Laura E. Pascal

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE)

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.

Regenerated Luminal Epithelial Cells Are Derived from Preexisting Luminal Epithelial Cells in Adult Mouse Prostate

Determining the source of regenerated luminal epithelial cells in the adult prostate during androgen deprivation and replacement will provide insights into the origin of prostate cancer cells and their fate during androgen deprivation therapy. Prostate stem cells in the epithelial layer have been suggested to give rise to luminal epithelium. However, the extent of stem cell participation to prostate regrowth is not clear. In this report, using prostate-specific antigen-CreER(T2)-based genetic lineage marking/tracing in mice, preexisting luminal epithelial cells were shown to be a source of regenerated luminal epithelial cells in the adult prostate. Prostatic luminal epithelial cells could survive androgen deprivation and were capable of proliferating upon androgen replacement. Prostate cancer cells, typically exhibiting a luminal epithelial phenotype, may retain this intrinsic capability to survive and regenerate in response to changes in androgen signaling, providing part of the mechanism for the ultimate failure of androgen deprivation therapy in prostate cancer.

Molecular and cellular characterization of ABCG2 in the prostate

BackgroundIdentification and characterization of the prostate stem cell is important for understanding normal prostate development and carcinogenesis. The flow cytometry-based side population (SP) technique has been developed to isolate putative adult stem cells in several human tissue types including the prostate. This phenotype is mainly mediated by the ATP-binding cassette membrane transporter ABCG2.MethodsImmunolocalization of ABCG2 was performed on normal prostate tissue obtained from radical prostatectomies. Normal human prostate SP cells and ABCG2+ cells were isolated and gene expression was determined with DNA array analysis and RT-PCR. Endothelial cells were removed by pre-sorting with CD31.ResultsABCG2 positive cells were localized to the prostate basal epithelium and endothelium. ABCG2+ cells in the basal epithelium constituted less than 1% of the total basal cell population. SP cells constituted 0.5–3% of the total epithelial fraction. The SP transcriptome was essentially the same as ABCG2+ and both populations expressed genes indicative of a stem cell phenotype, however, the cells also expressed many genes in common with endothelial cells.ConclusionThese results provide gene expression profiles for the prostate SP and ABCG2+ cells that will be critical for studying normal development and carcinogenesis, in particular as related to the cancer stem cell concept.

Gene expression relationship between prostate cancer cells of Gleason 3, 4 and normal epithelial cells as revealed by cell type-specific transcriptomes

BackgroundProstate cancer cells in primary tumors have been typed CD10-/CD13-/CD24hi/CD26+/CD38lo/CD44-/CD104-. This CD phenotype suggests a lineage relationship between cancer cells and luminal cells. The Gleason grade of tumors is a descriptive of tumor glandular differentiation. Higher Gleason scores are associated with treatment failure.MethodsCD26+ cancer cells were isolated from Gleason 3+3 (G3) and Gleason 4+4 (G4) tumors by cell sorting, and their gene expression or transcriptome was determined by Affymetrix DNA array analysis. Dataset analysis was used to determine gene expression similarities and differences between G3 and G4 as well as to prostate cancer cell lines and histologically normal prostate luminal cells.ResultsThe G3 and G4 transcriptomes were compared to those of prostatic cell types of non-cancer, which included luminal, basal, stromal fibromuscular, and endothelial. A principal components analysis of the various transcriptome datasets indicated a closer relationship between luminal and G3 than luminal and G4. Dataset comparison also showed that the cancer transcriptomes differed substantially from those of prostate cancer cell lines.ConclusionsGenes differentially expressed in cancer are potential biomarkers for cancer detection, and those differentially expressed between G3 and G4 are potential biomarkers for disease stratification given that G4 cancer is associated with poor outcomes. Differentially expressed genes likely contribute to the prostate cancer phenotype and constitute the signatures of these particular cancer cell types.

