Expression data from mouse breast cancer tissue: Serglycin heterozygous and knock-out conditions
ABSTRACT: Serglycin proteoglycans contribute to proper storage and secretion of inflammatory mediators in hematopoietic cells. Serglycin is also expressed in cancer cells where increased expression has been linked to poor prognosis. In the present study we report that serglycin proteoglycan is absolutely required for metastasis in the MMTV-PyMT-driven mouse breast cancer model. Serglycin seems to play a role in promoting epithelial to mesenchymal transition, cancer-related inflammation and extravasation. Our results suggest that serglycin and serglycin-dependent mediators are potential drug targets to prevent metastatic disease/dissemination of cancer. 6 total breast tumor samples were analyzed. 3 of SG+/- and 3 of SG-/- tumour tissue. Raw data was normalized using the robust multi-array average (RMA) method. To identify potential serglycin-regulated mediators of metastasis, we performed a microarray expression analysis of RNA isolated from SG+/- and SG-/- breast tumor tissue. The expression analysis identified 672 genes with a significantly altered expression level, at log2 fold >±1,2. Strikingly, only six genes were up-regulated in the SG-/- PyMT+ tumor cells compared to SG+/- PyMT+ tumor cells while 666 were significantly down-regulated.
Project description:The transcription factor Zinc finger protein 148 (Zfp148) interacts physically with the tumor suppressor p53, but the siginficance of this interaction is not known. We recently showed that knockout of Zfp148 in mice leads to ectopic activation of p53 in tissues and cultured fibroblasts, suggesting that Zfp148 represses p53 activity. Here we hypothesized that targeting Zfp148 would unleash p53 activity and protect against cancer development, and test this idea in the APCMin/+ mouse model of intestinal adenomas. Crypt-enriched tissues were isolated by laser microdissection (PALM) from the small intestines (proximal) of Zfp148gt/+APCMin/+ and Zfp148+/+APCMin/+ mice for RNA extraction and hybridization to Affymetrix microarrays.
Project description:To better understand the impact of integrin beta3 signaling in myeloid cells on the tumor microenvironment, we compared the gene expression profiles of FACS isolated GFP+ PyMT-BO1 MFP tumor cells and also M2 TAMs (CD11b+Gr1-F4/80+CD206+) from tumor tissue of WT mice and b3ΚΟΜ mice. PyMT-BO1-GFP-Luc tumor cells (100,000) were injected into mammary fat pad (MFP) tissue from WT and b3KOM mice. PyMT-BO1-GFP-Luc cells and CD206hi tumor-associated macrophages (TAMs) were FACS-sorted from day 10 MFP tumor tissue. FACS-sorted cells directly used for RNA extraction and hybridization on Affymetrix microarrays.
Project description:In a transcriptome study of psoriatic (PP) vs. normal (NN) skin, we found a co-expressed gene module (N5) enriched 11.5-fold for lipid biosynthetic genes. We also observed fewer visible hairs in PP skin, compared to uninvolved (PN) or NN skin (p<0.0001). To ask whether these findings might be due to abnormalities of the pilosebaceous unit, we carried out 3D morphometric analysis of paired PP and PN biopsies. Sebaceous glands (SG) were markedly atrophic in PP vs. PN skin (91% average reduction in volume, p=0.031). Module N5 genes were strongly downregulated in PP vs. NN skin (fold-change [FC] < 0.25, 44.4-fold), and strongly up-regulated in sebaceous hyperplasia (SH, FC > 4, 54.1-fold). The intersection of PP-downregulated and SH-upregulated gene lists generated a gene expression signature consisting solely of module N5 genes, whose expression in PP vs. NN skin was inversely correlated with the signature of IL17-stimuated keratinocytes. Despite loss of visible hairs, morphometry identified elongated follicles in PP vs. PN skin (average 1.7 vs. 1.2 Jm, p=0.020). These results document SG atrophy in non-scalp psoriasis, identify a cytokine-regulated set of SG signature genes, and suggest that loss of visible hair in PP skin may result from abnormal SG function. Gene expression was compared between sebaceous hyperplasia lesions (n = 5) and normal skin (n = 3) from control subjects.
Project description:The environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, hypoxia. It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In the associated study we performed a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on four different cell lines in conditions of normoxia, hypoxia and anoxia. A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA). Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs. Gene expression analysis was performed on MCF-7 cells after 90 hours in either anoxic or hypoxic conditions, and compared to cells grown in a regular cell incubator. The gene expression analysis was performed to validate that the cells were hypoxic/anoxic and showed the characteristic hypoxia response. Microarray based mRNA profiling was used to charactarize cells grown in hypoxia and anoxia. In the associated study we performed a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on four different cell lines in conditions of normoxia, hypoxia and anoxia. We fin that hypoxia/anoxia render cancer cells both more resistant and more sensistive, depending of the type of drug used. The gene expression analysis was performed to validate that the cells really were hypoxic/anoxic and showed the characteristic hypoxia response. The cell line used for the gene expression analysis was MCF-7.
Project description:Primary cultures of patient tumor cells (PCPTC) were used in a cell-based cytotoxicity screen. Microarray-based mRNA profiling was used to identify the mechanism-of-action for the small molecule VLX 50. MCF7 cells were treated with the PCPTC screening hit VLX 50 or DMSO control for 6h prior to RNA isolation. One sample per treatment. Data were analyzed using both MAS5.0 and RMA.
Project description:Histidine-rich glycoprotein (HRG) is a 75 kDa heparin-binding plasma protein which has been implicated in regulation of tumor angiogenesis and growth. To exert some of its biological functions, HRG acts on macrophages.This study was performed to assess changes in gene expression in peritoneal macrophages treated with HRG using oligonucleotide microarrays A total of 8 samples were analyzed. Peritoneal macrophages were pooled from 10 wt C57/BL6 mice and treated with recombinant HRG (1ug/ml) for 6 and 24 hours in duplicates
Project description:Breast cancer metastases develop in the bone more frequently than any other site, and are a common cause of morbidity in the form of bone pain, pathological fractures, nerve compression, and life-threatening hypercalcemia. Despite ongoing research efforts, the molecular and cellular mechanisms that regulate breast cancer cell homing to and colonization of the bone as well as resultant pathological bone alteration remain poorly understood. To identify key mediators promoting breast cancer metastasis to bone, we utilized an immunocompetent, syngeneic murine model of breast cancer metastasis employing the mammary tumor cell line NT2.5. Following intracardiac injection of NT2.5 cells in neu-N mice, metastases developed in the bone, liver, and lung, closely mimicking the anatomical distribution of metastases in breast cancer patients. Using an in vivo selection process, we established NT2.5 sub-lines demonstrating an enhanced ability to colonize the bone and liver. Genome-wide cDNA microarray analysis comparing gene expression between parental NT2.5 cells and established sub-lines was performed. Individual samples of RNA from parental NT2.5 cells, early passage (BO3 and LI1) sub-lines, and final passage (BO6 and LI3) sub-lines were compared using GeneChip® Mouse Genome 430 2.0 Arrays
Project description:The mechanisms involved in epithelium-stroma interactions remain poorly understood, despite the importance of the microenvironment during tumorigenesis. Here, we studied the role of the Ets2 transcription factor in tumor-associated fibroblasts in the MMTV-PyMT mammary tumor model. Inactivation of Ets2 specifically in fibroblasts using Fsp-cre significantly reduced tumor growth, in contrast to Ets2 inactivation in epithelium, in which no differences in tumor growth were observed. Microarray analysis on isolated fibroblasts demonstrated the important role of Ets2 in remodeling of the extracellular matrix and angiogenesis in these cells. At the molecular level, Ets2 regulated Mmp9 expression through direct binding to the promoter region. Tumors lacking Ets2 in fibroblasts had diminished blood vessels. We also found a significant correlation between phosphorylation of ETS2 and MMP9 levels in stroma of human breast cancer samples. Collectively, our results suggest Ets2 uniquely contributes to angiogenesis from fibroblasts in the tumor microenvironment. Primary mammary fibroblasts were isolated from mice without the PyMT oncogene with or without Ets2, and from mice with the PyMT oncogene with or without Ets2. RNA was extracted and samples were submitted for Affymetrix gene expression arrays.
Project description:Gene expression profiling of disseminated tumor cells in lung, lung metastatses and residual tumor cells in the MMTV-PyMT breast cancer model. Profiling gene expression change between disseminated tumor cells, lung metastases and residual tumor cells from the MMTV-PyMT breast cancer model.
Project description:Microarray based mRNA profiling was used to identify the mechanism of action for the small molecule VLX60. MCF7 cells were treated with the drug candidate VLX60 for 6h prior to RNA isolation