Transcription factor Ets1 cooperates with estrogen receptor α to stimulate estradiol-dependent growth in breast cancer cells and tumors.

The purpose of this study was to explore the role of transcription factor Ets1 in estrogen receptor α (ERα)-positive breast cancer progression. We expressed human Ets1 or empty vector in four human ERα-positive breast cancer cell lines and observed increased colony formation. Further examination of cellular responses in stable Ets1-expressing MCF7 clones displayed increased proliferation, migration, and invasion. Ets1-expressing MCF7 tumors grown in the mammary fat pads of nude mice exhibited increased rates of tumor growth (7.36±2.47 mm(3)/day) compared to control MCF7 tumors (2.52±1.70 mm(3)/day), but maintained their dependence on estradiol for tumor growth. Proliferation marker Ki-67 staining was not different between control and Ets1-expressing tumors, but Ets1-expressing tumors exhibited large necrotic centers and elevated apoptotic staining. Ets1 was shown to cooperate with ERα and the p160 nuclear receptor coactivator (NCOA/SRC) family to increase activation of a consensus estrogen response element luciferase reporter construct. Ets1-expressing MCF7 cells also exhibited elevated expression of the ERα target genes, progesterone receptor and trefoil factor 1. Using GST-pulldown assays, Ets1 formed stable complexes containing both ERα and p160 nuclear receptor coactivators. Taken together, these data suggest that the Ets1-dependent estradiol sensitization of breast cancer cells and tumors may be partially due to the ability of Ets1 to cooperate with ERα and nuclear receptor coactivators to stimulate transcriptional activity of estrogen-dependent genes.

Polymerase chain reaction-based species verification and microsatellite analysis for canine cell line validation.

Cell line cross-contamination as well as genetic drift during passaging have been acknowledged as widespread problems since the 1960s. Improper cell line identification can invalidate results and, if not discovered, pollute the scientific community's body of knowledge with regard to cancer cell lines, their gene expression, and their drug susceptibilities. Despite the obvious need, validation of cell line identity is not yet widely required, and the problem persists. A highly sensitive polymerase chain reaction (PCR)-based approach and short tandem repeat (STR) profiling were used to examine the prevalence of inter- and intraspecies cell line contamination in a veterinary research setting. First, 60 cell lines from 6 laboratories were tested with multiplex species-specific PCR capable of identifying 6 commonly used species. Of these, 3 were determined to be misidentified by species. Second, to identify intraspecies contamination among canine cancer cell lines, 29 canine lines from 3 different laboratories were analyzed with STR fingerprinting. Using this methodology, 3 canine cell lines were determined to be misidentified or cross-contaminated by other canine cell lines. Finally, genetic drift was observed within 1 cell line obtained from different laboratories. These findings emphasize the importance of cell line validation as a critical component of "good cell culture practice." A database of the STR profiles obtained in the current study has been established for future comparison and validation of canine cell lines by investigators at Colorado State University and other institutions.

Expression profiling in canine osteosarcoma: identification of biomarkers and pathways associated with outcome.

BACKGROUND: Osteosarcoma (OSA) spontaneously arises in the appendicular skeleton of large breed dogs and shares many physiological and molecular biological characteristics with human OSA. The standard treatment for OSA in both species is amputation or limb-sparing surgery, followed by chemotherapy. Unfortunately, OSA is an aggressive cancer with a high metastatic rate. Characterization of OSA with regard to its metastatic potential and chemotherapeutic resistance will improve both prognostic capabilities and treatment modalities. METHODS: We analyzed archived primary OSA tissue from dogs treated with limb amputation followed by doxorubicin or platinum-based drug chemotherapy. Samples were selected from two groups: dogs with disease free intervals (DFI) of less than 100 days (n = 8) and greater than 300 days (n = 7). Gene expression was assessed with Affymetrix Canine 2.0 microarrays and analyzed with a two-tailed t-test. A subset of genes was confirmed using qRT-PCR and used in classification analysis to predict prognosis. Systems-based gene ontology analysis was conducted on genes selected using a standard J5 metric. The genes identified using this approach were converted to their human homologues and assigned to functional pathways using the GeneGo MetaCore platform. RESULTS: Potential biomarkers were identified using gene expression microarray analysis and 11 differentially expressed (p < 0.05) genes were validated with qRT-PCR (n = 10/group). Statistical classification models using the qRT-PCR profiles predicted patient outcomes with 100% accuracy in the training set and up to 90% accuracy upon stratified cross validation. Pathway analysis revealed alterations in pathways associated with oxidative phosphorylation, hedgehog and parathyroid hormone signaling, cAMP/Protein Kinase A (PKA) signaling, immune responses, cytoskeletal remodeling and focal adhesion. CONCLUSIONS: This profiling study has identified potential new biomarkers to predict patient outcome in OSA and new pathways that may be targeted for therapeutic intervention.