Project description:Prostate primary epithelial and stromal cells were isolated from normal donorhuman prostate tissue and patient prostate tumors (>Gleason 8) using in vitroselective media conditions and differential substrate adhesion properties.Tumor cells specific to the prostate epithelial cell component attached tomatrigel while epithelial cells from donors preferentially attached to gelatinsubstrate. The prostate tumor cells did not proliferate over time in culturebut cells of normal epithelial phenotype in comparison to the donor tissueeventually emerged from this cell compartment. The epithelial componentof both donor and tumor specimens produced stem cell colonies thatexpressed OCT4 in growth factor reduced media but subsequently formedembryoid bodies in hanging drop cultures which differentiated into multiplegerm line lineages under the influence of growth factors. To determineif prostate tumor-derived stem cells were capable of forming tumors invivo, we compared the behavior of the donor and tumor stem cells aftertransplantation as tissue recombinants under the renal capsule of SCID micein the presence of rat urogenital mesenchyme (rUGM). Stem cells from bothdonor and tumor sources produced glandular structures that secreted prostatespecific antigen (PSA) while no glandular structures were formed from tissue recombinants of normal epithelial or tumor epithelial cells plus rUGM or fromrUGM alone. Serial transplantation of the stem cell recombinants from tumor specimens yielded subrenal structures reflecting an abundance of glandswith morphological features typical of Prostatic Intraepithelial Neoplasia(PIN). In order to determine whether intrinsic differences existed amongthe donor-derived and tumor-derived stem cells, array based microRNAexpression profiling was performed on all cell types obtained after in vitro isolation. Sample Labeling Key: PC-Prostate Cancer PD-Prostate Donor (Normal Tissue) DNE-Donor Normal Epithelial NS-Donor Normal Stem TNE-Tumor Normal Epithelial TE-Tumor Epithelial (TNE and TE samples with corresponding numbers are matched pairs) TS-Tumor Stem Cell S-Stromal PC 73 TNE was run in all 6 batches as a control for batch effect. The number in parentheses corresponds with the batch the sample was run in.

Project description:Prostate primary epithelial and stromal cells were isolated from normal donorhuman prostate tissue and patient prostate tumors (>Gleason 8) using in vitroselective media conditions and differential substrate adhesion properties.Tumor cells specific to the prostate epithelial cell component attached tomatrigel while epithelial cells from donors preferentially attached to gelatinsubstrate. The prostate tumor cells did not proliferate over time in culturebut cells of normal epithelial phenotype in comparison to the donor tissueeventually emerged from this cell compartment. The epithelial componentof both donor and tumor specimens produced stem cell colonies thatexpressed OCT4 in growth factor reduced media but subsequently formedembryoid bodies in hanging drop cultures which differentiated into multiplegerm line lineages under the influence of growth factors. To determineif prostate tumor-derived stem cells were capable of forming tumors invivo, we compared the behavior of the donor and tumor stem cells aftertransplantation as tissue recombinants under the renal capsule of SCID micein the presence of rat urogenital mesenchyme (rUGM). Stem cells from bothdonor and tumor sources produced glandular structures that secreted prostatespecific antigen (PSA) while no glandular structures were formed from tissuerecombinants of normal epithelial or tumor epithelial cells plus rUGM or fromrUGM alone. Serial transplantation of the stem cell recombinants from tumorspecimens yielded subrenal structures reflecting an abundance of glands with morphological features typical of Prostatic Intraepithelial Neoplasia(PIN). In order to determine whether intrinsic differences existed amongthe donor-derived and tumor-derived stem cells, array based microRNAexpression profiling was performed on all cell types obtained after in vitro isolation. Sample Labeling Key: PC-Prostate Cancer PD-Prostate Donor (Normal Tissue) DNE-Donor Normal Epithelial NS-Donor Normal Stem TNE-Tumor Normal Epithelial TE-Tumor Epithelial (TNE and TE samples with corresponding numbers are matched pairs) TS-Tumor Stem Cell S-Stromal PC 73 TNE was run in all 6 batches as a control for batch effect. The number in parentheses corresponds with the batch the sample was run in.

Project description:Renal cancer accounts for 2% to 3% of all adult malignancies in the US. In 2011 more than 64,770 new cases and 13,000 deaths were reported and the incidence is steadily increasing by 2.5% per year. Clear cell renal carcinoma (ccRCC) represents the majority (85% to 90%) of adult kidney cancers and is the most malignant. This cancer is characterized by an early loss of the von Hippel-Lindau tumor suppressor (VHL) on short arm of chromosome 3 in the majority of tumors (80%). It is a classic knowledge that loss of VHL results in an increased accumulation and activity of the hypoxia-inducible transcription factor (HIF), which in turns activates expression of genes promoting tumor growth. In that respect, induction of angiogenic factors induces blood flow through the tumors and delivery of nutrients and oxygen to cancer cells, while induction of genes inducing anaerobic glycolysis supports adaptation to reduced nutrients availability and shifts towards metabolic pathways promoting tumor growth. Recently we have discovered another tumor suppressing pathway regulated by VHL. We found that VHL is a major regulator of the process of macroautophagy (autophagy) (ref). We identified that VHL induces expression of a microRNA, miR-204, which in turn inhibits activity of a major regulator required for the formation of autophagosome, LC3B. LC3B-mediated autophagy is necessary for the ccRCC tumor growth, and inhibition of this process by VHL and miR-204 significantly contributes to the tumor suppression. Most importantly, we discovered that VHL by inhibiting activity of HIF, induced expression and activity of an LC3B paralog, LC3C. In contrast to LC3B, LC3C acts as a tumor suppressor, indicating the specificity and complexity of different autophagic programs. Integrating these novel data with the established role of VHL in regulating angiogenesis produces a more global picture of VHL as a master tumor suppressor that regulates nutrient access for renal cancer cells from both extracellular and intracellular sources. In the process of searching for other genes induced by VHL that could participate in the tumor suppressing activity of VHL, we discovered that reconstitution of VHL in RCC cells induces enrichment of genes expressed from the Smith-Magenis locus (SM), located on the short arm of chromosome 17p11.2. Smith-Magenis syndrome is a complex neurobehevioural disorder, characterized by intellectual impairment, craniofacial and skeletal anomalies and sleep disturbance. Recently, deletions or mutations of one specific gene in the SM locus, RAI1, (retinoic acid induced 1), were shown to be responsible for the syndrome. Interestingly, this locus contains another kidney tumor suppressor gene, folliculin (FLCN), of which activity is lost in the genetic dominant disorder, Birt-Hogg-Dube syndrome (BHD). BHD is characterized by skin fibrofolliculomas, pulmonary cysts and spontaneous pneumothorax, as well as renal cancer of mixed histological types, including chromophobe, oncocytic, clear cell and papillary type. Here we show that VHL regulates expression of FLCN, and in turn FLCN contributes to the VHL-mediated suppression of tumor growth. Two-condition experiment, VHL(-) vs. VHL(+). 4 biological replicates in each group. Two dual-channel arrays with VHL(+) on Cy5 and VHL(-) on Cy3, and two dual-channel arrays with flipped samples.

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