Characterizing the Contribution of Stem/Progenitor Cells to Tumorigenesis in the Pten^−/−TP53^−/− Prostate Cancer Model

Abstract

Loss of PTEN is one of the most common mutations in prostate cancer, and loss of wild-type TP53 is associated with prostate cancer progression and castrate resistance. Modeling prostate cancer in the mouse has shown that while Pten deletion in prostate epithelial cells leads to adenocarcinoma, combined loss of Pten and TP53 results in rapidly developing disease with greater tumor burden and early death. TP53 contributes significantly to the regulation of stem cell self-renewal, and we hypothesized that loss of Pten/TP53 would result in measurable changes in prostate cancer stem/progenitor cell properties. Clonogenic assays that isolate progenitor function in primary prostate epithelial cells were used to measure self-renewal, differentiation, and tumorigenic potential. Pten/TP53 null as compared with wild-type protospheres showed increased self-renewal activity and modified lineage commitment. Orthotopic transplantation of Pten/TP53 null cells derived from protospheres produced invasive Prostatic Intraepithelial Neoplasia (PIN)/adenocarcinoma, recapitulating the pathology seen in primary tumors. Pten/TP53 null progenitors relative to wild type also demonstrated increased dependence on the AKT/mammalian target of rapamycin complex 1 (mTORC1) and androgen receptor (AR) pathways for clonogenic and tumorigenic growth. These data demonstrate roles for Pten/TP53 in prostate epithelial stem/progenitor cell function, and moreover, as seen in patients with castrate-resistant prostate cancer, suggest for the involvement of an AR-dependent axis in the clonogenic expansion of prostate cancer stem cells.

Introduction

Prostate cancers display a range of clinical behavior, from slow-growing tumors of no clinical significance to aggressively metastatic and ultimately lethal disease. Human prostate adenocarcinoma has a mature luminal phenotype characterized by androgen receptor (AR) expression and PSA production. Progressive prostate cancer (PC) is almost always treated with androgen deprivation therapy, but despite such treatment, approximately 10% of prostate cancers progress to metastatic disease [1]. A major factor in the determination of aggressiveness is thought to result from the accumulation of particular somatic genetic alterations in one or more types of prostate stem/progenitor cells [2]. One of the most common genetic alterations in prostate cancer is deletion of at least one copy of the PTEN tumor suppressor, which occurs in approximately 70% of human prostate cancers. Biallelic deletion of PTEN and the associated increase in AKT phosphorylation, which occurs in roughly 25% of PC, is correlated with resistance to androgen-deprivation therapy (commonly referred to as castrate-resistant PC) [3]. A recent genomic profiling study of mostly primary prostate cancers demonstrated that 21% of cases had either a heterozygous or homozygous copy number loss of TP53 [4]. Other large-scale studies using combined immunohistochemistry and sequencing approaches have shown that TP53 mutations occur in approximately 5% of primary tumors and at much higher frequencies in lymph node metastases (16%) and castrate-resistant (26%) tumors [5, 6]. Additionally, TP53 mutations were found to be independent predictors of tumor recurrence in low and intermediate grade cancers. Thus, loss of PTEN and mutation of TP53 are implicated in aggressive forms of human PC [6].

Cancer stem cells (CSCs) are an experimentally defined subpopulations of self-renewing cancer cells that are capable of initiating tumor formation on transplantation. The phenotype and the relative content of CSCs have been shown to significantly contribute to tumor pathological features in hematopoietic malignancies and in solid tumors such as breast cancer [7, 8]. CSCs express stem/progenitor markers and can exhibit stem cell properties such as multilineage differentiation potential. In this respect, it is of interest that PC metastases tend to have a poorly differentiated morphology and not infrequently are composed of admixtures of mature luminal cells with intermediate and/or neuroendocrine cells [1, 9]. As one approach to gaining a mechanistic understanding of PC progression, here we have begun defining the functional characteristics of prostate CSCs carrying specific mutations associated with aggressiveness.

Knowledge of normal lineage differentiation in the prostate is necessary for analyzing the cell of origin and the CSCs for PC. The three cell types that compose prostate glands are basal, secretory luminal, and neuroendocrine cells. The existence of a tripotential stem cell has been established in mice following the reconstitution of prostatic acini containing all three cell types from individual prostate stem cells [10]. Recently, a progenitor cell has been observed in mice following castration that is distinct from the tripotential stem cell discussed earlier [11]. These cells, referred to as castration-resistant Nkx3.1 expressing (CARNs), display an AR luminal phenotype and have the capacity to produce basal, luminal, and neuroendocrine progeny. Starting from a stem cell, the hierarchical relationships and phenotypes of cells that form intermediate steps in the differentiation sequence leading to the mature lineages that constitute prostate glands are not precisely known. However, cells with intermediate phenotypes that express combinations of basal, luminal, and/or neuroendocrine markers have been described in histological sections of normal human and mouse prostates, and at least some of the intermediate cell types are thought to be transit amplifying common progenitor cells [12–16].

