Pten deletion leads to the expansion of a prostatic stem/progenitor cell subpopulation and tumor initiation

Abstract

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a potent tumor suppressor gene frequently mutated in human prostate cancers. Deletion of Pten in a murine model of prostate cancer recapitulates the disease progression seen in humans. Using defined cell lineage markers, we demonstrate that PTEN negatively regulates p63-positive prostatic basal cell proliferation without blocking differentiation. Concomitant with basal cell proliferation is the expansion of a prostate stem/progenitor-like subpopulation as evidenced by the progressive increase of stem cell antigen-1 (Sca-1)- and BCL-2-positive cells. This observation provides strong evidence that basal cell proliferation can be an initiating event for precancerous lesions. Sca-1(+) and BCL-2(+) progenitors may serve as cancer-initiating cells in this model.

Fig. 1.

Characterization of basal and luminal epithelial cells in the normal murine prostate, and localization of Cre expression. Immunofluorescence analyses were carried out by using sections from the proximal region of dorsolateral lobes of WT adult mice. Each section was double-stained by two independent cell markers, and some sections were counterstained with DAPI to visualize the nuclei. Only overlaid results are shown here. The WT adult prostatic acini are composed of two major cell types. The luminal cells are immunoreactive to antibodies against CK8 (A, arrows), p27 (D), epithelial cadherin (E-cad) (F), and androgen receptor (AR) (G). By contrast, the basal cells form a discontinuous cell layer scattered along the basement membrane and are positive for cellular markers CK5 (A, arrowheads), p63 (B), AR (G), and occasionally BCL-2 (E) but are negative for p27 (D), E-cad (F), and Ki67 (C). Cre is expressed in both basal (H) and luminal epithelial cell compartments (shown in green). (Inset) A high-power image of F. (Scale bar, 20 μm.)

Fig. 2.

Increased p63⁺ cell number and altered morphology along with tumor initiation and progression. (A) A schematic illustration of the kinetics of prostate cancer development caused by Pten conditional knockout. (B) Increased p63⁺ cell number and altered morphology in the Pten-null prostate epithelium. Immunohistochemical analysis was performed with anti-p63 antibody. Note that scattered p63⁺ cells lie flat on the basement membrane of WT epithelium (Left). p63⁺ cells in Pten-null prostate show altered morphology, including bigger cell size, changed polarity, and altered localization (Center). Most p63⁺ cells are organized into individual small clusters. Red arrowheads point to p63⁺ cells that appear to “bud-out” from the basal cell layer through asymmetric-like cell division. (Scale bar, 20 μm, 40 μm for Insets.) (Right) Quantification of p63⁺ cells. p63-stained sections at each time point, with five animals per point, were quantified for the density of p63⁺ cells along the basement membrane (BM). The values are presented as mean ± SD. (C) Cre-mediated Pten deletion in CK5⁺ basal cells leads to change in cell morphology. Arrows (Right) show Cre and CK5 double-positive Pten-null cells.

Fig. 3.

Pten deletion in the basal cell compartment leads to increased cell proliferation. (A) Immunofluorescence analysis demonstrates that most Ki67⁺ proliferating cells are associated with or near the basal cell compartment: an overlaid image of Ki67⁺p63⁺ cells in white or light yellow is shown (Right). (Scale bar, 50 μm.) (B) a and c show the localization of CK8⁺ (green) and p63⁺ cells (red). Arrows in C point to two adjacent p63⁺ cells along the basement membrane. b, d, and e illustrate BrdUrd⁺ cells (green) after 24-h (b and d) and 72-h (e) chases. (a and b) Control prostate. (c-e) Mutant prostate. Arrows in d point to BrdUrd-labeled p63⁺ cells undergoing symmetric cell division. (Scale bar, 20 μm.) (C) Quantification of BrdUrd⁺ and BrdUrd/p63 double-positive cells after a 24-h chase. The number of each cell type was counted from five microscopic fields of dorsolateral prostate from each of two individual mice, and the data are presented as mean ± SD.

Fig. 4.

Pten deletion leads to increased basal and TA/IC populations without blocking cell differentiation. (A) Immunofluorescence analysis illustrates asymmetric-like cell division events in Pten-null prostate glands. (Scale bar, 20 μm.) (B) Pten deletion does not perturb cell differentiation. From basal layer (BC) to terminally differentiated luminal cell layer (LC), the cancerous acini are composed of localized p63⁺;CK5⁺ basal cells (Left), TA/IC cells that coexpress both CK5 basal and CK8 luminal cell markers, and CK8 single-positive luminal cells (second panel from the left). Most of the proliferative Ki67⁺ cells are AR- and BCL-2-positive in the basal and TA/IC regions. (Scale bar, 50 μm.)

Fig. 5.

PTEN loss leads to an increase in Sca-1⁺ stem/progenitor subpopulations. (A) FACS analysis demonstrates increased Sca-1⁺ populations in Pten-null prostate gland, as compared with their age- and genetic background-matched littermate controls (n = 3). (B) Quantitative PCR analysis on mRNA samples prepared from Sca-1⁺ and Sca-1^- populations. Relative expression levels compared with a G3PDH control are illustrated. The experiment was repeated three times and one representative result is shown.