Prostatic inflammation enhances basal-to-luminal differentiation and accelerates initiation of prostate cancer with a basal cell origin

Significance

Inflammation promotes the initiation of various malignancies by inducing genetic and epigenetic changes. Here we show that bacterial infection-induced prostatitis results in microenvironmental changes that enhance the differentiation of prostate basal cells into luminal cells, a cellular process that rarely occurs under normal physiological conditions. Previously, we showed in a mouse model that disease initiation for prostate cancer with a basal cell origin requires and is limited by basal-to-luminal differentiation and that prostatic inflammation induced by bacterial infection accelerates disease initiation by enhancing basal-to-luminal differentiation. Collectively, our results show that inflammation-induced microenvironmental changes alter the prostate epithelial lineage differentiation program, and we propose this alteration as a distinct and complementary process through which inflammation promotes tumor initiation.

Abstract

Chronic inflammation has been shown to promote the initiation and progression of diverse malignancies by inducing genetic and epigenetic alterations. In this study, we investigate an alternative mechanism through which inflammation promotes the initiation of prostate cancer. Adult murine prostate epithelia are composed predominantly of basal and luminal cells. Previous studies revealed that the two lineages are largely self-sustained when residing in their native microenvironment. To interrogate whether tissue inflammation alters the differentiation program of basal cells, we conducted lineage tracing of basal cells using a K14-CreER;mTmG model in concert with a murine model of prostatitis induced by infection from the uropathogenic bacteria CP9. We show that acute prostatitis causes tissue damage and creates a tissue microenvironment that induces the differentiation of basal cells into luminal cells, an alteration that rarely occurs under normal physiological conditions. Previously we showed that a mouse model with prostate basal cell-specific deletion of Phosphatase and tensin homolog (K14-CreER;Pten^fl/fl) develops prostate cancer with a long latency, because disease initiation in this model requires and is limited by the differentiation of transformation-resistant basal cells into transformation-competent luminal cells. Here, we show that CP9-induced prostatitis significantly accelerates the initiation of prostatic intraepithelial neoplasia in this model. Our results demonstrate that inflammation results in a tissue microenvironment that alters the normal prostate epithelial cell differentiation program and that through this cellular process inflammation accelerates the initiation of prostate cancer with a basal cell origin.

Chronic inflammation is a significant contributor to the initiation and progression of a wide spectrum of malignancies, including prostate cancer (1). Loss-of-function mutations and certain single-nucleotide polymorphisms in inflammation-associated genes and a history of prostatitis have been positively correlated with prostate cancer risk (2–5). In addition, De Marzo et al. (6) have described regions of prostatic atrophy termed “proliferative inflammatory atrophy” (PIA) that often are associated with inflammatory cell infiltrates in human samples and have proposed that these areas are direct precursors of prostatic intraepithelial neoplasia (PIN) or adenocarcinoma. Finally, genetic studies using mouse models have demonstrated that inflammatory signals in the prostate can either induce or synergize with oncogenic signaling to promote the initiation and progression of prostate cancer (7, 8). Currently, it is widely accepted that chronic inflammation promotes tumor initiation and progression through the induction of various genetic and epigenetic alterations. For example, inflammation enhances the production of reactive oxygen species, up-regulates the expression of the enzyme that induces nucleotide alterations (9), and modulates the activity of DNA methyltransferases (10). Interestingly, inflammation also has been shown to deregulate self-renewal and differentiation of hematopoietic stem cells (11). However, it remains to be determined whether altering cell lineage differentiation programs per se serves as a mechanism through which inflammation promotes tumor initiation and progression.

Human prostate epithelia are composed of a layer of columnar luminal epithelial cells that rest on a continuous cuboidal layer of basal epithelial cells. In comparison, rodent prostates have fewer basal cells that instead usually form a punctuated layer (12). A third type of epithelial cell, neuroendocrine cells, is rare and hence often is not investigated as intensively as the other two lineages (13). Using a lineage-tracing approach, we and several other groups showed that adult murine prostate basal and luminal cell lineages are largely self-sustained when residing in their native microenvironment, although in two of the studies basal cells were shown to generate luminal cells at an extremely rare frequency (14–18). In stark contrast, other studies have shown that basal cells are able to differentiate very efficiently into luminal cells and neuroendocrine cells in a dissociated prostate-cell regeneration assay (19–25). In the prostate-cell regeneration assay, prostate basal epithelial cells were removed from their native environment, dissociated into single cells, and stimulated with embryonic urogenital sinus mesenchymal (UGSM) cells that have strong reprogramming activity (26–28). These experimental conditions recapitulate some processes that also occur during tissue inflammation. For example, cellular dissociation resembles tissue damage, whereas UGSM cells may mediate signaling that also can be induced by reactive stroma (29). Therefore, we reasoned that prostatic inflammation may induce functional plasticity in prostate basal cells in vivo and stimulate their capacity to differentiate into luminal cells. Previously, we and others have shown that basal-to-luminal differentiation is very rare under physiological conditions and serves as a barrier for disease initiation in mouse models of prostate cancer with a basal cell origin (14–16, 30). Therefore, we also hypothesized that inflammation can accelerate the initiation of prostate cancer with a basal cell origin by stimulating basal-to-luminal differentiation.

