Project description:CXCL12 and IGF1 are key secreting molecules produced by cancer-associated fibroblasts in breast cancer. These factors promote the survival of disseminated cancer cells in the bone marrow. To assess the combined responses elicited by CXCL12 and IGF1, we examined the translating transcriptome of cancer cells in response to these two factors by Translating Ribosome Affinity Purification (TRAP)-RNAseq. MDA-MB-231 cells were engineered to express an EGFP-tagged version of ribosomal protein L10a. This allows the retrieval of polysome-associated mRNA by anti-GFP pull down (TRAP) and profiling the translating transcriptome by RNAseq. EGFP-L10a+ cancer cells were serum starved (0.2% serum) for 24 hours, and then treated with CXCL12 (30ng/mL) + IGF1 (10ng/mL) or CXCL12 (300ng/mL) + IGF1 (100ng/mL) for 6hrs. Two biological replicates were profiled for each condition.
Project description:CXCL12 and IGF1 are key secreting molecules produced by cancer-associated fibroblasts in breast cancer. These factors promote the survival of disseminated cancer cells in the bone marrow. To assess the combined responses elicited by CXCL12 and IGF1, we examined the translating transcriptome of cancer cells in response to these two factors by Translating Ribosome Affinity Purification (TRAP)-RNAseq.
Project description:CXCL12 and IGF1 confer on cancer cells survival advantage. Src potentiates cancer cells' reponse to CXCL12 and IGF1 by strengthening AKT activation. Long-term incubation of CXCL12 and IGF1 select for cancer cells with enhanced Src activity and bone metastasis potential. MDA-MB-231 cells were incubated for three weeks in growth medium of reduced serum concentration (0.2%) with or without CXCL12 (30 ng/ml) and IGF1 (10 ng/ml). Cell populations survive under these condidtions are expanded by regular growth medium with 10% serum. Two biological replicates were profiled for each condition.
Project description:CXCL12 and IGF1 confer on cancer cells survival advantage. Src potentiates cancer cells' reponse to CXCL12 and IGF1 by strengthening AKT activation. Long-term incubation of CXCL12 and IGF1 select for cancer cells with enhanced Src activity and bone metastasis potential.
Project description:How organ-specific metastatic traits accumulate in primary tumors remains unknown. We identified a role of the primary tumor stroma in selecting breast cancer cells that are primed for metastasis in the bone. A fibroblast-rich stroma in breast tumors creates a microenvironment that is similar to that of bone metastases in its abundance of the cytokines CXCL12 and IGF1. Heterogeneous breast cancer cell populations growing in such mesenchymal environment evolve towards a preponderance of clones that thrive on CXCL12 and IGF1. Fibroblast-driven selection of bone metastatic clones in mammary tumors is suppressed by CXCL12 and IGF1 receptor inhibition. Thus, a fibroblast-rich stroma in breast tumors can pre-select bone metastatic seeds, promoting the evolution of metastatic traits and the interplay between a primary tumor and its distant metastases. Affymetrix U133 Plus2 arrays were hybridized according to the manufacturer's procedure using RNA extracted from 47 primary breast tumors. Specific gene sets were evaluated in this cohort.
Project description:How organ-specific metastatic traits accumulate in primary tumors remains unknown. We identified a role of the primary tumor stroma in selecting breast cancer cells that are primed for metastasis in the bone. A fibroblast-rich stroma in breast tumors creates a microenvironment that is similar to that of bone metastases in its abundance of the cytokines CXCL12 and IGF1. Heterogeneous breast cancer cell populations growing in such mesenchymal environment evolve towards a preponderance of clones that thrive on CXCL12 and IGF1. Fibroblast-driven selection of bone metastatic clones in mammary tumors is suppressed by CXCL12 and IGF1 receptor inhibition. Thus, a fibroblast-rich stroma in breast tumors can pre-select bone metastatic seeds, promoting the evolution of metastatic traits and the interplay between a primary tumor and its distant metastases.
Project description:To identify RBPs with previously unappreciated roles in translational regulation, we performed polysome proteomics (polysome profiling and label-free mass spectrometry) to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and changes in association during two acute stresses that induce the integrated stress response (ISR): oxidative stress (0.45 mM H2O2 treatment for 15 min) and amino acid starvation (10 mM 3-AT treatment for 15 min). Five fractions were isolated from sucrose gradients prepared from each extract, corresponding to monosomes (F1) and polysomes with increasing numbers of ribosomes per mRNA (F2-F5). Unfractionated cyotplasmic lysates (totals) were analysed for each sample. Four replicate extracts were prepared and analysed for each condition.
Project description:Polysome-profiling is commonly used to study translatomes, i.e. transcriptome-wide patterns of translational efficiency. The standard approach for collecting efficiently translated polysome-associated RNA results in laborious extraction of RNA from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts such as primary cells or frozen tissues. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for mRNAs associated with >3 ribosomes in only one or two fractions, thereby reducing sample handling 5-10 fold. The technique can be applied to frozen tissue samples from biobanks, and generates polysome-associated RNA with a quality reflecting the starting material. When coupled with smart-seq2, a single-cell RNA sequencing technique, translatomes from small tissue samples can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associated with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation to maintain protein levels constant. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in bio banks.
Project description:Chronic obstructive pulmonary disease (COPD) is an independent risk factor for lung cancer, suggesting that COPD stroma favors cancer initiation. Therefore, we used proteomics and polysome-profiling to identify gene expression programs that distinguish stroma of patients harboring lung cancer from those that do not, with varied COPD severities. This profiling unveiled distinct COPD-dependent cancer-associated gene expression programs predominantly manifesting as alterations in mRNA translation. Mechanistically, such programs are downstream of the mammalian target of rapamycin pathway in mild COPD and pathological extracellular matrix in more severe COPD; and both programs parallel activation of distinct pro-cancer fibroblast-derived secretomes. Therefore, depending upon COPD severity, the lung stroma can exist in two states favoring cancer initiation, which likely result in distinct disease entities.