Differential gene expression profiling of functionally and developmentally distinct human prostate epithelial populations

Abstract

BACKGROUNDHuman fetal prostate buds appear in the 10th gestational week as solid cords, which branch and form lumens in response to androgen 1. Previous in vivo analysis of prostate epithelia isolated from benign prostatectomy specimens indicated that Epcam+CD44−CD49fHi basal cells possess efficient tubule initiation capability relative to other subpopulations 2. Stromal interactions and branching morphogenesis displayed by adult tubule-initiating cells (TIC) are reminiscent of fetal prostate development. In the current study, we evaluated in vivo tubule initiation by human fetal prostate cells and determined expression profiles of fetal and adult epithelial subpopulations in an effort to identify pathways used by TIC.METHODSImmunostaining and FACS analysis based on Epcam, CD44, and CD49f expression demonstrated the majority (99.9%) of fetal prostate epithelial cells (FC) were Epcam+CD44− with variable levels of CD49f expression. Fetal populations isolated via cell sorting were implanted into immunocompromised mice. Total RNA isolation from Epcam+CD44−CD49fHi FC, adult Epcam+CD44−CD49fHi TIC, Epcam+CD44+CD49fHi basal cells (BC), and Epcam+CD44−CD49fLo luminal cells (LC) was performed, followed by microarray analysis of 19 samples using the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data was analyzed using Partek Genomics Suite Version 6.4. Genes selected showed >2-fold difference in expression and P < 5.00E-2. Results were validated with RT-PCR.RESULTSGrafts retrieved from Epcam+CD44− fetal cell implants displayed tubule formation with differentiation into basal and luminal compartments, while only stromal outgrowths were recovered from Epcam- fetal cell implants. Hierarchical clustering revealed four distinct groups determined by antigenic profile (TIC, BC, LC) and developmental stage (FC). TIC and BC displayed basal gene expression profiles, while LC expressed secretory genes. FC had a unique profile with the most similarities to adult TIC. Functional, network, and canonical pathway identification using Ingenuity Pathway Analysis Version 7.6 compiled genes with the highest differential expression (TIC relative to BC or LC). Many of these genes were found to be significantly associated with prostate tumorigenesis.CONCLUSIONSOur results demonstrate clustering gene expression profiles of FC and adult TIC. Pathways associated with TIC are known to be deregulated in cancer, suggesting a cell-of-origin role for TIC versus re-emergence of pathways common to these cells in tumorigenesis. Prostate 75: 764–776, 2015. © The Authors. The Prostate, published by Wiley Periodicals, Inc.

Integrated Classification of Prostate Cancer Reveals a Novel Luminal Subtype with Poor Outcome

“…We observed a strong association (FDR<0.001; fold change>1.5) between luminal genes and PCS1 and PCS2, and basal genes and PCS3. To verify this observation, we used 2 independent datasets derived from luminal and basal cells from human (40) and mouse (GSE39509) (37) prostates. The assignment of a basal designation to PCS3 is further supported by the highly significant enrichment in PCS3, in comparison to the other 2 subtypes, of a recently described prostate basal cell signature derived from CD49f-Hi versus CD49f-Lo benign and malignant prostate epithelial cells (Fig.…”

Keratin 13 Is Enriched in Prostate Tubule-Initiating Cells and May Identify Primary Prostate Tumors that Metastasize to the Bone

“…Archival tissue obtained in an anonymous fashion without identifiers did not require written or verbal consent, as determined, by the IRBs at UCLA and GLA-VA. Fetal prostate tissue samples (approximately 14–18 weeks gestation) were obtained in accordance with federal and state guidelines from the Center for AIDS Research at UCLA. Prostate tissue was preserved in frozen/paraffin blocks or dissociated to obtain single cell suspensions as previously described [ 6 ]. For benign adult prostate cell fractionation studies, surgical specimens were allocated for research by pathology and frozen sections of adjacent tissue were prepared in order to confirm areas of benign prostate glands.…”

“…RNA was extracted from fractionated cells using Qiagen RNAeasy ® Micro Kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions. After RNA extraction, all quantitation and microarray experiments were performed at the UCLA Department of Pathology Clinical Microarray Core Laboratory as previously described using Affymetrix Gene Chip U133Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA) [ 6 ]. The Partek Genomics Suite Version 6.4 (Partek, St Louis, MO, USA) was employed and conducted using Partek default settings.…”

“…The Partek Genomics Suite Version 6.4 (Partek, St Louis, MO, USA) was employed and conducted using Partek default settings. RNA was extracted from FC, TIC, BC, and LC fractions as previously described[ 6 ]. A comparison of TIC versus BC and LC differentially expressed genes (n = 853) were selected at ≥ 2-fold difference.…”

“…Prostate development begins following the outgrowth of the stem cell rich urogenital sinus, which stimulates outgrowth of small glandular buds [ 5 ]. These epithelial cord-like structures display a basal profile, marked, in part by predominant expression of KRT5, KRT6, and KRT14 [ 6 , 7 ]. Branching morphogenesis and maturation of prostatic buds generates ducts and acini composed of distinct basal and luminal layers.…”