Breast Cancer Cell Density: Imaging assay of the density- and context-dependence of small molecule perturbagen response in breast cancer cell lines. Dataset 1 of 3: Cell count and normalized growth rate inhibition values.
Project description:Breast Cancer (BC) is one of the most common cancers and one of the most common causes for cancer- related mortality. Discovery of protein biomarkers associated with cancer, is considered as important for early diagnosis and prediction of the cancer risk. Protein biomarkers could be investigated by large-scale protein investigation or proteomics, using mass spectrometry (MS)- based techniques. Our group applies MS-based proteomics to study the protein pattern in human breast milk from women with BC and controls and investigates the alterations and dysregula-tions of breast milk proteins in comparison pairs of BC versus control. These dysregulated pro-teins might be considered as potential future biomarkers of BC. Identification of potential bi-omarkers in breast milk may benefit young women without BC, but who could collect the milk for future assessment of BC risk. Previously we identified several dysregulated proteins in dif-ferent sets of human breast milk samples from BC patients and controls using gel-based protein separation coupled with MS. Here, we performed two-dimensional polyacrylamide gel electro-phoresis (2D-PAGE) coupled with nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) in a small-scale study on a set of 6 human breast milk samples (3 BC samples vs. 3 controls) and we identified several dysregulated proteins which have potential roles in cancer progression and might be considered as potential BC biomarkers in the future.
Project description:In previous studies, we identified a distantly related rhomboid homologue gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly overexpressed in the advanced stages of the breast and colorectal cancer diseases. In order to identify RHBDD2 modulated pathways, we analyzed two breast cancer cell lines (MCF7 and T47D) from control and RHBDD2-siRNA transient gene silencing followed by gene expression profiling analysis using the whole genome Toray 3D-GeneTM Human Oligo Chip. Statistical analysis of the Toray's 3D gene expression profiling data identified 566 commonly differentially expressed genes in association to the RHBDD2 knockdown in both breast cancer cell lines. Among the statistically significant over-represented biological process, we found the apoptosis, cell cycle and response to DNA damage process related genes. In addition, categories of genes found in the ubiquitin-proteasome and oxidative phosphorylation were also highly enriched related genes in the commonly deregulated gene list. We further used a lentivirus-based system (shRNA-pLKO.1) for stable silencing of RHBDD2 mRNA in the T47D breast cancer cell line. Using a staurosporine-induced apoptosis model, we demonstrate that RHBDD2 abrogation resulted in an apoptosis-resistant phenotype of T47D breast cancer cell line. These data are in line with a recent study, suggesting that RHBDD2 expression could be up-modulated in response to 5FU-induced apoptosis in colorectal cancer cells. Taken together, these data suggest that RHBDD2 could be involved in the modulation of the programmed cell death in cancer cells. In order to analyze differential gene expression profiling of RHBDD2 silencing and control cells, total RNA was isolated from replicate experiments from two breast cancer cell lines (MCF7 and T47D) derived from the negative control-siRNA and the RHBDD2-siRNA treatments in duplicate experiments.
Project description:Molecular mechanisms of cell cycle exit are poorly understood. A group of genes required for cell cycle exit and maintenance of cell quiescence in human fibroblasts following serum deprivation has been recently identified. Studies on lymphocytes following growth factor deprivation-induced cell cycle exit have predominantly focused on the initiation of apoptosis. A set of genes involved in lymphocyte quiescence have also been identified among genes highly expressed in resting lymphocytes and down-regulated after cell activation. In our study, proliferating IL-2-dependent human T cells were forced to exit cell cycle by growth factor withdrawal, and their gene expression profiles were examined. The differential gene expression analysis was performed in primary and immortalized IL-2-dependent T lymphocytes. Cell samples were collected directly from the IL-2-containing cultures and 8-hrs following IL-2 withdrawal, before apoptosis could be evidenced by the Annexin-V staining. The three primary T lymphoblast cell populations were obtained from the peripheral blood mononuclear cells (PBMC) stimulated for 24h by wheat germ agglutinin and cultured in the presence of IL-2 up to 4-8 population doublings. As shown by the cell surface analysis, these populations were composed of T cells exclusively. Samples of these cell populations were subsequently analyzed as biological replicates. Two spontaneously immortalized IL-2-dependent T cell lines were derived from normal spleen and from PBMC derived from Nijmegen Breakage Syndrome patient. Gene expression was assessed by the Affymetrix microarray HG-U133 2.0 Plus that detects 38,500 genes. The expression