Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics

The epithelium of the adult prostate contains 3 distinct cell types: basal, luminal, and neuroendocrine. Tissue-regenerative activity has been identified predominantly from the basal cells, isolated by expression of CD49f and stem cell antigen-1 (Sca-1). An important question for the field is whether all basal cells have stem cell characteristics. Prostate-specific microarray databases were interrogated to find candidate surface antigens that could subfractionate the basal cell population. Tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1/GA733-1) was identified because it was enriched after castration, in prostate sphere cells and in the basal fraction. In the murine prostate, Trop2 shows progenitor characteristics such as localization to the region of the gland proximal to the urethra and enrichment for sphere-forming and colony-forming cells. Trop2 subfractionates the basal cells into 2 populations, both of which express characteristic basal cell markers by quantitative PCR. However, only the basal cells expressing high levels of Trop2 were able to efficiently form spheres in vitro. In the human prostate, where Sca-1 is not expressed, sphere-forming progenitor cells were also isolated based on high expression of Trop2 and CD49f. Trop2-expressing murine basal cells could regenerate prostatic tubules in vivo, whereas the remaining basal cells had minimal activity. Evidence was found for basal, luminal, and neuroendocrine cells in prostatic tubules regenerated from Trop2^hi basal cells. In summary, functionally distinct populations of cells exist within the prostate basal compartment and an epithelial progenitor can give rise to neuroendocrine cells in vivo.     

Suppressor role of androgen receptor in proliferation of prostate basal epithelial and progenitor cells

Early studies have reported the differential roles of androgen receptor (AR) in different types (luminal, basal intermediate, and stromal) of prostate cancer cells. In vivo mouse model tumor studies using the total prostate epithelial knockout mice (pes-ARKO) also revealed that AR played a suppressive role in proliferation of the CK5(+)/CK8(+) progenitor/intermediate cells but a positive role in the CK5(-)/CK8(+) luminal epithelial cells. Using three different resources (one human basal epithelial cell line, one mouse basal epithelial originated progenitor cell line, and a basal epithelium-specific ARKO mouse model), we here demonstrated that the AR in basal epithelial cells of normal prostate plays a suppressive role in their proliferation but a positive role in differentiation into luminal epithelial cells. These results led us to conclude that ARs may play a negative role to suppress CK5(+) basal epithelial and progenitor cell proliferation, yet play an essential role to drive basal epithelial cells into more differentiated states. These results may explain why differential AR expression in different cell types within normal prostate is needed and suggest that ARs in prostate basal epithelial cells, although expressed at a very low level, are necessary to maintain the balance between progenitor cells and differentiated luminal epithelial cells.     

Isolation and functional characterization of murine prostate stem cells

The ability to isolate prostate stem cells is essential to explore their role in prostate development and disease. In vitro prostate colony- and sphere-forming assays were used to quantitatively measure murine prostate stem/progenitor cell enrichment and self-renewal. Cell surface markers were screened for their ability to positively or negatively enrich for cells with enhanced growth potential in these assays. Immunohistochemical and FACS analyses demonstrate that specific cell surface markers can be used to discriminate prostate stromal (CD34(+)), luminal epithelial (CD24(+)CD49f(-)), basal epithelial (CD24(+)CD49f(+)), hematopoietic (CD45(+), Ter119(+)), and endothelial (CD31(+)) lineages. Sorting for cells with a CD45(-)CD31(-)Ter119(-)Sca-1(+)CD49f(+) antigenic profile results in a 60-fold enrichment for colony- and sphere-forming cells. These cells can self-renew and expand to form spheres for many generations and can differentiate to produce prostatic tubule structures containing both basal and luminal cells in vivo. These cells also localize to the basal cell layer within the region of the gland that is proximal to the urethra, which has been identified as the prostate stem cell niche. Prostate stem cells can be isolated to a purity of up to 1 in 35 by using this antigenic profile. The remarkable similarity in cell surface profile between prostate and mammary gland stem cells suggests these markers may be conserved among epithelial stem cell populations.     