Deletion of Pten initiated by pan prostate epithelial cell expression of Probasin (Pb)-Cre leads to the expansion of basal cells and the development of adenocarcinoma with an intermediate/luminal phenotype [17]. Previous investigations into the effect of combined genetic deficiencies in mouse models of prostate cancer have demonstrated that prostate epithelial cell-specific loss of Pten and TP53 resulted in significantly more penetrant and rapidly developing PC than Pten deletion alone, while TP53 loss only did not lead to any notable phenotype [18]. Pten/TP53 null prostate tumors demonstrated many fewer senescent tumor cells than Pten null tumors, and it was proposed that the synergistic effect of Pten/TP53 deletion results from a loss of TP53-dependent cellular senescence secondary to Pten loss [18]. We hypothesized that in addition to abrogating a senescence response in the bulk tumor cells, Pten and TP53 play a crucial role in regulating the biological properties of prostate stem/progenitor cells. Similarly, recent investigations in glioblastoma stem cell function have demonstrated that the combined functions of Pten and TP53 control glioma self-renewal and differentiation [19]. Here, we report that mouse prostate stem/progenitor cells lacking both PTEN and TP53 as compared with similarly functionally defined populations of wild-type cells have higher intrinsic self-renewal activity, execute an altered lineage differentiation program and initiate invasive tumors on orthotopic transplantation. In addition, there exists a novel population of Pten/TP53 null progenitor cells not observed in wild-type progenitor populations whose clonogenic and tumorigenic growth is prevented by AKT or mTORC or AR inhibitors.

Materials and Methods

Cell Culture

Reagents.

Prostate epithelial cell basal media (PrEGM) with supplements was from Lonza (Walkersville, MD, http://www.lonza.com), Dulbecco’s-modified Eagle’s medium containing 10% fetal bovine serum (FBS) and antibiotic reagents, dispase, collagenase type II, and trypsin were from Invitrogen (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). BD Matrigel was purchased from BD Biosciences (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Drug inhibitors used were as follows: Triciribine from Cayman Chemical (Ann Arbor, MI, http://www.caymanchem.com), rapamycin from Calbiochem (Gibbstown, NJ, http://www.calbiotech.com), and Bicalutamide and nilutamide from Sigma-Aldrich (Saint Louis, MO, http://www.sigmaaldrich.com). Flutamide pellets and CCI-779 were from LC Laboratories (Woburn, MA, http://www.lclabs.com).

Preparation of Primary Mouse Prostate Epithelial Cells.

Three- to five-month-old Probasin (Pb)-Cre⁴⁺;Pten^fl/fl; TP53^fl/fl;Luc (hereafter referred to as Pten^−/−TP53^−/−) and Pb-Cre⁴⁻; Pten^fl/fl;TP53^fl/fl;Luc (hereafter referred to as wt) mice were euthanized by carbon dioxide inhalation and the entire lower urogenital tract aseptically removed and processed to single-cell suspensions, essentially as described by others [20] with the following modifications: after collagenase type II and trypsin treatment, cells were carefully passed through 19-, 23-, 25-, 27-, and 30.5-gauge needles. After washing, cells were passed through a 40-µm cell strainer and centrifuged for 6 minutes at 1,000 rpm. Finally, the pellet was resuspended in 1 ml of PrEGM and the number of viable cells was counted using the trypan blue exclusion method.

Colony Formation Assay.

Single-cell suspensions obtained as described earlier were seeded in 12-well plates in serum-free PrEGM with or without the indicated drug at a density of 10,000 cells/well in triplicate and incubated for 2 days at 37°C in a humidified incubator. After 2 days, half the media was gently aspirated and replaced with fresh media. Colonies were counted between days 5 and 7 after plating.

Three-Day Cultures and Sphere Formation Assay.

Single cells were suspended in Matrigel/serum-free PrEGM (1:1) at a concentration of 10,000 cells/well in a total volume of 100 µl, in triplicate. The solution was plated gently around the rim of individual wells of a 12-well plate and allowed to solidify for 1 hour at 37°C in a humidified incubator. Serum-free PrEGM, with or without the indicated drug, was added gently to the center of each well and the media was changed every 2–3 days. Spheres were counted between days 12 and 15 after plating. Spheres were propagated as described previously [20] with the modifications described earlier for the preparation of single-cell suspensions. Such methodology measures the self-renewal of bulk populations of progenitor cells, not of individual stem/progenitor cells, the self-renewal capacity of which has been shown to be variable from sphere to sphere [20].

Immunofluorescence and Confocal Microscopy

Antibodies and Reagents.

Antibodies and stains used in this study were as follows: mouse monoclonal anti-CK8 and rabbit polyclonal anti-CK5 from Covance (San Diego, CA, http://www.covance.com); rabbit polyclonal anti-β3 tubulin from Millipore (Temecula, CA, http://www.millipore.com); rabbit polyclonal anti-synaptophysin, mouse monoclonal anti-prostate secretory protein (PSP), rabbit polyclonal anti-chromogranin A, and rabbit polyclonal anti-Ki67 from Abcam (Cambridge, MA, http://www.abcam.com); rabbit monoclonal anti-phospho AKT, rabbit monoclonal anti-PTEN from Cell Signaling Technology (Danvers, MA, http://www.cellsignal.com); mouse monoclonal anti-p63, rabbit polyclonal anti-AR from Santa Cruz Biotechnology (Santa Cruz, CA, http://www.scbt.com); and Alexa 488 goat anti-mouse, goat anti-rabbit, Alexa 568 goat anti-mouse, goat anti-rabbit from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). Fluoro-gel II with 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Electron Microscopy Sciences (Hatfield, PA, http://www.emsdiasum.com).

Staining Procedures for Monolayer Cells.

Adherent cells were fixed in 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS) for 10 minutes, followed by permeabilization with 0.5% Triton X-100 in PBS for 2 minutes. Nonspecific sites were blocked by incubation in 2% bovine serum albumin (BSA) in PBS for 30 minutes. Cells were then incubated overnight at 4°C with the specified antibodies in 2% BSA/PBS. Cells were washed with PBS containing 0.1% Tween-20, incubated with Alexa-488 and/or 568 conjugated IgG in 2% BSA for 30 minutes at room temperature, and finally washed and mounted using the antifade reagent Fluoro-gel II with DAPI. Fluorescent signals and bright-field images were captured using an inverted and/or upright fluorescent Zeiss Axioplan microscope. For cytospun cells, single-cell suspensions were washed twice with PBS, and 1 × 10⁴ cells were deposited on glass slides in PBS by centrifugation at 1,000 rpm for 2 minutes using a cytospin system from Thermo Shandon (Pittsburgh, PA, http://www.thermoscientific.com). Cells were fixed and stained as described earlier. Using a ×63 objective of the upright fluorescent Zeiss Axioplan microscope, at least 300 cells were manually quantified and all numbers were plotted as percent of total cells counted.

Staining Procedures for Protospheres.

Cells were grown in 35-mm glass bottom culture plates with 10-mm microwell from MatTek Cultureware (Ashland, MA, http://www.mattek.com) in Matrigel-containing media as described earlier. Spheres were fixed in situ in 4% PFA at room temperature for 20 minutes. The PFA was aspirated gently and spheres were permeabilized with 0.5% Triton X-100 for 30 minutes at room temperature. After carefully aspirating the permeabilization solution, spheres were blocked using the sphere-blocking buffer (0.1% BSA, 0.2% Triton X-100, 0.05% Tween-20, and 10% normal goat serum in PBS) for 2 hours at room temperature. Spheres were incubated overnight with primary antibodies at 4°C. After gentle washing with PBS containing 0.1% Tween-20, spheres were incubated with Alexa-488 and/or 568 conjugated IgG for 2 hours at room temperature. The culture plates were then washed gently, and 50 µl of the antifade reagent Fluoro-gel II with DAPI was added directly on the microwell and a 12-mm glass coverslip was gently mounted. Confocal microscopic analyses were performed using Zeiss LSM 510 Meta Mk4 confocal microscope and images were acquired and analyzed using the Zeiss LSM image software.

Histology and Immunohistochemistry

Generation 1 (G1) spheres, grown for 12–15 days, were fixed with 4% PFA for 2 hours, washed once, and treated with dispase for 30 minutes. Spheres were collected, embedded in 4% agarose gel, and placed on ice for 10 minutes. The spheres were subsequently fixed with 4% PFA overnight prior to standard histological processing, sectioning, and staining (Histoserve Inc., Fredrick, MD, http://www.histoservinc.com). Bright-field images were taken using an upright fluorescent Zeiss Axioplan microscope.

Orthotopic tumors were fixed in 4% PFA overnight and subjected to standard processing to obtain paraffin-embedded sections. Antigen retrieval was performed in a citrate buffer (DAKO-targeted antigen retrieval solution) in a steamer at 100°C for 15 minutes followed by 15 minutes incubation at room temperature. Blocking was performed with Cyto Q Background Buster reagent (Innovex Biosciences, Richmond, CA, http://www.innvx.com) for 30 minutes at room temperature. Primary antibody incubation was performed overnight at 4°C, followed by secondary antibody incubation at room temperature for 30 minutes. The ABC peroxidase kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) was used followed by DAB (Dako, Carpinteria, CA, http://www.dako.com) for chromogen visualization. All slides were counterstained with hematoxylin.

Orthotopic Transplantation

Ten- to fourteen-week-old BALB/c nu/nu male mice were used. Cell suspensions in 10 µl prostate harvest media were injected using a 30 gauge needle or a 50 µl Hamilton syringe used with a surgical micromanipulator etc. with a microsyringe pump (World Precision Instruments, Sarasota, FL, http://www.wpiinc.com) into one of the dorsal prostatic lobes. Either 100,000 or 500,000 cells were inoculated. The initiating cell number affected the lag time to palpable tumor development but not the distribution of histological types.

Subcutaneous Transplantation and Drug Treatment

Seven- to eight-week-old non-obese diabetic-severe combined immunodeficiency (NOD-SCID) male mice were inoculated subcutaneously with a single-cell suspension of 1.5 × 10⁶ Pten^−/−TP53^−/− primary mouse prostate cells and then randomly assigned to treatment groups. After a palpable tumor was detected, mice were treated with 20 mg/kg CCI-779 (intraperitoneally four times a week), 100 mg Flutamide pellet (implanted, 60-day release), or vehicle only. Mice were weighed and tumor volumes were measured weekly. Drug treatments were performed for 6 weeks.

Cell Labeling and FACS Sorting

For labeling reactions, cells were resuspended in PBS (without Mg²⁺ or Ca²⁺) containing 1% heat-inactivated FBS and 0.09% (w/v) sodium azide. Fcγ III/II receptors were blocked using anti-CD16/CD32 antibody for 15 minutes at 4°C. Cells were stained with the fluorescein isothiocyanate (FITC)-conjugated lineage markers (CD45, CD31, and Ter-119), APC- Sca-1, and PE-CD49f antibodies for 30 minutes at 4°C. 7-Aminoactinomycin D (7-AAD; 100 µg ml⁻¹; Sigma) was added prior to analysis and viable lineage negative fractions were collected. Cell sorting was performed on FACSVantage and FACSAria cell sorters (Becton Dickinson) using FACSDiva software. Postsort purity of individual fractions was assessed and purities of >90% were routinely achieved.

Data Analysis

The significance of the data was analyzed using a Student’s t test, and differences between two means with a p < .05 were considered significant.

Results

We have analyzed the effect of combined loss of Pten and TP53 on the properties of prostate stem/progenitor cell self-renewal, differentiation, tumorigenicity, and signal transduction pathway dependence. Stem/progenitor cell populations were analyzed from Pten^−/−TP53^−/− and wt mice. In this model of prostate cancer, 100% of mice display prostatic intraepithelial neoplasia (PIN) by 10 weeks of age, and 100% of mice display adenocarcinoma by 15 weeks of age (Philip Martin, personal communication). The prostates used in this study were obtained from animals between 14 and 22 weeks of age unless specified otherwise.

To isolate and characterize stem/progenitor cells contained in tumor-bearing and wt prostates, single-cell suspensions were produced from prostate tissue and cultured in serum-free media at low densities, conditions that have been shown to select for the growth of prostate epithelial stem/progenitor cells (see Fig. 1) [20]. Individual cells suspended in semisolid basement membrane (Matrigel)-containing media form spherical structures, so-called protospheres, which contain a mixture of immature and mature cell types, including daughter stem/progenitor cells that can be serially passaged. A second assay involves plating individual cells on plastic in the absence of Matrigel. The colonies formed are mostly composed of cells displaying an intermediate or transit-amplifying phenotype, characterized by coexpression of basal (CK5) and luminal (CK8)-type cytokeratins. Unlike cells derived from spheres, cells derived from colonies lose clongenic self-renewal ability, perhaps as a result of differentiation.

Figure 1.

The prostate sphere- and colony-forming assays demonstrate the presence of stem/progenitor cells in freshly isolated prostates. (A): A schematic illustration of the sphere formation assay in 3-day basement membrane culture. A single-cell suspension of unfractionated prostate cells was mixed with Matrigel (1:1) and plated at a density of 10,000 cells/well. Media was changed every 2–3 days and bright-field images were taken at days 12–15. A representative bright-field image is shown. Scale bar = 100 µm. (B): A schematic illustration of the colony formation assay in 2D culture. A single-cell suspension of unfractionated prostate cells was plated at a density of 10,000 cells/well. Media was changed every 2–3 days and bright-field images were taken at days 5–7. A representative bright-field image of a prostate epithelial colony is shown. Scale bar = 100 µm.

Pten/TP53-Deleted Epithelial Stem/Progenitor Cells Produce Protospheres with Different Morphologies as Compared with wt

A morphological comparison of Pten^−/−TP53^−/− and wt protospheres is shown in Figure 2. Protospheres from both populations demonstrated some morphological heterogeneity, which was observed as a range in protosphere sizes and the presence or absence of an obvious lumen, with lumen-containing spheres dominating in both populations. Despite the heterogeneity, differences between the normal and transformed spheres were readily apparent. Most strikingly, prostate spheres generated from Pten^−/−TP53^−/− cells were on average approximately three times larger than those formed by wt cells (Fig. 2A, 2B). In addition, the transformed spheres formed irregular ovals with bulging areas unlike the smooth round spheres formed by the normal cells. To determine whether the increased size of Pten^−/−TP53^−/− protospheres was due to additional cell divisions, the average numbers of cells/sphere were determined. As shown in Figure 2C, the average numbers of cells/sphere were 400 and 650 for wt and Pten^−/−TP53^−/− spheres, respectively. Consistent with this, the number of proliferating cells (based on Ki67 staining) in Pten^−/−TP53^−/− spheres was twofold higher than that of wt cells (Fig. 2D, 2E). In addition, inspection of stained histological sections from protospheres showed that Pten^−/−TP53^−/− cells appear larger on average than wt cells and that the transformed spheres contain occasional large, multinucleated cells that interrupt cell packing (see Fig. 4C). Dissociation of spheres and measurement of cell diameters on cytospin preparations of cells quantified the average diameter of cells-forming Pten^−/−TP53^−/− spheres to be approximately 30% larger than wt cells (Fig. 2F). Therefore, increased cell numbers and size in addition to decreased compaction of protosphere layers are likely to contribute to increased sphere size generated by Pten^−/−TP53^−/− cells.

Figure 2.

Pten^−/−TP53^−/− prostate spheres are significantly larger than their wt counterpart. (A): Representative bright-field and H&E images of wt and Pten^−/−TP53^−/− prostate spheres are shown. Scale bar = 100 µm. (B): Quantification of the average diameter of wt and Pten^−/−TP53^−/− prostate spheres. The data are reported as mean ± SEM (*, p < .05). (C): Quantification of the average number of cells/wt and Pten^−/−TP53^−/− prostate spheres. The data are reported as mean ± SEM (*, p < .05). (D): Cytospun cells derived from wt and Pten^−/−TP53^−/− protospheres were labeled for Ki67 and quantified. At least 300 cells were counted. The data are reported as mean ± SEM (*, p < .05). (E): Immunofluorescent images of wt and Pten^−/−TP53^−/− protospheres stained for Ki67 are shown. Scale bar 50 µm. (F): Quantification of the average diameter of prostate epithelial cells from dissociated wt and Pten^−/−TP53^−/− protospheres. The data are reported as mean ± SEM (*, p < .05). Abbreviation: wt, Pten^fl/fl;TP53^fl/f;^lLuc+.

Figure 4.

Immunophenotype of wt and Pten/TP53 null protospheres and prostate epithelial colonies. (A): Immunofluorescent images of confocal cross sections from wt and Pten^−/−TP53^−/− protospheres stained for PTEN (upper panel), p-AKT (middle panel), and P63 (lower panel). Scale bar = 100 µm. (B): Immunofluorescent images of confocal cross section wt and Pten^−/−TP53^−/− protospheres stained for CK5 and CK8 (top panel), CK5 (middle panel), and β3 tubulin (lower panel). Scale bar = 50 µm. (C): Immunohistochemical images of serial sections from wt and Pten^−/−TP53^−/− protospheres stained for CK5 (upper panel) and CK8 (lower panel). Scale bar = 50 µm. (D): Immunofluorescent images of wt and Pten^−/−TP53^−/− prostate epithelial colonies stained for PTEN (upper panel) and p-AKT (lower panel). Scale bar = 100 µm. (E): Immunofluorescent images of wt and Pten^−/−TP53^−/− prostate epithelial colonies stained for CK5 and CK8 (upper panel) and β3 tubulin (lower panel). Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; p-AKT, phosphorylated AKT; PTEN, phosphatase and tensin homolog; wt, Pten^fl/fl;TP53^fl/f;^lLuc+.

Stem/Progenitor Cells Isolated from Pten^−/−TP53^−/− Prostates Demonstrate More Intrinsic Self-Renewal

To determine whether Pten and TP53 loss affects the self-renewal properties of prostate stem/progenitor cells, primary sphere-forming units (SFU) were established for wt and Pten^−/−TP53^−/− cell suspensions prepared from prostate tissue. There were approximately twofold more SFU per 10,000 nucleated cells in the tumorigenic as compared with the wt prostate (Fig. 3A). Notably, when serial dilutions of wt and Pten^−/−TP53^−/− primary prostate cells were plated, the number of spheres, as well as colonies, was proportional to the number of input cells even at a 10-fold lower input (Supporting Information Fig. 1). Furthermore, using the cell surface markers CD49f and Sca-1 to isolate the different subpopulations of lineage-negative cells, we determined that the great majority of sphere- and colony-forming cells were contained in the previously identified CD49f⁺/Sca-1⁺ stem/progenitor fraction [21, 22] (Supporting Information Fig. 3). This suggests that the sphere- and colony-forming cells from unfractionated prostate cells appropriately measures relative progenitor activity. The ability to form daughter spheres on passage in vitro measures intrinsic self-renewal activity. As determined relative to a constant cell number assayed from dissociated spheres, Pten^−/−TP53^−/− spheres showed 2–6 times greater self-renewal activity compared with wt over five additional passages. In addition, at each sphere generation, dissociated cells were assayed for colony-forming units (CFU) as an independent measure of progenitor activity (Fig. 3B). The number of SFU and CFU were comparable, suggesting that the cells initiating sphere and colony formation are highly overlapping. Importantly, following the expansion of a de fined starting population of 10,000 cells over several generations, we showed that the number of Pten^−/−TP53^−/− protospheres and total number of cells at G6 were approximately 50 and approximately180-fold, respectively, more than that of wt (Fig. 3C, 3D), reflecting both sustained Pten^−/−TP53^−/− and declining wt self-renewal capacity.

Figure 3.

Pten^−/−TP53^−/− primary prostate epithelial cells demonstrate higher self-renewal activity. (A): wt and Pten/TP53 null primary prostate epithelial cells were plated in Matrigel at a density of 10,000 cells/well for sphere formation assay. Spheres generated from primary cells are referred to as G1 spheres. Sphere-forming units obtained from serially passaged protospheres are shown. A representative analysis of four independent experiments is shown. The data are reported as mean ± SD (*, p < .05). (B): wt and Pten/p53 null primary prostate epithelial cells were seeded at a density of 10,000 cells/well for colony formation assay. The initial colonies formed from primary cells are referred to as P0. The subsequent colonies were generated by cells dissociated from spheres. A representative analysis of four independent experiments is shown. The data are reported as mean ± SD (*, p < .05). (C): wt and Pten/TP53 null primary prostate epithelial cells were plated in Matrigel at a density of 10,000 cells, and the accumulated expansion of the spheres was enumerated for six generations. (D): Starting from the same population as in (C), the number of cells from each generation of spheres was determined by counting an aliquot of dissociated spheres. The experiment was repeated twice, and a representative experiment is shown. Abbreviations: G1, Generation 1; wt, Pten^fl/fl;TP53^fl/f;^lLuc+.

Pten/TP53 Deletion Alters the Lineage Distribution of Progeny Generated In Vitro from Prostate Stem/Progenitor Cells

The capacity for multilineage differentiation is a characteristic of stem/progenitor cells, and multipotency or plasticity in differentiation potential is often associated with CSCs. To analyze the differentiation potential of normal and transformed spheres, we determined the phenotypes of cells within spheres using immunofluorescent staining for signaling pathway and lineage markers. We optimized staining intact spheres in situ as an approach to observe the relative organization of the various cell types within the sphere and to maximize the observation of rare cellular phenotypes.

Loss of PTEN expression was confirmed in spheres formed by prostate cells taken from Pten^−/−TP53^−/− mice as shown by representative confocal images from stained spheres (Fig. 4A, upper panel). In addition, spheres were stained with antibody directed against phospo-AKT to verify the expected downstream activation of AKT in Pten-deleted prostate epithelium (Fig. 4A, middle panel). Notably, the pattern of PTEN loss and AKT activation was seen in the vast majority of Pten^−/−TP53^−/− prostate-derived spheres and CFU as well (Fig. 4A, 4D). P63 basal cells make contact with the basement membrane in normal prostate glands, and p63 cells previously have been described as the outermost layer in normal protospheres grown in reconstituted basement membrane [20]. As shown in representative confocal sections through the middle of wt and Pten^−/−TP53^−/− spheres, p63 cells uniformly compose the outermost layer in both types of spheres (Fig. 4A, lower panel).