Stromal mesenchyme cell genes of the human prostate and bladder

BackgroundStromal mesenchyme cells play an important role in epithelial differentiation and likely in cancer as well. Induction of epithelial differentiation is organ-specific, and the genes responsible could be identified through a comparative genomic analysis of the stromal cells from two different organs. These genes might be aberrantly expressed in cancer since cancer could be viewed as due to a defect in stromal signaling. We propose to identify the prostate stromal genes by analysis of differentially expressed genes between prostate and bladder stromal cells, and to examine their expression in prostate cancer.MethodsImmunohistochemistry using antibodies to cluster designation (CD) cell surface antigens was first used to characterize the stromas of the prostate and bladder. Stromal cells were prepared from either prostate or bladder tissue for cell culture. RNA was isolated from the cultured cells and analyzed by DNA microarrays. Expression of candidate genes in normal prostate and prostate cancer was examined by RT-PCR.ResultsThe bladder stroma was phenotypically different from that of the prostate. Most notable was the presence of a layer of CD13+ cells adjacent to the urothelium. This structural feature was also seen in the mouse bladder. The prostate stroma was uniformly CD13-. A number of differentially expressed genes between prostate and bladder stromal cells were identified. One prostate gene, proenkephalin (PENK), was of interest because it encodes a hormone. Secreted proteins such as hormones and bioactive peptides are known to mediate cell-cell signaling. Prostate stromal expression of PENK was verified by an antibody raised against a PENK peptide, by RT-PCR analysis of laser-capture microdissected stromal cells, and by database analysis. Gene expression analysis showed that PENK expression was down-regulated in prostate cancer.ConclusionOur findings show that the histologically similar stromas of the prostate and bladder are phenotypically different, and express organ-specific genes. The importance of these genes in epithelial development is suggested by their abnormal expression in cancer. Among the candidates is the hormone PENK and the down-regulation of PENK expression in cancer suggests a possible association with cancer development.

Gene expression down-regulation in CD90+ prostate tumor-associated stromal cells involves potential organ-specific genes

BackgroundThe prostate stroma is a key mediator of epithelial differentiation and development, and potentially plays a role in the initiation and progression of prostate cancer. The tumor-associated stroma is marked by increased expression of CD90/THY1. Isolation and characterization of these stromal cells could provide valuable insight into the biology of the tumor microenvironment.MethodsProstate CD90+ stromal fibromuscular cells from tumor specimens were isolated by cell-sorting and analyzed by DNA microarray. Dataset analysis was used to compare gene expression between histologically normal and tumor-associated stromal cells. For comparison, stromal cells were also isolated and analyzed from the urinary bladder.ResultsThe tumor-associated stromal cells were found to have decreased expression of genes involved in smooth muscle differentiation, and those detected in prostate but not bladder. Other differential expression between the stromal cell types included that of the CXC-chemokine genes.ConclusionCD90+ prostate tumor-associated stromal cells differed from their normal counterpart in expression of multiple genes, some of which are potentially involved in organ development.

Androgen receptor gene mutation, rearrangement, polymorphism

Genetic aberrations of the androgen receptor (AR) caused by mutations, rearrangements, and polymorphisms result in a mutant receptor that has varied functions compared to wild type AR. To date, over 1,000 mutations have been reported in the AR with most of these being associated with androgen insensitivity syndrome (AIS). While mutations of AR associated with prostate cancer occur less often in early stage localized disease, mutations in castration-resistant prostate cancer (CRPC) patients treated with anti-androgens occur more frequently with 10-30% of these patients having some form of mutation in the AR. Resistance to anti-androgen therapy usually results from gain-of-function mutations in the LBD such as is seen with bicalutamide and more recently with enzalutamide (MDV3100). Thus, it is crucial to investigate these new AR mutations arising from drug resistance to anti-androgens and other small molecule pharmacological agents.