Cell–cell interaction in prostate gene regulation and cytodifferentiation

To examine the role of intercellular interaction on cell differentiation and gene expression in human prostate, we separated the two major epithelial cell populations and studied them in isolation and in combination with stromal cells. The epithelial cells were separated by flow cytometry using antibodies against differentially expressed cell-surface markers CD44 and CD57. Basal epithelial cells express CD44, and luminal epithelial cells express CD57. The CD57⁺ luminal cells are the terminally differentiated secretory cells of the gland that synthesize prostate-specific antigen (PSA). Expression of PSA is regulated by androgen, and PSA mRNA is one of the abundant messages in these cells. We show that PSA expression by the CD57⁺ cells is abolished after prostate tissue is dispersed by collagenase into single cells. Expression is restored when CD57⁺ cells are reconstituted with stromal cells. The CD44⁺ basal cells possess characteristics of stem cells and are the candidate progenitors of luminal cells. Differentiation, as reflected by PSA production, can be detected when CD44⁺ cells are cocultured with stromal cells. Our studies show that cell–cell interaction plays an important role in prostatic cytodifferentiation and the maintenance of the differentiated state.     

Androgen receptor is a tumor suppressor and proliferator in prostate cancer

Targeting androgens/androgen receptor (AR) functions via androgen deprivation therapy (ADT) remains the standard treatment for prostate cancer. However, most tumors eventually recur despite ADT. Here we demonstrate that the prostate AR may function as both a suppressor and a proliferator to suppress or promote prostate cancer metastasis. Results from orthotopically recombining stromal WPMY1 cells with epithelial PC3 prostate cancer cells in mice demonstrated that restoring AR in epithelial PC3 cells or knockdown of AR in stromal WPMY1 cells suppressed prostate cancer metastasis. Knockdown of the AR in epithelial CWR22rv1 prostate cancer cells also resulted in increased cell invasion in vitro and in vivo. Restoring AR in PC3 cells (PC3-AR9) results in decreased invasion in bone lesion assays and in vivo mouse models. Mice lacking the prostate epithelial AR have increased apoptosis in epithelial luminal cells and increased proliferation in epithelial basal cells. The consequences of these two contrasting results led to the expansion of CK5/CK8-positive intermediate cells, and mice developed larger and more invasive metastatic tumors in lymph nodes and died earlier than wild-type littermates. Mechanistic dissection suggested that androgens/AR might directly or indirectly modulate metastasis-related genes and suppression of TGFβ1 signals results in the partial inhibition of AR-mediated metastasis. Collectively, our understanding of these opposing roles of prostatic AR may revolutionize the way we combat prostate cancer, and allow the development of new and better therapies by targeting only the proliferative role of AR.     

Effect of androgen ablation on prostatic cell differentiation in dogs

This study was conducted to gain further insight into the role of androgen in maintaining a balanced prostate gland growth in dogs. Effects of castration on prostatic cell were assessed by comparing the expression level of high molecular weight cytokeratin (HMW), alpha-actin, and vimentin in intact and castrated dogs. Mature dogs were castrated while they were under general anesthesia and were killed after 1 month. Mature prostate gland structures from intact dogs are characterized by the presence of differentiated columnar secretary epithelial cells and progenitor basal cells that are located within acini and ducts embedded in a thin fibromuscular tissue. Basal cells were distinguished from secretory epithelial cells by HMW cytokeratin immunostaining, which is expressed specifically by basal cells but not by epithelial cells. Castration-induced secretory epithelial cell death, leave the basal cells intact to form a continuous layer lining the atrophied acini. However, the survived basal cells lost their capacity to differentiate to secretory epithelial cells. In addition, androgen ablation induced remarkable reorganization of the cellular components of the fibromuscular compartment. In intact dogs, this compartment of prostate gland is composed mainly of differentiated smooth muscles and scattered mesenchymal muscles as reflected by the high and low actin and vimentin expressions, respectively. Castration for 1 month induced a progressive shift toward mesenchymal cells, which appeared to occupy most of the fibromuscular compartment. Based on these findings, it appears that androgen acts to maintain a steady state of prostate gland by driving the differentiation of prostatic cells and by maintaining its fully differentiated state.     

Expression of AC133 and CD117 on candidate normal stem cell populations in childhood B-cell precursor acute lymphoblastic leukaemia

To identify residual candidate normal progenitor/stem cell populations in childhood B-cell precursor acute lymphoblastic leukaemia (ALL), expression of AC133 and CD117 was analysed on the leukaemic cell clone and on immature B-lineage-negative CD34+CD19- bone marrow cells. 10/25 patients (40%) had no detectable expression of AC133 within the leukaemic cell clone. 24/26 patients (92%) lacked expression of CD117 on the leukaemic blast cell population. In contrast, a distinct AC133-positive cell population was found in 8/8 children with AC133-negative ALL and a CD117-positive cell population could be identified in 12/12 children with CD117-negative ALL, within the CD34+CD19- progenitor/stem cell compartment. These observations provide further evidence that in B-cell precursor ALL, unlike in acute myelogenous leukaemia, it may be possible to distinguish residual normal progenitor/stem cells from the leukaemic cell clone.     

Estrogen-initiated transformation of prostate epithelium derived from normal human prostate stem-progenitor cells

The present study sought to determine whether estrogens with testosterone support are sufficient to transform the normal human prostate epithelium and promote progression to invasive adenocarcinoma using a novel chimeric prostate model. Adult prostate stem/early progenitor cells were isolated from normal human prostates through prostasphere formation in three-dimensional culture. The stem/early progenitor cell status and clonality of prostasphere cells was confirmed by immunocytochemistry and Hoechst staining. Normal prostate progenitor cells were found to express estrogen receptor α, estrogen receptor β, and G protein-coupled receptor 30 mRNA and protein and were responsive to 1 nm estradiol-17β with increased numbers and prostasphere size, implicating them as direct estrogen targets. Recombinants of human prostate progenitor cells with rat urogenital sinus mesenchyme formed chimeric prostate tissue in vivo under the renal capsule of nude mice. Cytodifferentiation of human prostate progenitor cells in chimeric tissues was confirmed by immunohistochemistry using epithelial cell markers (p63, cytokeratin 8/18, and androgen receptor), whereas human origin and functional differentiation were confirmed by expression of human nuclear antigen and prostate-specific antigen, respectively. Once mature tissues formed, the hosts were exposed to elevated testosterone and estradiol-17β for 1-4 months, and prostate pathology was longitudinally monitored. Induction of prostate cancer in the human stem/progenitor cell-generated prostatic tissue was observed over time, progressing from normal histology to epithelial hyperplasia, prostate intraepithelial neoplasia, and prostate cancer with local renal invasion. These findings provide the first direct evidence that human prostate progenitor cells are estrogen targets and that estradiol in an androgen-supported milieu is a carcinogen for human prostate epithelium.     

Actions of estrogens and endocrine disrupting chemicals on human prostate stem/progenitor cells and prostate cancer risk

Estrogen reprogramming of the prostate gland as a function of developmental exposures (aka developmental estrogenization) results in permanent alterations in structure and gene expression that lead to an increased incidence of prostatic lesions with aging. Endocrine disrupting chemicals (EDCs) with estrogenic activity have been similarly linked to an increased prostate cancer risk. Since it has been suggested that stem cells and cancer stem cells are potential targets of cancer initiation and disease management, it is highly possible that estrogens and EDCs influence the development and progression of prostate cancer through reprogramming and transforming the prostate stem and early stage progenitor cells. In this article, we review recent literature highlighting the effects of estrogens and EDCs on prostate cancer risk and discuss recent advances in prostate stem/progenitor cell research. Our laboratory has recently developed a novel prostasphere model using normal human prostate stem/progenitor cells and established that these cells express estrogen receptors (ERs) and are direct targets of estrogen action. Further, using a chimeric in vivo prostate model derived from these normal human prostate progenitor cells, we demonstrated for the first time that estrogens initiate and promote prostatic carcinogenesis in an androgen-supported environment. We herein discuss these findings and highlight new evidence using our in vitro human prostasphere assay for perturbations in human prostate stem cell self-renewal and differentiation by natural steroids as well as EDCs. These findings support the hypothesis that tissue stem cells may be direct EDC targets which may underlie life-long reprogramming as a consequence of developmental and/or transient adult exposures.     

The Sca-1 cell surface marker enriches for a prostate-regenerating cell subpopulation that can initiate prostate tumorigenesis

Sca-1 (stem cell antigen-1) enriches for murine prostate cells capable of regenerating tubular structures containing basal and luminal cell lineages in a dissociated cell prostate regeneration system. Sca-1(+) fractions are enriched for cells at the G(0) stage of the cell cycle, and Sca-1(+) cells cluster in the proximal region of prostatic tubules where replication-quiescent cells have been localized. Castration-induced enrichment for androgen-independent cells results in a concomitant enrichment for Sca-1(+) cells. Genetic perturbations of PTEN/AKT signaling in prostate-regenerating cells leads to the initiation of tumorigenesis, and cancer progression is associated with a dramatic increase in Sca-1(+) cells. Sca-1-enriched prostate-regenerating cells possess multiple stem/progenitor cell properties and can serve as targets for cancer initiation.     