To test these hypotheses, we took advantage of a mouse model of bacterial infection-induced prostatitis. One reason for chronic inflammation in the prostate is bacterial colonization in the prostate via reflux of urine into the prostatic ducts of the peripheral zone (31). A reproducible mouse model of bacterial prostatitis has been established in which transurethral inoculation of uropathogenic strains of Escherichia coli into the prostate results in massive inflammation and reactive hyperplasia in the prostate (7, 32–34). As reported here, we established this mouse model for prostatitis using a uropathogenic E. coli strain CP9 that was isolated from the blood of a patient with pyelonephrititis (35). Using this model, we demonstrate that acute inflammation induced by bacterial infection causes tissue damage and results in a tissue microenvironment that stimulates the differentiation of basal cells to luminal cells and accelerates disease initiation in a mouse model for prostate cancer with a basal cell origin.

Results

Prostate Inflammation Induced by Bacterial Infection Results in Hyperplasia Characterized by High Proliferation and Reactive Stroma.

To interrogate how the prostate epithelial lineage hierarchy is maintained in response to acute inflammation, we first characterized a mouse model for prostatitis induced by infection with a uropathogenic E. coli strain, CP9. As described in detail in Materials and Methods, the CP9 E. coli and PBS control solutions were inoculated transurethrally into 9-wk-old wild-type C57BL/6 male mice. Consistent with previous reports, there were significantly variable degrees of inflammation among experimental mice and among different prostatic lobes (7, 32). In addition, inflamed and noninflamed ducts often were observed concomitantly in all different lobes. Inflammation occurred in anterior and dorsolateral prostate lobes at high penetrance but was observed less frequently in ventral prostate lobes (Table S1). Epithelial hyperplasia and immune cell infiltrations were commonly observed in the inflamed area 5 d after bacteria instillation and persisted 3 mo later (Fig. 1A).

Fig. 1.

The mouse model for CP9-induced prostatitis. (A) H&E staining of anterior (AP), dorsolateral (DLP), and ventral (VP) prostate lobes of mice at 5 d, 1 mo, and 3 mo after intraurethral instillation of PBS and CP9 inoculum. (B) Immunostaining of K5 and K8. (C) IHC analysis of smooth muscle actin (SMA) and vimentin. (D) Masson’s trichrome staining of prostate tissues at 3 mo after intraurethral instillation of PBS and CP9. The black arrow points to red staining for smooth muscle cells; the yellow arrow indicates blue collagenous stroma. (E) Coimmunostaining of Ki67, K14, and K8. Data shown in the bar graphs represent means and SD from four mice. (F and G) FACS plots of prostatic lineage composition in PBS- and CP9-treated mice 2 wk posttreatment. Data shown in the bar graph represent means and SD from three mice. (H–J) FACS plots of CD45⁺ leukocytes in prostates of PBS- and CP9-treated mice 2 wk posttreatment. Data shown in the bar (H and J) and pie (I) graphs represent means and SD from three mice. *P < 0.05; **P < 0.01; ***P < 0.001.

The PBS-treated control mice generally showed normal prostatic histology (Fig. 1A). As shown in Fig. 1B and Fig. S1, a single layer of luminal cells that express cytokeratin 8 (K8) and the androgen receptor (AR) surround the prostatic luminal space, whereas the basal cells that express cytokeratin 5 (K5) and p63 form a punctuated layer between luminal cells and the basement membrane. Transit-amplifying cells (36) that are positive for both K5 and K8 were rare. In contrast, there was a dramatic increase in basal cells and transit-amplifying cells in the inflamed prostatic lobes of the CP9-treated mice 5 d after bacteria inoculation, and this increase persisted 3 mo later (Fig. 1B and Fig. S1). This observation is similar to the report that intermediate cells in human prostate epithelium are enriched in PIA (37). Finally, piling up of the K8-expressing luminal epithelial cells also was prominent in the inflamed prostates at different time points.

