Project description:The goal of the study was to examine the transcriptional profile of pancreatic cancer cell lines and assess if the molecular subtypes observed in tumor samples were represented in existing cell line models. Cell line models allow us to investigate if the molecular subtype observed in tumor have unique sensitivity profiles to anticancer drugs. 29 pancreatic cancer cell lines were compared to a mixed reference pool of 30 pancreatic cancer cell lines to identify cell line specific gene expression.
Project description:The goal of the study was to examine the transcriptional profile of pancreatic tumors to identify molecular subtypes in order to develop validated clinically useful gene expression signature with the potential to guide therapy decision. Tissue was obtained by snap freezing in liquid nitrogen as soon as removed. All tissue samples were stored at -80C. Samples were embedded in OCT and a 4ug section was taken for H/E staining. After QC procedure to ensure high quality RNA (RIN>7) and confirm PDAC histology, 78 samples were subjected to microarray analysis. All patients signed an Institutional Review Board approved consent for bio-banking, clinical data extraction and molecular analysis. 85 samples (77 tumors, 3 normal and 5 pancreatitis) were compared to a mixed reference pool of 66 tumor samples to identify gene expression patterns.
Project description:About half of all melanomas harbor a constitutively active mutant BRAFV600E/K kinase that can be selectively inhibited by targeted BRAF inhibitors (BRAFi). While patients treated with BRAFi initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. We observe significant elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein are also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction in levels of endogenous WNT5A in melanoma decreases cell growth, increases apoptosis in response to BRAFi challenge, and decreases the activity of pro-survival AKT signaling. Overexpression of WNT5A conversely promotes melanoma growth and tumorigenesis and activates AKT signaling. Similar to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibits growth, sensitizes melanoma cells to BRAFi, and reduces AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which then acts through FZD7 and RYK to promote AKT signaling, leading to increased growth and therapeutic resistance. Increased WNT5A expression in BRAFi-resistant melanomas also correlates with an associated transcriptional signature, which identifies potential therapeutic targets to reduce clinical resistance to BRAFi. Expression of WNT5A-correlated genes was compared in melanoma cell lines generated to be resistant to PLX4032 and the their associated naïve parental line Basal expression of the WNT5A-correlated genes was also measured in experiments comparing each naïve line to a mixed reference pool containing equal amounts of 47 melanoma cell lines.
Project description:Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by Glycogen Synthase Kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This prompted us to examine the relationship between MITF, endolysosomal biogenesis and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the Tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-Catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies (MVBs) into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in MVB biosynthesis, which in turn increased Wnt signaling, generating a positive feed-back loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer. Expression of selected Lysosomal genes and CLEAR element plus MITF were compared in 51 melanoma cell lines to a mixed reference pool containing equal amounts of 47 melanoma cell lines.
Project description:The goal of the study was to identify molecular subgroups that might predict clinical outcomes in serous epithelial ovarian cancer (EOC) patients. A second objective was to identify potential therapeutic targets for serous EOC based on improved understanding of the molecular diversity of the disease. Ovarian tissues and matched peripheral blood samples were prospectively obtained from sequential patients undergoing planned gynecologic surgery at Cedars-Sinai Medical Center between 1989 and 2005. All patients underwent surgery and received adjuvant chemotherapy with a contemporaneous standard-of-care regimen. Ovarian tissue samples (n=172) were compared to a reference pool of 106 ovarian samples. Mixed reference includes normal, benign, borderline, and malignant samples.
Project description:Selected genes within the 1 Mb minimal amplified region on 20q13.3 driven by ADRM1 in gastric cancer cell line AGS were analyzed by Gene expression Microarray. Gene Expression of ADRM1, HRH3, MTG2, LAMA5 and OSBPL2 were then compared in 16 Gastric cancer cell lines to a reference composed of mixed gastric cancer cell lines.
Project description:Male and female CD-1 mice were administered dietary Phenobarbital for 2 or 7 days. In-life, enzyme activity, cell proliferation, genomic analysis, and Bench-mark dose modeling was carried out. The goal was to exmaine low-dose PB effects on early key events in CAR-mediated hepatocarcinogenesis. PB at 0, 0.15, 1.5, 15, 75, or 150 mg/kg-day for 2 or 7 days to characterize multiple apical and molecular endpoints.
Project description:All established protocols for differentiation of mouse and human pluripotent stem cells into specific neural subpopulations generate a considerable cellular heterogeneity that hampers experimental and clinical progress. In order to obtain a homogenous population of neuronal precursor cells and to streamline the differentiation of embryonic stem cells (ESCs), we assessed PSA-NCAM, a surface glycoprotein that is specifically expressed on immature neurons. We developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from differentiated ESC cultures and characterized their neuronal differentiation potential in vitro. PSA-NCAM enrichment at an early step of neural differentiation increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. The in vivo potential of in vitro derived PSA-NCAM+ enriched precursors was functionally characterized by grafting into the forebrain of adult mice. Analysis for several neuronal and glia markers at 10 or 40 days post graft showed a distinct differentiation pattern. While unsorted control cells gave rise to a mixed population composed of immature precursors, early postmitotic neurons or glial cells, the majority of PSA-NCAM+ enriched cells differentiated into NeuN positive neurons. Furthermore, when in contact with the rostral migratory stream, higher numbers of cells integrated into the stream and migrated towards the olfactory bulb when the PSA-NCAM enriched population was grafted. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells. Two conditions (step 4, step 5), each represented by three biological replicates of control and enriched cells (Cy5); mESC was used as common reference (Cy3)
Project description:Adult mouse subventricular zone (SVZ) neural progenitor cells (NPCs) retain the capacity to generate multiple lineages in vitro and in vivo. Thus far, the mechanisms involved in the regulation of these cells have not been well elucidated. We have carried out RNA profiling of adult SVZ cell cultures undergoing differentiation, to identify pathways that regulate progenitor cell proliferation and to define a set of transcripts that can be used as molecular tools in the drug discovery process. We carried out a stepwise stratification of the results to identify transcripts specifically enriched in NPCs and validated some of these using comparative literature analysis, quantitative PCR and immunological techniques. The results show a set of transcription factors, secreted molecules, and plasma membrane markers which are differentially regulated during differentiation. Pathway analysis highlights alterations in IGF, Wnt and TGFbeta signalling cascades. Further characterization of these components could provide greater insight into the mechanisms involved in the regulation of neurogenesis in the adult brain. Mouse subventricular zone neural stem cells cultures were prepared from C57Bl6/J mice (Johansson et al.,1999) and cultured in suspension in DFEM/F12 1:1 supplemented with PSF, B27 supplement and EGF/ FGF-2 at 20ng/ml. RNA was isolated from either expanding neurospheres in the presence of FGF2 and EGF (Reynolds and Weiss, 1992; Gritti et al., 1999), or from cultures induced to differentiate upon growth factor withdrawal or serum exposure, either after 24 hours (in suspension, to avoid changes induced by substrate attachment) or 5 days (as adherent cultures in poly-lysine and laminin-coated plates). Experiments were conducted in triplicate from independent batches of SVZ preparations. As a control for potential effects of media changes, a set of identical cultures were included where cells were grown for a further 24 hours in fresh media containing FGF2 and EGF (20 ng/ml each, normal growth media, NGM). Fluorescently labelled aRNA from individual samples were competitively hybridised against a pool of labelled aRNA from undifferentiated reference (starting) material on custom Agilent microarrays representing ~22,500 mouse transcripts. Technical, fluor-reversed hybridisations were performed for every sample.