Androgen receptor signalling in prostate: effects of stromal factors on normal and cancer stem cells

The prostate gland is the most common site for cancer in males within the developed world. Androgens play a vital role in prostate development, maintenance of tissue function and pathogenesis of prostate disease. The androgen receptor signalling pathway facilitates that role in both the epithelial compartment and in the underlying stroma. Stroma is a key mediator of androgenic effects upon the epithelium and can regulate both the fate of the epithelial stem cell and potentially the initiation and progression of prostate cancer. Different groups of growth factors are expressed by stroma, which control proliferation, and differentiation of prostate epithelium demonstrating a critical role for stroma in epithelial growth and homeostasis. Paracrine stromal proteins may offer the possibility to control tumour stem cell growth and could permit prostate specific targeting of both therapies and of androgen responsive proteins. The effect of 5alpha-dihydrotestosterone, the more potent metabolite of testosterone, on expression of androgen-regulated genes in stroma from benign prostatic hyperplasia is a key mediator of epithelial cell fate. Global gene expression arrays have recently identified new candidate genes in androgen responsive stroma, some of which have androgen receptor binding sites in their promoter regions. Some of these genes have direct androgen receptor binding ability.     

In vivo regeneration of murine prostate from dissociated cell populations of postnatal epithelia and urogenital sinus mesenchyme

The existence of a postnatal prostate stem cell is supported by several types of evidence. Withdrawal of androgen leads to involution of the gland, but readdition can rapidly stimulate regeneration. Tissue fragments derived from mouse or rat prostatic epithelia from midgestation embryos or adult mice, when combined with tissue fragments from urogenital sinus mesenchyme and grafted under the kidney capsule, can regenerate prostatic structures. Indirect evidence supports that the stem cell population is contained within the basal layer. Purified prostatic stem cell preparations would be useful to define the physical and functional properties required for regeneration and to compare with cells that accumulate during abnormal growth states, like prostate cancer. We have developed a regeneration system using dissociated cell populations of postnatal prostate epithelia and embryonic urogenital sinus mesenchyme. Efficient in vivo regeneration of prostatic structures in the subcapsular space of the kidney was observed within 4-8 wk with as few as 103 epithelial cells from prostates derived from donors 10 d to 6 wk of age. The regenerated structures show a branching tubular epithelial morphology, with expression of a panel of markers consistent with prostate development. Donor epithelial populations can be readily infected with GFP expressing lentiviral vectors to provide integration markers and easy visualization. The cell preparations of urogenital sinus mesenchyme can be expanded in short-term in vitro culture while their inductive capabilities are retained. Further definition of the subpopulation of prostate epithelial cells containing the regeneration activity should be possible with such technologies.     

Functions of normal and malignant prostatic stem/progenitor cells in tissue regeneration and cancer progression and novel targeting therapies

This review summarizes the recent advancements that have improved our understanding of the functions of prostatic stem/progenitor cells in maintaining homeostasis of the prostate gland. We also describe the oncogenic events that may contribute to their malignant transformation into prostatic cancer stem/progenitor cells during cancer initiation and progression to metastatic disease stages. The molecular mechanisms that may contribute to the intrinsic or the acquisition of a resistant phenotype by the prostatic cancer stem/progenitor cells and their differentiated progenies with a luminal phenotype to the current therapies and disease relapse are also reviewed. The emphasis is on the critical functions of distinct tumorigenic signaling cascades induced through the epidermal growth factor system, hedgehog, Wnt/beta-catenin, and/or stromal cell-derived factor-1/CXC chemokine receptor-4 pathways as well as the deregulated apoptotic signaling elements and ATP-binding cassette multidrug transporter. Of particular therapeutic interest, we also discuss the potential beneficial effects associated with the targeting of these signaling elements to overcome the resistance to current treatments and prostate cancer recurrence. The combined targeted strategies toward distinct oncogenic signaling cascades in prostatic cancer stem/progenitor cells and their progenies as well as their local microenvironment, which could improve the efficacy of current clinical chemotherapeutic treatments against incurable, androgen-independent, and metastatic prostate cancers, are also described.     