Tumor suppressor U19/EAF2 regulates thrombospondin-1 expression via p53

Inactivation of U19/EAF2 has been shown previously to lead to tumorigenesis in multiple organs; however, the mechanism of U19/EAF2 tumor suppression remains unclear. In this paper, we report that the expression of an anti-angiogenic protein, thrombospondin-1 (TSP-1) is down-regulated in the prostate and liver of U19/EAF2 knockout mouse. The U19/EAF2 knockout liver displayed increased CD31-positive blood vessels, suggesting that the TSP-1 down-regulation can contribute to increased angiogenesis. TSP-1 is reported to be a p53-target gene and p53 is a known binding partner of ELL, which binds to U19/EAF2. Here, we show that U19/EAF2 can co-localize and co-immunoprecipitate with p53 in transfected cells. In a TSP-1 promoter-driven luciferase reporter assay, p53 transfection suppressed the TSP-1 promoter activity and U19/EAF2 co-transfection blocked the p53 suppression of TSP-1 promoter. However, U19/EAF2 transfection alone had little or no effect on the TSP-1 promoter. The above observations together suggest that U19/EAF2 regulates the expression of TSP-1 via blocking p53 repression of the TSP-1 promoter.

The urologic epithelial stem cell database (UESC) - a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder.

BackgroundPublic databases are crucial for analysis of high-dimensional gene and protein expression data. The Urologic Epithelial Stem Cells (UESC) database http://scgap.systemsbiology.net/ is a public database that contains gene and protein information for the major cell types of the prostate, prostate cancer cell lines, and a cancer cell type isolated from a primary tumor. Similarly, such information is available for urinary bladder cell types.DescriptionTwo major data types were archived in the database, protein abundance localization data from immunohistochemistry images, and transcript abundance data principally from DNA microarray analysis. Data results were organized in modules that were made to operate independently but built upon a core functionality. Gene array data and immunostaining images for human and mouse prostate and bladder were made available for interrogation. Data analysis capabilities include: (1) CD (cluster designation) cell surface protein data. For each cluster designation molecule, a data summary allows easy retrieval of images (at multiple magnifications). (2) Microarray data. Single gene or batch search can be initiated with Affymetrix Probeset ID, Gene Name, or Accession Number together with options of coalescing probesets and/or replicates.ConclusionDatabases are invaluable for biomedical research, and their utility depends on data quality and user friendliness. UESC provides for database queries and tools to examine cell type-specific gene expression (normal vs. cancer), whereas most other databases contain only whole tissue expression datasets. The UESC database provides a valuable tool in the analysis of differential gene expression in prostate cancer genes in cancer progression.

Lineage relationship of prostate cancer cell types based on gene expression

BackgroundProstate tumor heterogeneity is a major factor in disease management. Heterogeneity could be due to multiple cancer cell types with distinct gene expression. Of clinical importance is the so-called cancer stem cell type. Cell type-specific transcriptomes are used to examine lineage relationship among cancer cell types and their expression similarity to normal cell types including stem/progenitor cells.MethodsTranscriptomes were determined by Affymetrix DNA array analysis for the following cell types. Putative prostate progenitor cell populations were characterized and isolated by expression of the membrane transporter ABCG2. Stem cells were represented by embryonic stem and embryonal carcinoma cells. The cancer cell types were Gleason pattern 3 (glandular histomorphology) and pattern 4 (aglandular) sorted from primary tumors, cultured prostate cancer cell lines originally established from metastatic lesions, xenografts LuCaP 35 (adenocarcinoma phenotype) and LuCaP 49 (neuroendocrine/small cell carcinoma) grown in mice. No detectable gene expression differences were detected among serial passages of the LuCaP xenografts.ResultsBased on transcriptomes, the different cancer cell types could be clustered into a luminal-like grouping and a non-luminal-like (also not basal-like) grouping. The non-luminal-like types showed expression more similar to that of stem/progenitor cells than the luminal-like types. However, none showed expression of stem cell genes known to maintain stemness.ConclusionsNon-luminal-like types are all representatives of aggressive disease, and this could be attributed to the similarity in overall gene expression to stem and progenitor cell types.