There also were considerable changes in prostatic stroma of the CP9-treated mice. Prostate epithelial ducts of the control PBS-treated mice are encapsulated by a thin and continuous layer of smooth muscle actin-positive stromal cells, which becomes slightly thicker upon aging (Fig. 1C). In comparison, in the CP9-treated group this smooth muscle cell layer often was disrupted by 1 mo after bacteria inoculation, indicating damage in tissue structures. Meanwhile, at the peri-glandular spaces there was an expansion of myofibroblast stromal cells that were positive for both smooth muscle actin and vimentin, which are frequently reported during wound healing and in reactive stroma associated with prostate cancer (29). Masson’s trichrome staining further confirmed a reduction of peri-glandular smooth muscle cells and increased collagen deposition in the prostate tissues in the CP9-treated group, as shown by the fainter red staining (black arrows, Fig. 1D and Fig. S2) and darker blue staining (yellow arrow, Fig. 1D and Fig. S2). Collectively, these observations suggest that CP9-induced inflammation induces stromal fibrosis and instigates a reactive stromal environment.

Immunostaining for the proliferation marker Ki67 showed that the proliferation indices of the basal and luminal cells in inflamed prostatic lobes of the CP9-treated mice increased by more than 31.4- and 10.6-fold, respectively, as compared with those of the cells in PBS-treated control mice (Fig. 1E). The urogenital organs of the CP9-treated mice appeared smaller than those of the PBS-treated mice, but the average weight of the prostate tissues in the two groups was similar (Fig. S3). We dissociated prostate tissues to quantify the absolute cell numbers of individual cell lineages by FACS analysis as described previously (21). FACS analysis corroborated that the basal cell and the stromal cell percentages increased by 1.5- and 1.2-fold, respectively, in the prostates of CP9-treated mice (Fig. 1F). The absolute number of basal cells and stromal cells per prostate also increased (basal cells: 7,745.0 ± 991.7 in the control group versus 20,088.3 ± 1,968.1 in the CP9 group, P < 0.01; stromal cells: 21,281.7 ± 625.0 in the control group versus 44,326.6 ± 5,343.9 in the CP9 group, P < 0.05) (Fig. 1G). Luminal cell number also increased but did not reach statistical significance (41,998.3 ± 3,573.5 in the control group versus 55,305.0 ± 4,682.4 in the CP9 group). The increase in cell numbers is consistent with the increased proliferation indices in these lineages and suggests that the decreased tissue volumes in the CP9-treated group may be caused by the impaired secretory function.

FACS analysis also revealed a fivefold increase in the percentage of CD45⁺ leukocytes within CP9-treated mouse prostates (5.93% in the control group versus 27.53% in the CP9-treated group, P < 0.001) (Fig. 1H). FACS analysis also showed that macrophages and T cells were the major leukocytes in the prostate in the control group, whereas neutrophils, macrophages, and T cells became the dominant leukocytes in the CP9-treated group (Fig. 1I). Fig. 1J shows that each of the different types of leukocytes examined increased in the prostates of CP9-treated mice (116-fold for neutrophils, 8.0-fold for T cells, 3.9-fold for macrophages, and 10.2-fold for B cells). Collectively, these data corroborate the notion that transurethral instillation of the CP9 bacteria induces prostatic inflammation and results in reactive hyperplasia.

Inflammatory Prostatic Microenvironment Induces Basal-to-Luminal Differentiation.

Previously, several different transgenic mouse models have been used to mark prostatic basal cells specifically with fluorescent proteins (14–16, 18). We bred a K14-CreER line that expresses tamoxifen-responding CreER driven by the basal cell-specific cytokeratin 14 (K14) promoter with an mTmG fluorescent reporter line (38) that expresses GFP after Cre-LoxP–mediated homologous recombination (14). When K14-CreER;mTmG bigenic mice are treated with tamoxifen, GFP is expressed only in prostate basal cells. With this approach, adult murine prostate basal cells seldom generate other cell lineages under their normal physiological conditions (14–16).

We sought to investigate whether bacteria-induced tissue inflammation affects the in vivo differentiation program of prostate basal cells. As schematically illustrated in Fig. 2A and described in Materials and Methods, 5-wk-old K14-CreER;mTmG bigenic male mice were treated with tamoxifen to label prostate basal cells with GFP. Three weeks later, a group of mice was inoculated transurethrally with CP9 bacteria. Control mice either were instilled transurethrally with the PBS solution or underwent no treatment. Prostate tissues were collected and examined for the expression of GFP and lineage markers 1 and 3 mo later (n = 8 per group per time point).