Noncanonical Wnt signaling mediates androgen-dependent tumor growth in a mouse model of prostate cancer

Prostate cancer development is associated with hyperactive androgen signaling. However, the molecular link between androgen receptor (AR) function and humoral factors remains elusive. A prostate cancer mouse model was generated by selectively mutating the AR threonine 877 into alanine in prostatic epithelial cells through Cre-ERT2-mediated targeted somatic mutagenesis. Such AR point mutant mice (ARpe-T877A/Y) developed hypertrophic prostates with responses to both an androgen antagonist and estrogen, although no prostatic tumor was seen. In prostate cancer model transgenic mice, the onset of prostatic tumorigenesis as well as tumor growth was significantly potentiated by introduction of the AR T877A mutation into the prostate. Genetic screening of mice identified Wnt-5a as an activator. Enhanced Wnt-5a expression was detected in the malignant prostate tumors of patients, whereas in benign prostatic hyperplasia such aberrant up-regulation was not obvious. These findings suggest that a noncanonical Wnt signal stimulates development of prostatic tumors with AR hyperfunction.     

Androgen induces differentiation of a human papillomavirus 16 E6/E7 immortalized prostate epithelial cell line.

Androgen signaling is crucial for the growth and development, as well as for tumorigenesis of the prostate. However, many of the prostate epithelial cell lines developed previously, either normal or tumorigenic, do not express androgen receptor (AR) or respond to androgen. In order to advance our understanding on how androgen signaling regulates the growth and the differentiation status, and affects tumorigenicity of the epithelial cell, we performed experiments on HPr-1, a prostate cell line recently immortalized from normal human prostate epithelial cells. In the present study, AR was stably transfected into HPr-1 cells by replication-defective retrovirus. Treatment of HPr-1AR cells with androgen resulted in cell differentiation and growth retardation accompanied with up-regulation of cytokeratins K8 and K18, prostate specific antigen, p21 and p27, and down-regulation of c-myc, bcl-2 and telomerase activity. Our results suggest that androgen promotes the process of differentiation in a human papillomavirus 16 E6/E7 immortalized prostate epithelial cell line which may reflect the normal effects of androgen on prostate cells.     

Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma

Like many epithelial tumors, head and neck squamous cell carcinoma (HNSCC) contains a heterogeneous population of cancer cells. We developed an immunodeficient mouse model to test the tumorigenic potential of different populations of cancer cells derived from primary, unmanipulated human HNSCC samples. We show that a minority population of CD44(+) cancer cells, which typically comprise <10% of the cells in a HNSCC tumor, but not the CD44(-) cancer cells, gave rise to new tumors in vivo. Immunohistochemistry revealed that the CD44(+) cancer cells have a primitive cellular morphology and costain with the basal cell marker Cytokeratin 5/14, whereas the CD44(-) cancer cells resemble differentiated squamous epithelium and express the differentiation marker Involucrin. The tumors that arose from purified CD44(+) cells reproduced the original tumor heterogeneity and could be serially passaged, thus demonstrating the two defining properties of stem cells: ability to self-renew and to differentiate. Furthermore, the tumorigenic CD44(+) cells differentially express the BMI1 gene, at both the RNA and protein levels. By immunohistochemical analysis, the CD44(+) cells in the tumor express high levels of nuclear BMI1, and are arrayed in characteristic tumor microdomains. BMI1 has been demonstrated to play a role in self-renewal in other stem cell types and to be involved in tumorigenesis. Taken together, these data demonstrate that cells within the CD44(+) population of human HNSCC possess the unique properties of cancer stem cells in functional assays for cancer stem cell self-renewal and differentiation and form unique histological microdomains that may aid in cancer diagnosis.     

Detection of tumor stem cell markers in pancreatic carcinoma cell lines

IntroductionT he cell population of most tumors is hetero- geneous with regard to its proliferation capacity, apoptosis-resistance mechanisms, and ability to reconstitute the tumor upon xeno-transplantation. This phenomenon arises as the result of accumulation of multiple genetic and epigenetic changes. Current evidence suggests that only a few cells within the tumor, the cancer stem cells, possess unlimited proliferative capacity and give rise to tumors that phenotypically resemble their init…