Protospheres were stained for lineage markers including CK5, CK8, and β3 tubulin, which are characteristically expressed in basal, luminal, and neuroendocrine cells, respectively. Confocal images through representative normal and transformed protospheres costained for CK5 and CK8 are shown in Figure 4B (top panel). The outermost layers of cells stain strongly for CK5 (Fig. 4B, middle panel), whereas inner layers of cells at or near the sphere lumen express both CK5 and CK8 or occasionally CK8 only (Fig. 4B, 4C). This pattern of CK5/CK8 expression was similarly observed on immunohistochemical staining in serial sections of fixed spheres (Fig. 4C). Finally, rare wt and Pten^−/−TP53^−/− protospheres contained cells staining positive for the neuroendocrine marker, β3 tubulin (Fig. 4B, lower panel). Altogether, the staining pattern for protospheres indicates that the hierarchical organization has been maintained in Pten^−/−TP53^−/− spheres. In contrast to protospheres, CFU are almost entirely composed of CK5⁺/CK8⁺ cells; however, similar to protospheres, rare CFU contained β3 tubulin-positive cells (Fig. 4E). Rare chromagranin A-positive cells were also observed (Supporting Information Fig. 2).

To quantify protosphere cells expressing the various lineage markers, single-cell suspensions of dissociated spheres were distributed by cytospinning onto slides, fixed, and immunostained simultaneously for CK5 and CK8. In addition, single-cell suspensions prepared from wt prostates or adenocarcinoma-bearing prostates were similarly stained. Interestingly, such staining revealed a significant range of expression for CK5 in particular (see Fig. 5A). Single-cell staining observed with a ×63 objective was highly sensitive and specific for staining a cytoskeleton structure. Staining with antibody dilutions between 1/200 and 1/1,000 showed different intensities of staining but with a continuous range of heterogeneous expression. Cells displaying a specific staining pattern regardless of intensity were considered positive (Fig. 5A).

Figure 5.

Pten and TP53 deletion alters the lineage hierarchical distribution of prostate epithelial cells in protospheres. (A): Immunofluorescent images of cytospin preparations of wt and Pten^−/−TP53^−/− primary prostate cells stained for CK5 (upper panel) or CK5 and CK8 from Pten^−/−TP53^−/− only (lower panel). The arrows indicate the different levels of CK5 expression. (B): Quantification of the percentage of CK5+, CK8+, and CK5+/CK8+ cells from cytospin preparations of prostate cells isolated from primary prostate tissues, protospheres, and prostate epithelial colonies. The average of three independent experiments is shown. The data are reported as mean ± SEM (*, p < .05). (C): Quantification of the percentage of β3 tubulin+ cells from cytospin preparations of prostate cells isolated from primary prostate tissues, protospheres, and prostate epithelial colonies. The average of three independent experiments is shown. The data are reported as mean ± SEM (*, p < .05). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; wt, Pten^fl/fl;TP53^fl/f;^lLuc+.

The distribution of cytokeratin phenotypes in single-cell suspensions from Pten^−/−TP53^−/− prostates as compared with wt prostates revealed a relative loss of CK5 cells and a gain of CK8 and CK5/8 double-positive cells, consistent with the expectation for adenocarcinoma (Fig. 5B). Approximately 20% of wild-type cells score as CK5/8 double-positive, which is significantly more than has been appreciated by other staining methods. The sensitivity of staining single dissociated cells allows for a range of cytokeratin expression to be seen that is not normally observable when staining formalin-fixed intact tissue sections. These CK5/8 double-positive cells may represent immature or recently differentiated epithelial cells.

Cells recovered from protospheres were predominantly CK5+, consistent with the staining observed in whole and sectioned spheres and with previous reports [20, 23]. Despite the fact that protosphere culture conditions appear to favor expansion of basal cells, cells recovered from Pten^−/−TP53^−/− spheres as compared with wt spheres have a measurable propensity to differentiate toward CK8 intermediate and luminal lineages. Consistent with this, there were more Pten^−/−TP53^−/− than wt cells staining for PSP (Supporting Information Fig. 2). In addition, there were approximately three and two times more cells that expressed β3 tubulin and chromagranin A, respectively, in Pten^−/−TP53^−/− as compared with wt cell populations derived from protospheres (Fig. 5C and Supporting Information Fig. 2). Staining single-cell suspensions made from CFU confirmed that most cells expressed both CK5 and CK8 (Fig. 5B). In agreement with increased neuroendocrine marker expression in protospheres, Pten^−/−TP53^−/− cells derived from CFU stained about five times more frequently for β3 tubulin as compared with wt (Figs. 4E, 5C). Altogether, the differentiation marker profiles in protospheres and CFU imply that Pten and TP53 influence differentiation potential and that loss of Pten and/or TP53 leads to enhanced differentiation of prostate progenitors toward the luminal and neuroendocrine lineages.

Pten^−/−TP53^−/− Protospheres Contain Tumor-Initiating Cells

We sought to determine the tumorigenic properties of the cell populations contained in Pten^−/−TP53^−/− protospheres. Pten^−/−TP53^−/− G1 spheres were grown for 7 days, and single-cell suspensions of cells derived from the spheres were injected orthotopically into the prostates of nu/nu mice. The transplanted epithelial cells expressed transgenic luciferase and were monitored for growth over time with bioluminescent imaging. Twelve of twenty-three (52%) mice demonstrated tumor growth on monitoring for 12 months, although most tumors developed within 4 months. Mice were sacrificed for pathological analyses on signs of morbidity. Two histological types of tumors were observed, orthotopic PIN (oPIN)/adenocarcinoma and basal squamous carcinoma, in approximately equal amounts overall, although in some hosts one type of tumor predominated (Fig. 6A, 6B). Interestingly, significant lung metastases were observed in one animal.

