=0.0007) between genes associated previously with endoplasmic reticulum stress, calcium regulation and histone modifications.
Conclusion
Our results provide genetic evidence supporting associations between hippocampal volume and MDD, which may reflect underlying cellular stress responses. Our study provides evidence of biological mechanisms that should be further explored in the search for disease-modifying therapeutic targets for depression
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Identification of the global miR-130a targetome reveals a role for TBL1XR1 in hematopoietic stem cell self-renewal and t(8;21) AML
Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSCs) point to shared core stemness properties. However, discordance between mRNA and protein signatures highlights an important role for post-transcriptional regulation by microRNAs (miRNAs) in governing this critical nexus. Here, we identify miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impairs B lymphoid differentiation and expands long-term HSCs. Integration of protein mass spectrometry and chimeric AGO2 crosslinking and immunoprecipitation (CLIP) identifies TBL1XR1 as a primary miR-130a target, whose loss of function phenocopies miR-130a overexpression. Moreover, we report that miR-130a is highly expressed in t(8;21) acute myeloid leukemia (AML), where it is critical for maintaining the oncogenic molecular program mediated by the AML1-ETO complex. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of genes and molecular networks underpinning stemness properties of normal and leukemic cells
miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells
SummaryTo investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance
The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase
Tumour metastasis is a complex process involving reciprocal interplay
between cancer cells and host stroma at both primary and secondary
sites, and is strongly influenced by microenvironmental
factors such as hypoxia. Tumour-secreted proteins play a crucial role
in these interactions and present strategic therapeutic potential.
Metastasis of breast cancer to the bone affects approximately 85%
of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed,
lead to debilitating skeletal complications and increased patient morbidity
and mortality. The molecular interactions governing the
early events of osteolytic lesion formation are currently unclear.
Here we show hypoxia to be specifically associated with bone relapse
in patients with oestrogen-receptor negative breast cancer. Global
quantitative analysis of the hypoxic secretome identified lysyl oxidase
(LOX) as significantly associated with bone-tropism and relapse.
High expression of LOX in primary breast tumours or systemic delivery
of LOX leads to osteolytic lesion formation whereas silencing or
inhibition of LOX activity abrogates tumour-driven osteolytic lesion
formation. We identify LOX as a novel regulator of NFATc1-driven
osteoclastogenesis,independent of RANK ligand, which disrupts normal
bone homeostasisleading to the formation of focal pre-metastatic
lesions. We show that these lesions subsequently provide a platform
for circulating tumour cells to colonize and form bone metastases.
Our study identifies a novel mechanism of regulation of bone homeostasis
and metastasis, opening up opportunities for novel therapeutic
intervention with important clinical implications
Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics
Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed. 2014 American Chemical Societ
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