Ovarian cancer stem cell-like side populations are enriched following chemotherapy and overexpress EZH2.

Platinum-based chemotherapy, with cytoreductive surgery, is the cornerstone of treatment of advanced ovarian cancer; however, acquired drug resistance is a major clinical obstacle. It has been proposed that subpopulations of tumor cells with stem cell-like properties, such as so-called side populations (SP) that overexpress ABC drug transporters, can sustain the growth of drug-resistant tumor cells, leading to tumor recurrence following chemotherapy. The histone methyltransferase EZH2 is a key component of the polycomb-repressive complex 2 required for maintenance of a stem cell state, and overexpression has been implicated in drug resistance and shorter survival of ovarian cancer patients. We observed higher percentage SP in ascites from patients that have relapsed following chemotherapy compared with chemonaive patients, consistent with selection for this subpopulation during platinum-based chemotherapy. Furthermore, ABCB1 (P-glycoprotein) and EZH2 are consistently overexpressed in SP compared with non-SP from patients' tumor cells. The siRNA knockdown of EZH2 leads to loss of SP in ovarian tumor models, reduced anchorage-independent growth, and reduced tumor growth in vivo. Together, these data support a key role for EZH2 in the maintenance of a drug-resistant, tumor-sustaining subpopulation of cells in ovarian cancers undergoing chemotherapy. As such, EZH2 is an important target for anticancer drug development.

A novel, spontaneously immortalized, human prostate cancer cell line, Bob, offers a unique model for pre-clinical prostate cancer studies.

INTRODUCTION: New in vitro models of castration-resistant prostate cancer (CRPC) are urgently required. METHODS: Trans-rectal needle biopsies (TRBP) of the prostate were performed for research purposes on progressing CRPC patients who had not received prior treatment to the prostate. Biopsies were immediately digested with collagenase and plated onto collagen-coated flasks with a feeder layer of 3T6 cells and cultured in cytokine-supplemented keratinocyte serum-free medium. RESULTS: Biopsies from 25 patients were collected and one of these, following an initial period of crisis, spontaneously immortalized. A series of cell lines called Bob were then established from a clone that survived CD133-selection followed by 4 weeks under adhesion-independent conditions in methylcellulose. Gains and losses previously described in clinical prostate tumors, most notably loss of 8(p) and gain of 8(q), were identified on comparative genomic hybridization and long-term growth in culture, survival in methylcellulose and invasion through matrigel confirmed the malignant phenotype of Bob. Furthermore, Bob expressed high levels of p53 and markers of early differentiation, including K8, prostatic acid phosphatase and prostate stem cell antigen. There was, however, no in vivo growth and ERG and ETV1 were not rearranged. Growth in serum permitted some differentiation. CONCLUSION: This is the first spontaneously immortalized prostate cancer cell line to be established from a TRBP of a patient with CRPC. Bob is a novel pre-clinical model for functional studies in CRPC and especially for studying the CRPC "basal" phenotype.

Expression profiling of CD133+ and CD133- epithelial cells from human prostate.

BACKGROUND: Recent evidence suggests that prostate stem cells in benign and tumor tissue express the cell surface marker CD133, but these cells have not been well characterized. The aim of our study was to gene expression profile CD133-expressing cells. METHODS: We analyzed CD133-positive (CD133+) and -negative (CD133-) sub-populations of high-integrin expressing epithelial cells isolated from benign human prostate tissue and hormone-refractory prostate cancer (HRPC). RESULTS: CD133+ cells freshly isolated from benign prostate tissue exhibited an expression profile characteristic of a putative stem/progenitor cell population, with transcripts involved in biological processes ranging from development and ion homeostasis to cell communication. The profile of CD133- cells was consistent with that of a transit amplifying population, suggesting up-regulated proliferation and metabolism. Comparison of benign populations to those from HRPC showed some similarities between CD133+ profiles but also revealed significant differences that provide a tumor-specific pattern, which included evidence of increased metabolic activity and active proliferation. Subsequently, we demonstrated protein expression of a number of candidate genes in these cell populations and in benign tissue. In a novel observation we also found expression of some of these markers in prostate tumors, including the oligodendrocyte lineage transcription factor OLIG1. CONCLUSIONS: This study provides a unique genome-wide molecular signature of CD133+ and CD133- human prostate epithelial cells. This will provide a valuable resource for prostate stem cell biology research and the identification of novel therapeutic targets for the treatment of prostate cancer.

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation.

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.

Comparison of the zones of the human prostate with the seminal vesicle: morphology, immunohistochemistry, and cell kinetics.

BACKGROUND: The prostate contains three glandular zones (central, peripheral, transition) with widely differing susceptibilities to cancer and benign prostatic hyperplasia (BPH). Most of the prostate is derived from urogenital sinus, but the central zone may be derived from Wolffian duct, in common with the seminal vesicles (SV). The peripheral zone is the most frequent site of cancer and the transition zone is the almost exclusive site of BPH. METHOD: We compared the histology and immunohistochemistry of the SV with those of the prostate zones in order to identify differences associated with susceptibility to disease or different embryological origins. Sections from the prostates of nine organ donors (aged 15-36) were stained for tissue-specific markers, antigens previously shown to stain differentially between the zones and markers of cell proliferation and cell death. RESULTS: Neuroendocrine cells were absent from the SV and significantly fewer neuroendocrine cells were seen in the central zone compared to the peripheral zone. Most of the SV epithelium stained for lactoferrin, compared to approximately one-third of central zone and only 2% of peripheral zone epithelial cells. The proliferative index of the central zone was approximately 50% lower and the incidence of apoptotic cells approximately half that of the peripheral and transition zones. CONCLUSIONS: The central zone has features in common with both the SV and the other zones of the prostate. The higher incidence of proliferative diseases in the transition and peripheral zones may be associated with the higher rate of cell turnover observed in these zones.

Differential expression of CD44 during human prostate epithelial cell differentiation.

CD44 is a polymorphic transmembrane glycoprotein that binds hyaluronan and growth factors. Multiple isoforms of the protein can be generated by alternative splicing but little is known about the expression and function of these isoforms in normal development and differentiation. We have investigated the expression of CD44 during normal prostate epithelial cell differentiation. A conditionally immortalized prostate epithelial cell line, Pre2.8, was used as a model system. These cells proliferate at 33C but at 39C stop dividing and undergo changes consistent with early stages of cell differentiation. During the differentiation of these cells, the expression of the CD44 isoform v3-v10 was upregulated. Two layers of epithelial cells can clearly be distinguished in the human prostate, a basal layer expressing keratins 5/14 and a luminal layer expressing keratins 8/18. In prostate tissue the v3-v10 isoform was found predominantly in basal cells but also in keratin 14-negative, keratin 19-positive cells intermediate between the two layers. CD44 v3-v10 was also expressed in other keratin 14-negative prostate tissues, the ejaculatory ducts and prostatic urethra. Therefore, CD44 v3-v10 may be important as a cell surface marker for differentiating cells in the prostate epithelium.

Prostate epithelial stem cell culture.

The prostate gland is the site of the second most common cancer in men in the UK, with 9,280 deaths recorded in 2000. Another common disease of the prostate is benign prostatic hyperplasia and both conditions are believed to arise as a result of changes in the balance between cell proliferation and differentiation. There are three types of prostatic epithelial cell, proliferative basal, secretory luminal, and neuroendocrine. All three are believed to be derived from a common stem cell through differentiation along different pathways but the mechanisms behind these processes is poorly understood. In particular, there has until recently been very little information about prostate stem cell growth and differentiation. This review will discuss ways of distinguishing these prostate cell types using markers, such as keratins. Methods available for the culture of prostate epithelial cells and for the characterisation of stem cells both in monolayer and three-dimensional models are examined.