Figure 6.

Characterization of orthotopic tumors initiated from G1 sphere-derived cells. (A): Orthotopic prostate carcinoma histological patterns as a percent of total tumor area for each individual orthotopic tumor that was generated by injecting nude mice with cells from dissociated G1 protospheres (PIN/Adeno: orthotopic PIN and adenocarcinoma; Basal/Squam: basal squamous carcinoma). (B): Cross sections of orthotopic tumors stained with H&E showing typical PIN/adenocarcinoma and basal squamous carcinoma. The arrows indicate wild-type nude mouse prostate glands entrapped in the tumor area. Scale bar = 50 µm. (C): Serial sections of the adeno and basal/squam orthotopic tumors labeled with antibodies for CK8, CK5, Synaptophysin, and AR. Scale bar = 50 µm. Abbreviations: AR, androgen receptor; PIN, prostatic intraepithelial neoplasia.

Immunophenotyping of serial tumor sections (Fig. 6C) showed that oPIN/adenocarcinoma glands could be one to several cell layers thick, and the cells were primarily CK8⁺ or CK5⁺/CK8⁺. Cells expressing the neuroendocrine lineage marker synaptophysin were focally abundant and were also either CK8⁺ or CK5⁺/CK8⁺. Most cells were AR⁺. Nuclear AR staining intensities varied, and in general, the less differentiated and invasive adenocarcinoma had reduced levels of staining. Overall, oPIN/adenocarcinoma was heterogeneous, consisting of glands composed of cells with immature to mature luminal phenotypes. Orthotopic basal squamous carcinoma was composed of 100% CK5⁺ cells that very rarely coexpressed either CK8 or synaptophysin. Most tumors contained a significant number of AR⁺ cells, although the relative composition and staining intensities were variable.

In summary, G1 spheres appear to be heterogeneous with respect to the differentiated phenotype of tumor-initiating progenitors. Importantly, approximately half of the tumor-initiating cells contained in G1 spheres gave rise to adenocarcinoma, the predominant histology observed in primary tumors occurring in Pb-Cre-initiated Pten/TP53 deleted prostates. The high incidence of basal squamous carcinoma produced from G1 spheres is in contrast to the infrequent observation of squamous type in donor Pten^−/−TP53^−/− prostates or in orthotopic tumors derived from single-cell suspensions of tumorigenic prostates (P. Martin, manuscript in preparation). These data suggest an influence of the in vitro culturing conditions on the outcome of orthotopic histological tumor types that develop.

AKT/mTORC1 and AR Dependence Distinguish Pten^−/−TP53^−/− Stem/Progenitor Cells from wt

Using colony and sphere formation assays as functional reporters of progenitor activity in primary prostate cells, we examined the role of specific signaling pathways using different drugs. AKT is a major proximal target of Pten deletion, and mTORC is another target whose activation is AKT-regulated. AKT and mTORC1 are inhibited by triciribine and rapamycin, respectively. As expected, the relative number of colonies and spheres formed by Pten^−/−TP53^−/− progenitor cells was almost twofold more than that of wt (Fig. 7A, 7B; Supporting Information Fig. 4). Treatment with triciribine or with rapamycin at the initiation of culture significantly reduced (approximately 70%) the number of colonies or spheres produced by mutant progenitors, and importantly, there was no consistent effect on wt progenitor function. The Pten^−/−TP53^−/− colonies and spheres that grew in the presence of the inhibitors had normal sizes and morphologies. This suggests that the AKT/mTORC1 pathway plays a necessary role in Pten^−/−TP53^−/− prostate progenitor cell activity but is not required in wt. This finding generally parallels that in Pten-deleted HSC [24].

Figure 7.

AKT/mTORC1 and androgen receptor (AR) pathways play central roles in regulating the clonogenic and tumorigenic activity of Pten^−/−TP53^−/− stem/progenitor prostate epithelial cells. The activity of wt and Pten/TP53 null primary prostate epithelial stem/progenitor cells was tested in a colony formation assay (A) and sphere formation assay (B) in the absence or presence of drugs targeting the AKT/mTORC1 pathway (Rapamycin and Triciribine) and AR pathway (nilutamide and bicalutamide). A representative analysis of four independent experiments is shown. The data are reported as mean ± SEM (*, p < .05). (D): Pten/TP53 null primary prostate cells (1.5 × 10⁶ per mouse) were injected subcutaneously into NOD-SCID male mice. After a palpable tumor was detected (week 0), mice were implanted with a 100 mg flutamide pellet or treated i.p. four times a week with 20 mg/kg CCI-779 or with vehicle only. Tumor volume was measured every week and the average volume of four mice per group is plotted. The data are reported as mean ± SEM (*, p < .05). Abbreviations: DHT, dihydrotestosterone; wt, Pten^fl/fl;TP53^fl/f;^lLuc+; mTORC, mammalian target of rapamycin; NOD-SCID, non-obese diabetic-severe combined immunodeficiency.

Loss of Pten expression appears to be a common pathway whereby transformed prostate epithelial cells are readily selected in vivo for androgen-independent growth [3]. Various lines of evidence suggest that these androgen-independent cells are at least in part dependent on signaling from the AR for tumor formation [25]. To examine whether AR is required by primary progenitor cells, CFU and SFU assays were performed in the presence of the AR antagonists nilutamide and bicalutamide. Plating in the presence of a range of concentrations of AR antagonists reduced Pten^−/−TP53^−/− progenitor activity by greater than 50%, whereas wt activity was not changed (Supporting Information Fig. 4). By contrast, the presence of DHT had minimal effect on the numbers of CFU and SFU that were formed. The dependence of Pten/TP53 null cells on mTOR and AR signaling pathways to form spheres and colonies in vitro suggested that the same pathways might be required for tumor initiation in vivo. To determine the potential effect of mTOR and AR inhibition on tumor-initiating capacity of Pten/TP53 null cells, we evaluated the effect of CCI-779 (a rapalog mTOR inhibitor) and Flutamide (an AR antagonist). After establishing subcutaneous palpable tumors, mice were implanted with a Flutamide pellet (100 mg) or were treated i.p. four times a week for 6 weeks with 20 mg/kg CCI-779 or with vehicle alone. Treatment with CCI-779 or Flutamide significantly inhibited tumor progression when compared with the control group. Interestingly, CCI-779 treatment not only inhibited tumor growth but also reduced the size of tumors after 6 weeks of treatment. There were no significant effects on the weight of mice resulting from drug treatment (data not shown). Taken together, these studies demonstrate that the mTOR and AR signaling pathways are required for in vitro clonogenic and tumor initiating activity in Pten^−/−TP53^−/− prostate progenitor cells.

Discussion

In prostate cancer, homozygous deletion of the Pten tumor suppressor and loss of function of TP53 are associated with aggressive disease and metastasis [3, 6]. Many aggressive cancers have been suggested to reflect the numbers and/or properties of their CSCs [7, 8, 26]. TP53 has recently been shown to be important for negatively regulating the maintenance and amplification of the stem cell state [27, 28]. We demonstrate here using the protosphere assay that Pten/TP53 null prostate progenitors display tumor-initiating activity and express phenotypes of potential relevance to progressive human prostate cancer including altered drug sensitivity and increased self-renewal ability.

Protospheres are predominantly composed of the differentiated progeny generated from dividing stem/progenitor cells and of rare daughters with the ability to regenerate new spheres. Two lines of evidence suggest that Pten/TP53 null prostates relative to wt contain a progenitor population(s) with unique lineage properties. First, a significant proportion of Pten/TP53 null progenitors are inhibited in clonogenic growth by AR antagonists, a characteristic that is absent in wt progenitors. Second, relative to wt progenitors, Pten/TP53 progenitors produce different proportions of progeny expressing specific lineage markers. Despite the fact that protosphere-culturing conditions favor basal lineage differentiation, Pten/TP53 null progenitors give rise to a significant relative increase in neuroendocrine cells and an observable increase in intermediate and luminal cells (Fig. 5). The relationship of the progenitor cells described here to CARNs is of obvious interest, although addressing this question will require the definition of markers that distinguish prostate epithelial progenitor subpopulations. Pten/TP53 deletion has been shown to inhibit the differentiation capacity of neural stem cells, consistent with the histological features of glioblastoma multiforme [19]. Similarly, it is possible that Pten/TP53 deletion in prostate progenitors leads to the accumulation of a progenitor with propensity to differentiate toward luminal and neuroendocrine lineages, consistent with characteristics of progressive PC.

We suggest that the rapid and aggressive nature of the carcinoma resulting from the addition of TP53 loss of function to Pten deletion in prostate epithelial progenitors results in part from increased and sustained progenitor self-renewal activity as shown in Figure 3. By contrast, Probasin Cre-driven deletion of Pten alone resulted in protosphere self-renewal activity in serial passages that was roughly similar to wt [23]. TP53 contributes to fundamental regulation of stem cell divisions [27– 29]. Our findings are consistent with recent investigations using both transformed and nontransformed mammary stem cells showing that loss of TP53 leads to increased symmetric stem cell divisions, which amplifies stem cell numbers [27].

Morphological analyses of Pten/P53 null protospheres relative to wt are revealing, and one of the most distinguishing features is a threefold increase in size. This increase in sphere size can be attributed mostly to increased average cell size and increased cell numbers. An average increase in cell size is consistent with PTEN being a known regulator of cell size, mostly as a result of inhibiting the AKT-mTORC1 axis and associated anabolic activities [30]. TP53 is known to negatively regulate proliferative growth through apoptotic and senescence mechanisms [31]. As shown by the increased Ki67 labeling and increased cell numbers/protosphere, the loss of TP53 appears to affect proliferation in the differentiating protosphere progeny as well as CSCs. It is important to note that the basic organization of mutant and wt protospheres is maintained. Also, normal cell-cell and cell-matrix contacts were found to be intact as visualized by the degree and pattern of E-cadherin and integrin α6 staining (data not shown).

We have identified a progenitor population that is inhibited in its in vitro clonogenic and in vivo tumorigenic growth by AR antagonists and mTORC1 inhibitors. Importantly, concentrations of drugs that are inhibitory for the mutant progenitors in vitro do not decrease the stem/progenitor activity of wt cells. The apparent dependence on AKT/mTORC1 activity is an oncogene pathway addiction phenotype that has been described in hematopoietic and neural stem cells as well [19, 24]. The AR signaling dependence of the mutant progenitor population is of particular interest. Although the presence of exogenous androgen was not required to stimulate growth in vitro, AR antagonists inhibited growth. A significant fraction of CRPC shows a similar dependence on AR in an androgen-depleted environment [25]. The observation that CRPC have characteristics in common with the Pten/P53 null progenitor population suggests that isolation of the mutant progenitor population and further characterization of the signaling networks that regulate their growth and differentiation will reveal gene-specific mechanisms of relevance for CRPC.

Conclusion

Pten/TP53 loss in prostate epithelial stem/progenitor cells results in increased self-renewal, modified lineage commitment, and acquired dependence upon AR and AKT/mTORC1 signaling pathways.