Fig. 2.

CP9-induced prostatitis stimulates basal–luminal differentiation. (A) Schematic illustration of the experimental strategy. Tmx, tamoxifen. (B) GFP-labeled basal and luminal cells in tamoxifen-treated K14-mTmG mice. (C) Immunostaining of GFP, K14, and K8 in prostates of K14-mTmG mice. (D) Costaining of GFP, K14, and K8 in prostates of tamoxifen-treated K14-mTmG mice that did not undergo transurethral instillation (Tmx) or that underwent instillation of PBS (Tmx + PBS) or CP9 (Tmx + CP9), performed 1 or 3 mo after transurethral instillation. Red arrows point to GFP⁺ luminal cells. (E–G) Bar graphs show the percentages of K14⁺K8⁻, K14⁺K8⁺, and K14⁻K8⁺ cells in GFP⁺ cells in the three groups. Data were collected from eight mice. (H) Immuno-costaining of K8 and cleaved caspase 3 in the three groups. Data shown in the bar graphs represent means and SD from four mice. *P < 0.05; **P < 0.01.

Consistent with our previous studies (14), 5 d after tamoxifen treatment, a small percentage (∼9%) of basal cells were labeled with GFP under our experimental conditions (Fig. 2B), and all GFP⁺ cells expressed the basal cell marker K14 but not the luminal cell marker K8 (Fig. 2C; n = 1,390 GFP⁺ cells). In the group that received no transurethral instillation, GFP-expressing cells remained almost exclusively K14⁺K8⁻ (1,720 of 1,721 and 1,245 of 1,246 GFP⁺ cells examined 2 and 4 mo after tamoxifen treatment, respectively) (Fig. 2 D and E). This result corroborates previous findings that adult murine K14-expressing basal cells primarily generate basal cells in vivo (14–16). In the control group that received PBS, the majority of GFP⁺ cells remained as K14-expressing basal cells even 3 mo after instillation (Fig. 2 D and F). However, we were able to detect ∼1.05% K8-expressing cells within the GFP⁺ cells 1 mo after PBS instillation (Fig. S4), and the percentage rose to 1.70% 3 mo after instillation. In sharp contrast, in the CP9-treated group, many GFP⁺ cells (red arrows, Fig. 2D) were K8⁺ (21.05% and 20.45% for K8⁺K14⁺ cells at 1 mo and 3 mo after instillation, respectively; 17.85% and 33.39% for K8⁺K14⁻ cells at 1 mo and 3 mo postinstillation, respectively) (Fig. 2G). The increasing percentage of K8⁺ cells within total GFP⁺ cells during aging was not caused by the increased proliferation of luminal cells compared with basal cells, as shown by Ki67 staining (Fig. 1E), nor did fewer luminal cells than basal cells undergo apoptosis, as shown by immunostaining of cleaved caspase 3 (Fig. 2H). Most likely, this increased percentage reflects a continuous basal-to-luminal differentiation or the difference in the length of the cell cycle in basal and luminal cells. In conclusion, CP9 infection causes tissue damage and microenvironmental changes that stimulate the differentiation of basal cells into luminal cells.

The frequency of basal–luminal differentiation also was increased in the PBS-treated group as compared with the untreated group (Fig. 2 E and F). Intraurethral instillation of PBS may cause reflux of urine into the prostate, leading to chemical or physical trauma (39). Therefore, the increased basal–luminal differentiation in the PBS-treated group may reflect the occurrence of a mild inflammatory response, as reported previously (32).

We also investigated whether basal cells are capable of generating neuroendocrine cells during prostatitis. Neuroendocrine cells remained extremely rare within the prostates of the CP9-treated mice, suggesting that the inflammatory response does not induce neuroendocrine differentiation (Fig. S5). There was no evidence that basal cells can generate neuroendocrine cells, because none of the 187 neuroendocrine cells that we examined expressed GFP. One potential caveat regarding these studies is that we were able to GFP-label only ∼9% of the basal cells. Therefore, we cannot exclude the possibility that nonlabeled basal cells do possess a differentiation capacity or that GFP⁺ neuroendocrine cells might be detected if more neuroendocrine cells were examined.

CP9-Induced Prostatitis Accelerates Initiation of Prostate Cancer in the K14- Phosphatase and Tensin Homolog Model.

Previously, we examined the capacity of the prostate basal cells to serve as the cells of origin for prostate cancer using a K14-CreER;Pten^fl/fl (K14-Pten) model, through which Phosphatase and tensin homolog (Pten) can be ablated specifically in prostate basal cells upon tamoxifen induction (14). Pten deletion is not sufficient to transform basal cells. Instead, disease initiation in this model requires the differentiation of basal cells into transformation-competent luminal cells. However, basal-to-luminal differentiation rarely takes place; hence this mouse model develops prostate cancer with a long latency and low penetrance. Therefore, we sought to determine experimentally whether prostatitis-induced basal-to-luminal differentiation could accelerate disease initiation in the K14-Pten model.

We showed previously that at early times after tamoxifen treatment the K14-Pten mouse model develops rare and tiny PIN lesions that are difficult to detect (14). In this study, we generated K14-CreER^Tg/Tg;PTEN^fl/fl;mTmG^Tg/Tg (hereafter referred to as “K14-Pten-mTmG”) mice, reasoning that expression of GFP from the mTmG allele might facilitate a more sensitive and semiquantitative detection of multifocal tiny PIN lesions. The experimental design was similar to that shown in Fig. 2A. Briefly, PBS or bacteria solution was instilled transurethrally into K14-Pten-mTmG mice 3 wk after tamoxifen treatment. Prostate tissues were collected and examined 1 and 3 mo after instillation of PBS or CP9 (n = 8 per group per time point).

As shown in Fig. 3 A and B, GFP-expressing foci were detected in all experimental mice in both groups. However, on average, twice as many GFP foci per mouse were observed in the CP9-treated group (8.4 and 23.6, respectively, at 1 and 3 mo post-CP9 instillation) as in the PBS group (4.3 and 11.0, respectively, at 1 and 3 mo post-PBS instillation). The GFP foci in the CP9-treated group at 3 mo after transurethral instillation also were larger than those in the PBS group (Fig. 3A).

Fig. 3.

CP9-induced prostatitis accelerates disease initiation and progression in the K14-Pten model. (A) Fluorescent images of prostates from tamoxifen-treated K14-Pten-mTmG mice 1 mo and 3 mo after transurethral instillation of PBS (Tmx + PBS) or CP9 bacteria (Tmx + CP9). (B) Dot plots quantify numbers of GFP foci. Data represent means and SD from eight mice per group. (C) H&E staining and IHC analysis of GFP and pAKT in prostates from tamoxifen-treated K14-Pten-mTmG mice 1 mo and 3 mo after transurethral instillation of PBS. (D) H&E staining and IHC analysis of GFP and pAKT in prostates of tamoxifen-treated K14-Pten-mTmG mice 1 and 3 mo after transurethral instillation of CP9. (Upper) Representative images of pAKT⁻ lesions. (Lower) Representative images of pAKT⁺ lesions. (E) The table summarizes disease grades in PBS- and CP9-treated groups. Disease grade is defined by the most advanced GFP⁺ lesions detected in tissues. (F) IHC staining of Ki67 in GFP⁺ and GFP⁻ foci in murine prostates 1 mo after CP9 treatment. Data shown in the bar graph represent means and SD from eight mice. *P < 0.05; **P < 0.01.

H&E staining of the GFP foci revealed that five mice in the PBS-treated group developed PIN1/2 lesions (40) 1 mo after PBS treatment (Fig. 3 C, row 1, and E). The GFP foci in the other three mice contained only normal tissue with clusters of GFP-labeled basal cells (Fig. 3C, row 2). The GFP⁺ basal cells did not always express pAKT, suggesting that the efficiency of the tamoxifen-induced homologous recombination may be greater at the mTmG locus than at the Pten locus. PIN1/2 lesions were detected in four mice 3 mo after PBS treatment, and PIN3/4 lesions were detected in the other four mice (Fig. 3 C, rows 3 and 4, and E). All PIN lesions comprised mainly K8-expressing luminal cells (Fig. S6), as reported previously (14). Cells within these lesions stained positively for both GFP and pAKT, confirming that they were derived from Pten-null basal cells.

In contrast, multifocal lesions were observed in all the CP9-treated mice 1 mo after transurethral instillation (Fig. 3 D and E). The majority of these lesions did not stain positively for GFP or pAKT (Fig. 3D, Upper). Therefore these lesions likely were derived from cells that did not undergo homologous recombination and may have resulted directly from the prostatitis induced by CP9 infection. Nevertheless, PIN3/4 lesions consisting of cells that expressed both GFP and pAKT were detected in all the mice, albeit at a low frequency (three to nine foci per mouse) (Fig. 3 D and E). By 3 mo after CP9 instillation, GFP⁺pAKT⁺ PIN3/4 lesions were easily detectable in all experimental mice (Fig. 3 D and E). All these GFP⁺ PIN lesions were composed of K8-expressing cells (Fig. S6). As expected, we also observed lesions that contained a mixture of GFP⁺pAKT⁺ cells and wild-type cells (Fig. S7). The proliferation index in the GFP⁻pAKT⁻ lesions was less than that observed in the GFP⁺pAKT⁺ lesions, suggesting that Pten deletion confers a stronger or additive mitotic signaling (Fig. 3F). Collectively, these results demonstrate that CP9-induced prostatitis accelerates disease initiation and progression in the K14-Pten model, probably through the combined effects of enhanced basal-to-luminal differentiation and promitotic signaling induced by the inflammatory response.

Discussion

Inflammation can contribute to tumor initiation and progression by inducing genetic and epigenetic changes. Our study suggests that the inflammation also may accelerate tumor initiation by altering the tissue lineage differentiation program. Cells inclined to acquire oncogenic signaling within a tissue may be intrinsically resistant to transformation. However, tissue damage induced by various insults, including inflammation, may result in microenvironmental changes that are capable of stimulating the differentiation of these cells into other lineages. The prooncogenic signaling inherited by the daughter lineages may enhance their susceptibility to transformation induced by ensuing genetic changes. Of note, we believe that it is not the type of inflammation, but the inflammation- and tissue damage-induced signals, such as the NF-κB signaling, that drive basal-to-luminal differentiation. This hypothesis is supported by our observation that basal-to-luminal differentiation was slightly enhanced even in the PBS-treated group, probably because of the very mild inflammation caused by the urinal reflex-induced chemical and physical trauma.

Mouse Model for Bacterial Infection-Induced Prostatitis.

Two models have been used frequently in previous studies to investigate the prostate epithelial lineage hierarchy: the aging model and the model in which epithelial turnover is induced by alternate androgen ablation and replacement (14–18). A limitation of these models is that they do not consider some environmental factors, such as inflammation, that affect human prostate epithelial homeostasis. Laboratory mice usually do not develop prostatitis even during aging because they are maintained in aseptic vivaria and fed with a standard laboratory dietary regimen. On the other hand, even though the infiltration of inflammatory cells into the prostate was observed when mice were subjected to alternate androgen deprivation and replacement (41, 42), tissue damage such as the disruption or ulceration of acinar outlines and epithelial reactive hyperplasia that frequently are observed in human chronic prostatitis rarely occur.

The model of prostatitis induced by CP9 bacterial infection complements these models but is not without limitations. First, the bacterial-infection model is not representative of all types of human prostatitis, because many patients with chronic prostatitis and chronic pelvic pain syndrome have no evidence of urinary tract infection (43). In addition, E. coli is not the only source for bacterial infection (39, 44, 45). Second, transurethral instillation of uropathogenic bacteria usually causes an immediate phase of acute inflammation followed a few months later by a second phase of multifocal chronic inflammation (34). Because bacteria-induced prostatitis in this model causes the expansion of transit-amplifying cells that express K14 (Fig. 1), we had to label the basal cells in the K14-mTmG model before bacteria instillation. Consequently, we were not able to determine the basal cell differentiation program under the chronic inflammatory conditions. Therefore other models for prostatitis may be used to investigate the differentiation potential of basal cells during non-bacteria–induced prostatitis and during chronic prostatic inflammation (46–51).

Functional Plasticity of Prostate Basal Cells.

We and others showed previously that adult murine prostate basal cells primarily generate basal cells in vivo when residing in their native microenvironment (14–16). However, basal cells exhibit functional plasticity and possess the stem cell potential to differentiate into the other lineages in prostate epithelia. Rodent prostate basal epithelial cells are capable of adapting a luminal cell phenotype during in vitro culture (52). Several independent groups (19–25) have shown in a prostate regeneration assay that both human and rodent basal cells possess the capacity for multilineage differentiation. In the current study, we further showed that acute prostatic inflammation induced by bacterial infection significantly promoted basal-to-luminal differentiation in situ. Finally, in normal human prostate tissues, luminal cells have been shown to share the same genetic changes as basal cells; this observation also indirectly supports the occurrence of basal-to-luminal differentiation (53).

The functional plasticity of basal cells may be suppressed by residence in their natural niches or, alternatively, stimulated by basal cell-autonomous genetic and epigenetic changes or alterations in their native niches. The first mechanism is less likely because isolated basal cells are not able to differentiate readily into luminal or neuroendocrine cells when cultured in vitro. Hormones or defined growth factors are required for their differentiation (54–56). Therefore, future studies will focus on the identification of the basal cell-autonomous or niche-derived signaling that actively drives basal-to-luminal differentiation. During inflammation infiltrating immune cells such as macrophages, T cells, and myeloid-derived suppressor cells can play critical roles in tumor initiation and progression (1). However, they may not be directly necessary for basal-to-luminal differentiation, because basal cells can generate luminal cells efficiently in immune-deficient environments with the help of UGSM cells (19, 21). Therefore, it is more likely that prostate stromal cells were reprogramed by the inflammatory signaling so that they reactivated signaling shared by the UGSM cells, thereby acquiring the capacity to induce basal-to-luminal differentiation.

Cells of Origin for Human Prostate Cancer.

Recent molecular studies have demonstrated that multifocal human prostate cancer represents an independent group of diseases (57). These data suggest the existence of multiple cells-of-origin for individual prostate cancers. Treatment-naive human prostate cancer consists of cells that display a luminal cell phenotype. Therefore, prostate cancer has been proposed to have a luminal cell origin, so that disease is initiated directly from luminal cells that have acquired oncogenic genetic changes de novo. On the other hand, in human prostates, basal cells are more likely to acquire and maintain genetic changes, because they display a higher proliferation index but undergo less apoptosis than luminal cells (58–60). In normal human prostate tissues, luminal cells share the same genetic changes in mitochondria as basal cells, indicating that they have the same origin or that basal cells can generate luminal cells (53). Therefore, genetic alterations that contribute to the initiation of some prostate cancers also may take place in basal cells, so that these prostate cancers have a basal cell origin. The differentiation of basal cells with preexisting prooncogenic signaling into luminal cells would enhance the susceptibility of luminal cells to transformation by ensuing genetic changes, as demonstrated by our current study and by several previous studies using mouse models (14–16, 19, 61). A very recent study using the prostate regeneration model showed that basal cells even may serve as the origin of prostate cancer stem cells (62). Currently, because of technical limitations, no published study has directly demonstrated that basal cells and luminal cells within the same human PIN lesions share genetic changes. With improved technologies, future studies using laser-capture microscopy in concert with array comparative genomic hybridization should generate compelling results to validate this hypothesis. Finally, our study does not imply that all genetic changes associated with prostate cancer must take place in basal cells or that that only basal cells can serve as the cells of origin for human prostate cancer.

Materials and Methods

Mice.

C57BL/6 mice were from Charles River. The K14-CreER mice (63) were purchased from the Jackson Laboratory. The mTmG fluorescence reporter mice (38) were originally from Liqun Luo (Stanford University, Stanford, CA) and were internally transferred from Jeffery Rosen’s laboratory at the Baylor College of Medicine. The Pten^fl/fl mice (64) were made by Hong Wu (University of California, Los Angeles). Mice genotypes were verified by PCR using mouse genomic DNA from tail biopsy specimens. PCR products were separated electrophoretically on 1% agarose gels and visualized via ethidium bromide under UV light. All animal work was approved by and performed under the regulations of the Institutional Animal Care Committee of the Baylor College of Medicine.

E. coli Strain.

The uropathogenic E. coli strain CP9 was from Allison O’Brien (Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda). This strain is a human blood isolate that expresses cytotoxic necrotizing factor 1 and hemolysin and previously has been reported to induce a reproducible mouse model for prostatitis (7, 35). To prepare CP9 inoculum, a single CP9 colony was inoculated and cultured in 5 mL of static Luria broth for 24 h and subsequently was passaged in 50 mL of static Luria broth for 48 h at 37 °C. Bacteria were adjusted to an A₆₀₀ of 0.85 (bacterial density equal to 4.5 × 10⁸ cfu/mL), washed, and serially diluted by sterile PBS to a final concentration of 1 × 10⁷ cfu/mL.

Bacterial Infection and Tamoxifen Treatment.

Intraurethral instillation of bacterial inoculum was performed based on the procedures described previously (7, 34). For characterization of the inflammation model, 9-wk-old C57BL/6 mice were put under anesthesia (300 mg/kg Avertin; Sigma-Aldrich) and instilled with 200 µL of sterile PBS or CP9 solution (2 × 10⁶ cfu) intraurethrally using sterile polyethylene tubing (Jelco I.V; Catheter Radiopaque, Smith Medical). For lineage tracing and tumor initiation studies, 5-wk-old K14-mTmG and K14-Pten-mTmG mice first were treated with tamoxifen as described previously (14). Briefly, tamoxifen (Sigma-Aldrich) was dissolved in corn oil and was injected i.p. at a dosage of 9 mg⋅40g⁻¹⋅d⁻¹ for 4 d consecutively. Three weeks after the last tamoxifen injection, mice were intraurethrally instilled with either PBS or CP9 inoculum.

Dissociation of Primary Prostates and Flow Cytometry.

Dissociation of prostate cells and lineage fractionation were performed as described previously (55). For digestion, tissues were incubated with 10% (vol/vol) FBS DMEM containing collagenase/hyaluronidase (StemCell Technologies) for 3 h at 37 °C with rotation. After the medium was removed, 0.25% Trypsin-EDTA (Invitrogen) was added for 1-h additional digestion on ice. Subsequently, digested cells were resuspended in medium containing 5 mg/mL dispase (Invitrogen) and 1 mg/mL DNase I (Roche Applied Science) and were pipetted for 1 min. Finally, dissociated cells were filtered through 100-μm cell strainers (BD Biosciences) to collect single cells. For lineage fractionation, single murine prostate cells were stained with FITC-conjugated anti CD31, CD45, and Ter119 antibodies (eBioscience), PE-conjugated anti–Sca-1 antibody (eBioscience), and Alexa 647-conjugated anti CD49f antibody (Biolegend). To analyze the population of immune cells in prostate, digested prostate tissues were stained with FITC-conjugated anti-CD45, PE-conjugated anti-CD3, APC-conjugated anti-CD19, Pacific Blue-conjugated anti-F4/80, and PerCP/Cy5.5-conjugated anti-Ly-6G (Biolegend). The analyses were performed by using LSR II or LSR Fortessa (BD Biosciences).

Histology, Masson’s Trichrome Staining, and Immunohistochemical Analysis.

Collected prostates were fixed in 10% buffered formalin overnight at 4 °C and embedded with paraffin. Five-micrometer–sectioned slides were examined histologically by H&E. Masson’s trichrome stain was performed according to the manufacturer’s instructions (HT15-1KT; Sigma-Aldrich). For immunohistochemistry (IHC), sections were deparaffinized, and antigen retrieval was performed by steam heating in 0.01 M sodium citrate buffer (pH 6.0) for 10 min in a steamer. Cooled slides were incubated with 5% normal goat serum (Vector Labs) and with primary antibodies diluted in the 5% normal goat serum overnight at 4 °C. Primary antibodies used were rabbit anti-K5 (PRB-160P; Covance), mouse anti-K8 (MMS-162P; Covance), rat anti-K8 (Troma-1; Developmental Studies Hybridoma Bank), mouse anti-P63 (4A4; Santa Cruz Biotechnology), rabbit anti-AR (SC-816; Santa Cruz Biotechnology), mouse anti-smooth muscle actin (Sigma-Aldrich), rabbit anti-vimentin (5741; Cell Signaling Technology), rabbit anti-Ki67 (NCL-Ki67-P; Novocastra), mouse anti-K14 (LL002; Biogenex), mouse anti-GFP (JL8; Clontech), chicken anti-GFP (ab13970; Abcam), rabbit anti-synaptophysin (18-0130; Invitrogen), rabbit anti-cleaved caspase 3 (9661; Cell Signaling Technology), and rabbit anti-pAKT (3787; Cell Signaling Technology) Biotinylated secondary antibodies and streptavidin-conjugated HRP (Vector Laboratories) were used for chromatic visualization. For fluorescence visualization, slides were incubated with secondary antibodies (diluted 1:1,000 in PBS containing 0.05% Tween 20) labeled with Alexa-Fluor 488, 594, or 633 (Invitrogen). Sections were counterstained with DAPI (Sigma-Aldrich). Immunofluorescence staining was imaged with a fluorescence microscope (Olympus BX60; Olympus Optical Co Ltd.) or confocal microscope (Leica EL6000; Leica Microsystems). Cell counting was performed via Image J Software.

Statistics.

All experiments were performed using 3–11 mice in independent experiments. Data are presented as mean ± SD. A Student t test was used to determine significance between groups. For all statistical tests, the 0.05 level of confidence was accepted for statistical significance.