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Developing a Novel Two-Dimensional Culture System to Enrich Human Prostate Luminal Progenitors That Can Function as a Cell of Origin for Prostate Cancer

ABSTRACT: Elucidating the cell of origin of cancer has great significance in stratifying patients into appropriate treatment groups and for developing novel targeted therapies. Early studies demonstrate that only stem-like basal cells in the normal human prostate (NHP) can function as the cell of origin for prostate cancer (PCa). Here, we show that the organoids derived from bulkNHPluminal cells can also be tumorigenically transformed. We further show that the WIT medium, which is used to culture human mammary epithelial progenitor cells, when combined with the ROCK inhibitor, can readily propagate a population of progenitor-like cells from the primary NHP luminal cell isolates. Such functionally defined luminal progenitors can be transformed by distinct sets of genetic perturbations (i.e., AR+AKT/ERG or c-MYC+PTEN knockout) to form tumor glands. Genome-wide RNA-Seq analysis of freshly purified unperturbed human benign prostatic basal and luminal cells and culture-expanded lineage specific stem/progenitor populations reveals that the luminal progenitors possess a distinct gene expression profile that is greatly enriched in advanced, castration-resistant, and metastatic PCa, and it associates with poor patient survival. The ability of the simple two-dimensional culture system reported herein to greatly enrichNHPprogenitor-like cells should facilitate biological and biochemical studies as well as high-throughput screening in these cells and in progenitor-like PCa cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE74698 | GEO | 2016/09/25

SECONDARY ACCESSION(S): PRJNA301175

REPOSITORIES: GEO

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Project description:Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain incompletely understood. Here we first show that multiple primary strains of NHP cells are all immunophenotyped as CK5+/CK18+CD44+α2β1+p63+hTERT+ progenitor cells, which gradually lose progenitor markers as they lose proliferative capacity. NHP cell senescence involves loss of telomerase expression, upregulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and hTERT is required for unlimited proliferative lifespan. Hence, suppression of 16 alone significantly extends NHP cell lifespan but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, the immortalized NHP cells are normal progenitors that possess the ability to differentiate into functional prostatic glands containing both basal and luminal cells and, frequently, neuroendocrine cells. The immortalized NHP cells possess gene expression profiles characteristic of proliferating progenitor cells. Our studies shed light on the molecular mechanisms regulating the proliferative lifespan of NHP progenitor cells and provide direct evidence that basal-like progenitor cells can regenerate the entire prostatic glands in vivo. The availability of these cells should facilitate future studies of prostate cancer development. Further processed data linked below as Supplementary files. TestvsControl: corresponding GSM# 3vs1: GSM266720, 721, 722 4vs1: GSM266727, 730, 731 5vs1: GSM266732, 733, 734 7avs1: GSM266735, 736, 737 9vs1: GSM266772, 773, 812 11vs1: GSM266813, 816, 823 13vs1: GSM266824, 826, 828 Note: The 'TestvsControl' comparisons report the top 100 up- or down-regulated genes. The numeric designations (1 to 13) are used in the corresponding manuscript. The 'SignificantGenes' lists were used in clustering analysis. Keywords: Cell type comparison (senscent and immortalized NHP cells compared to young NHP progenitors)

Project description:The highly intra-tumoral heterogeneity and complex cell origination of PCa greatly limited the significance of traditional bulk RNA sequencing in finding better biomarker for disease stratification. In the context of these sequencing strategies, significant gene expression differences in specific cell population might be normalized or even shaded by genes in those less “important” but large in amount cells. Tissue specimens based single-cell RNA sequencing holds great promise for identification of novel stratification biomarkers. However, the preparation of enough viable cells for sequencing, in particular for prostate cancer (PCa), is very challenging when using this technique. In the current study, we, for the first time, employed single-cell RNA sequencing of PCa cells isolated from cancerous prostate tissues of two patients and identified fifteen cell groups including three luminal clusters with different expression profiles. One of these luminal clusters had the highest copy number variation level and marker genes mainly enriched in PCa-related metabolic process, thereafter, was considered as the malignant luminal cells in PCa. Further analysis indicates that the fifth subgroup of these cells highly expressed genes significantly correlated with PCa initiation and early development. In addition, we found this subgroup highly expressed PCa prognosis biomarkers such as AMACR and PCA3. These findings suggest that this cell subgroup might be a critical cell population for PCa diagnosis and stratification. Moreover, by further deciphering the gene expression of this subgroup, we identified another marker gene, HPN, with a 0.930 area under the curve (AUC) score distinguishing normal tissue from PCa lesion using the RNA-seq data from The Cancer Genome Atlas (TCGA). This finding was further validated by immunostaining of HPN in PCa tissue array. The expression of HPN in PCa with Gleason score > 6 is significantly higher than those in Gleason score = 6. Taken together, we provide a valuable resource for interpreting the tumor heterogeneity in PCa, and a novel candidate marker for PCa management.

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Developing a Novel Two-Dimensional Culture System to Enrich Human Prostate Luminal Progenitors That Can Function as a Cell of Origin for Prostate Cancer

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