of a selected number of genes was verified by the qRT-PCR method. We have identified a set of 53 genes that we called a â??T lymphocyte cell cycle exit signatureâ??, comprised of 13 up-regulated and 40 down-regulated genes. Genes linked to transcription, cell cycle, cell growth, proliferation and differentiation, cell adhesion and immune functions were found to be overrepresented among the differentially expressed, before and after IL-2 deprivation. Among those, PIM1, BCL2, IL-8, HBEGF, DUSP6, OSM, CISH, SOCS2, SOCS3, LIF and IL13 were down-regulated and RPS24, SQSTM1, TMEM1, LRRC8D, ECOP, YY1AP1, C1orf63, ASAH1, SLC25A46 and MIA3 were up-regulated. Identification of genes involved in cell cycle exit and quiescence, may provide new insights into the mechanisms of tissue repair and regeneration as well as of cancer development. Experiment Overall Design: Cell sources and cell sample preparation. Experiment Overall Design: Samples of three primary, IL-2-dependent T lymphoblast cell lines, derived from three healthy donors (j, 43, 6) were collected from IL-2-containing culture and 8-hrs following IL-2 withdrawal (three pairs, each sample was analyzed once = 6 samples). Experiment Overall Design: The two spontaneously immortalized IL-2-dependent T cell lines were derived from normal spleen (line5) and from PBMC derived from a Nijmegen Breakage Syndrome patient (S9). Samples of the two immortalized cell lines were collected in three biological replicates each, from the cultures with and without IL-2 (2 x 2 x 3 = 12 samples).
Project description:CD44 is a transmembrane glycoprotein playing a key role in cel adhesion to the extracellular matrix. CD44 expression is upregulated in various cancer cells and recognized as a molecular marker for tumor-initiating cancer cells. However, the intricate correlation between CD44 and underlying biological functions is yet to be fully disclosed at molecular levels. Here, we discovererd global proteome changes induced by CD44 knockdown in the four different breast cancer cell lines by TMT based quantitative proteomics.
Project description:Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent phospholipid peroxidation. It can be induced by inhibiting glutathione peroxidase 4 (GPX4), the key enzyme for efficiently reducing peroxides within phospholipid bilayers. Recent data suggest that cancer cells undergoing EMT (dedifferentiation) and those resistant to standard therapy expose a high vulnerability toward ferroptosis. Although recent studies have begun to identify and characterize the metabolic and genetic determinants underlying ferroptosis, many mechanisms that dictate ferroptosis sensitivity remain unknown. Here, we show that low cell density sensitizes primary mammary epithelial and breast cancer cells to ferroptosis induced by GPX4 inhibition, whereas high cell density confers resistance. These effects occur irrespective of oncogenic signaling, cellular phenotype and expression of the fatty acid ligase acyl-CoA synthetase long chain family member 4 (ACSL4). By contrast, we show that a massive accumulation of neutral triacylglycerides (TAG) enriched with polyunsaturated fatty acids (PUFA) is induced at low cell density. In addition, de novo lipogenesis and desaturation pathways were found to be reduced at low cell density, indicative of increased fatty acid uptake. Our study suggests that PUFA-mediated toxicity is limited by the enrichment in TAGs that in turn might pose a vulnerability towards ferroptosis. Conclusively, cell density regulates lipid metabolism of breast epithelial and cancer cells, which results in a ferroptosis-sensitive cell state with the potential to be exploited therapeutically during metastatic dissemination.
Project description:The following describes additional preliminary data to that outlined in Project PXD014022 and specifically describes the 12-hour co-exposure and P. aeruginosa 12 hour exposure preliminary experiments. Aspergillus fumigatus and Pseudomonas aeruginosa are the most prevalent fungal and bacterial pathogens associated with cystic fibrosis-related infections, respectively. P. aeruginosa eventually predominates as the primary pathogen, though it is unknown why this is the case. Label-free quantitative (LFQ) proteomics was employed to characterize the cellular response to co-exposure of A. fumigatus and P. aeruginosa using the type II alveolar epithelial cell line, A549, as a model of the alveolar surface. Proteomic data revealed that P. aeruginosa replication increased exponentially when co-cultured with A. fumigatus. Comparative analysis using LFQ proteomics revealed similarities and distinct differences in the response of A549 cells to A. fumigatus, or P. aeruginosa and sequential exposure to both pathogens. In total, 2264 proteins were identified. Analysis of statistically significant (p<0.05; ±1.5 fold change) differentially abundant (SSDA) proteins, revealed an increase in the relative abundance of proteins associated with biological processes common to all pathogen-exposed groups.
Project description:MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been previously reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome. The pre-treatment sera of 42 stage II–III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs.