Kenichiro Ishii

Forkhead box A1 regulates prostate ductal morphogenesis and promotes epithelial cell maturation

We have previously shown that a forkhead transcription factor Foxa1 interacts with androgen signaling and controls prostate differentiated response. Here, we show the mouse Foxa1 expression marks the entire embryonic urogenital sinus epithelium (UGE), contrasting with Shh and Foxa2, which are restricted to the basally located cells during prostate budding. The Foxa1-deficient mouse prostate shows a severely altered ductal pattern that resembles primitive epithelial cords surrounded by thick stromal layers. Characterization of these mutant cells indicates a population of basal-like cells similar to those found in the embryonic UGE, whereas no differentiated or mature luminal epithelial cells are found in Foxa1-deficient epithelium. These phenotypic changes are accompanied with molecular aberrations, including focal epithelial activation of Shh and elevated Foxa2 and Notch1 in the null epithelium. Perturbed epithelial-stromal interactions induced by Foxa1-deficient epithelium is evident, as demonstrated by the expansion of surrounding smooth muscle and elevated levels of stromal factors (Bmp4, Fgf7, Fgf10 and Gli). The prostatic homeobox protein Nkx3.1, a known proliferation inhibitor, was downregulated in Foxa1-deficient epithelial cells, while several prostate-specific androgen-regulated markers, including a novel Foxa1 target, are absent in the null prostate. These data indicate that Foxa1 plays a pivotal role in controlling prostate morphogenesis and cell differentiation.

Aminopeptidase N regulated by zinc in human prostate participates in tumor cell invasion

Aminopeptidase N (AP‐N) degrades collagen type IV and is proposed to play a role in tumor invasion. However, the precise functions of AP‐N in tumor cells and the relationship of AP‐N to prostate cancer remains unclear. In our study, we examined a possible role for zinc in the regulation of AP‐N enzymatic activity in relation to tumor cell invasion in human prostate. AP‐N purified from human prostate was irreversibly inhibited by low concentrations of zinc (Ki = 11.2 μM) and bestatin. AP‐N, which has zinc in the active center, was also inhibited by the chelating agents, EDTA, o‐phenanthroline and EGTA. EDTA was shown to remove zinc from the enzyme. When the effects of zinc and bestatin on invasion of PC‐3 cells were investigated in vitro using a Transwell cell‐culture chamber, zinc and bestatin effectively suppressed cell invasion into Matrigel at the concentration range of 50–100 μM. These results strongly suggest that the suppression of PC‐3 cell invasion by zinc is based on the inhibition of AP‐N activity by zinc. We also evaluated the expression of AP‐N to investigate the relationship with the progression of prostate disease in human cancerous prostate. AP‐N was found to be located at the cytoplasmic membranes of prostate gland epithelial cells and to be expressed more in prostate cancer, while the expression of prostate‐specific antigen (PSA), which is a useful marker for prostate cancer, was shown in normal and cancer tissues, suggesting that AP‐N is potentially a good histological marker of prostate cancer. Thus, highly expressed AP‐N in human cancerous prostate probably plays an important role in the invasion and metastasis of prostate cancer cells.

NE-10 neuroendocrine cancer promotes the LNCaP xenograft growth in castrated mice.

Increases in neuroendocrine (NE) cells and their secretory products are closely correlated with tumor progression and androgen-independent prostate cancer. However, the mechanisms by which NE cells influence prostate cancer growth and progression, especially after androgen ablation therapy, are poorly understood. To investigate the role of NE cells on prostate cancer growth, LNCaP xenograft tumors were implanted into nude mice. After the LNCaP tumors were established, the NE mouse prostate allograft (NE-10) was implanted on the opposite flank of these nude mice to test whether NE tumor-derived systemic factors can influence LNCaP growth. Mice bearing LNCaP tumors with or without NE allografts were castrated 2 weeks after NE tumor inoculation, and changes in LNCaP tumor growth rate and gene expression were investigated. After castration, LNCaP tumor growth decreased in mice bearing LNCaP tumors alone, and this was accompanied by a loss of nuclear androgen receptor (AR) localization. In contrast, in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continued to grow, had increased levels of nuclear AR, and secreted prostate-specific antigen. Therefore, in the absence of testicular androgens, NE secretions were sufficient to maintain LNCaP cell growth and androgen-regulated gene expression in vivo. Furthermore, in vitro experiments showed that NE secretions combined with low levels of androgens activated the AR, an effect that was blocked by the antiandrogen bicalutamide. Because an increase in AR level has been reported to be sufficient to account for hormone refractory prostate cancers, the NE cell population ability to increase AR level/activity can be another mechanism that allows prostate cancer to escape androgen ablation therapy.

Steroid hormones stimulate human prostate cancer progression and metastasis

Tissue recombinants (TRs) composed of mouse urogenital mesenchyme (mUGM) plus an immortalized nontumorigenic human prostatic epithelial cell line (BPH‐1) were grown under the kidney capsule of male athymic nude mice under different hormonal conditions. The objectives were to determine temporal plasma concentrations of testosterone (T) and estradiol‐17β (E2) that elicit progression of nontumorigenic human prostatic epithelial cells in vivo. Second, to determine whether mUGM+BPH‐1 TRs in [T+E2]‐treated hosts could progress to metastases. Control mouse hosts received no exogenous hormonal support, whereas treated mice received Silastic implants containing T and E2 for 1–4 months. Plasma from hormonally treated mice contained significantly higher (p < 0.01) concentrations of T at 1 month (11.7 vs. 0.9 ng/ml). Plasma levels of E2 in steroid implanted mice were significantly higher (p <0.05) at 2 months (104.5 vs. 25.6 ng/l) and 4 months (122.8 vs. 19.2 pg/ml). Wet weights of mUGM+BPH‐1 TRs from [T+E2]‐implanted mice were significantly larger (p < 0.001) than those from untreated hosts. Untreated mUGM+BPH‐1 TRs contained a well organized differentiated epithelium surrounded by smooth muscle stroma similar to developing prostate. In [T+E2]‐implanted mice, mUGM+BPH‐1 TRs formed carcinomas that contained a fibrous connective tissue stroma permeating the tumor; smooth muscle when present was associated with vasculature. Renal lymph nodes collected from [T+E2]‐treated mice, but not untreated mice, contained metastatic carcinoma cells. Moreover, metastases could be observed at distant sites including lung and liver. Epithelial cells isolated from untreated mUGM+BPH‐1 TRs exhibited benign histology and formed small nontumorigenic grafts when subsequently transplanted into athymic nude mice. In contrast, epithelial cells isolated from mUGM+BPH‐1 tumors of [T+E2]‐treated hosts formed large tumors that grew independent of stromal and hormonal support and developed lymph node metastases. We conclude that [T+E2]‐treatment promotes prostatic cancer progression in mUGM + BPH‐1 TRs. Use of mUGM in this system will allow future studies to utilize the power of mouse genetics to identify paracrine factors involved in human prostatic carcinogenesis.

Endocrine Disrupter Bisphenol A Increases In Situ Estrogen Production in the Mouse Urogenital Sinus

The balance between androgens and estrogens is very important in the development of the prostate, and even small changes in estrogen levels, including those of estrogen-mimicking chemicals, can lead to serious changes. Bisphenol A (BPA), an endocrine-disrupting chemical, is a well-known, ubiquitous, estrogenic chemical. To investigate the effects of fetal exposure to low-dose BPA on the development of the prostate, we examined alterations of the in situ sex steroid hormonal environment in the mouse urogenital sinus (UGS). In the BPA-treated UGS, estradiol (E2) levels and CYP19A1 (cytochrome P450 aromatase) activity were significantly increased compared with those of the untreated and diethylstilbestrol (DES)-treated UGS. The mRNAs of steroidogenic enzymes, Cyp19a1 and Cyp11a1, and the sex-determining gene, Nr5a1, were up-regulated specifically in the BPA-treated group. The up-regulation of mRNAs was observed in the mesenchymal component of the UGS as well as in the cerebellum, heart, kidney, and ovary but not in the testis. The number of aromatase-expressing mesenchymal cells in the BPA-treated UGS was approximately twice that in the untreated and DES-treated UGS. The up-regulation of Esrrg mRNA was observed in organs for which mRNAs of steroidogenic enzymes were also up-regulated. We demonstrate here that fetal exposure to low-dose BPA has the unique action of increasing in situ E2 levels and CYP19A1 (aromatase) activity in the mouse UGS. Our data suggest that BPA might interact with in situ steroidogenesis by altering tissue components, such as the accumulation of aromatase-expressing mesenchymal cells, in particular organs.

Naftopidil, a selective α-1 adrenoceptor antagonist, inhibits growth of human prostate cancer cells by G1 cell cycle arrest

α‐1 adrenoceptor antagonists are generally prescribed for benign prostate hyperplasia with lower urinary tract symptoms. Naftopidil, a selective α‐1 adrenoceptor antagonist, is frequently used in Japan because it has fewer side effects. Here we demonstrate for the first time that naftopidil has growth inhibitory effect in androgen‐sensitive and ‐insensitive human prostate cancer cell lines. The concentrations causing 50% inhibition (IC50) of cancer cell growth were 22.2 ± 4.0 μM in androgen‐sensitive LNCaP cells and 33.2 ± 1.1 μM in androgen‐insensitive PC‐3 cells. FACS analysis revealed that cell growth inhibition by naftopidil was due to the arrest of the G1 cell cycle. Expressions of p27kip1 and p21cip1 were significantly increased in LNCaP cells treated with naftopidil. In PC‐3 cells, naftopidil induced p21cip1 but not p27kip1. In vivo, oral administration of naftopidil to nude mice inhibited the growth of PC‐3 tumors as compared to vehicle‐treated controls. These results suggest that naftopidil may be useful in the chemoprevention of prostate cancer and the intervention of hormone refractory prostate cancer.

Decreases of Metallothionein and Aminopeptidase N in Renal Cancer Tissues

Abstract Good molecular markers for investigating the biochemical differences between renal cancer and surrounding tissues have not yet been developed. Sixteen kidney samples (clear cell RCC) were investigated to determine the differences in the protein components between renal cancer and surrounding tissues, using HPLC analysis. The metallothionein (MT) and zinc levels were consistently lower in renal cancer tissues compared with in surrounding tissues. The mean concentration of MT in normal tissues surrounding renal tumors was about 15 times higher than that in cancer tissues. An immunohistochemical study confirmed that the expression of MT in renal cancer tissues was lower than that in adjacent normal tissues. The activities of aminopeptidases (APs) were significantly decreased in renal cancer tissues compared with in adjacent normal tissues. An immunohistochemical study and Western blot analysis confirmed that the expression of AP-N in renal cancer tissues was also lower than in adjacent normal tissues. These results suggest that the immunohistochemical detection of MT and AP-N could provide useful information as a pathological diagnostic tool for classifying renal cancer and surrounding tissues.

Urothelial transdifferentiation to prostate epithelia is mediated by paracrine TGF-β signaling

The embryonic urogenital sinus mesenchyme (UGM) induces prostate epithelial morphogenesis in development. The molecular signals that drive UGM-mediated prostatic induction have not been defined. We hypothesized that the TGF-beta signaling directed the prostatic induction. UGM from TGF-beta type II receptor stromal conditional knockout mice (Tgfbr2(fspKO)) or control mice (Tgfbr2(floxE2/floxE2)) was recombined with wild-type adult mice bladder urothelial cells. The resulting urothelium associated with Tgfbr2(floxE2/floxE2) UGM was instructively differentiated into prostatic epithelium, as expected. In contrast, the urothelium associated with Tgfbr2(fspKO) UGM permissively maintained the phenotype of bladder epithelial cells. Microarray analysis of UGM tissues suggested the down-regulation of multiple Wnt ligands and the up-regulation of the Wnt antagonist, Wif 1, by the Tgfbr2(fspKO) UGM compared with Tgfbr2(floxE2/floxE2) UGM. The overexpression of Wif-1 by wild-type UGM resulted in the inhibition of prostatic induction. These data suggest that the stromal TGF-beta activity mediated by paracrine Wnt is necessary for the induction of prostatic differentiation. As Wnt ligands mediate differentiation and maintain the stem cell phenotype, the contribution of mouse stem cells and somatic cells to prostatic epithelium in the tissue recombination models was tested. The directed differentiation of mouse embryonic stem cells by UGM is suggested by a threshold number of mouse stem cells required in prostatic differentiation. To determine the contribution of somatic cells, the adult bladder epithelial compartment was labeled with green-fluorescent vital dye (CMFDA) and the stem-like cells marked by bromodeoxyuridine (BrdU) label-retention. The resulting prostatic epithelia of the tissue recombinants maintained the CMFDA dye, suggesting minimal cell division. Thus, the UGM can induce endoderm-derived epithelia and stem cells to form prostate through a transdifferentiation mechanism that requires stromal TGF-beta signaling to mediate epithelial Wnt activity.

Naftopidil, a Selective α1-Adrenoceptor Antagonist, Suppresses Human Prostate Tumor Growth by Altering Interactions between Tumor Cells and Stroma

In prostate cancer, tumor–stroma interactions play a critical role in the promotion of tumorigenesis, and thus the prevention of those interactions is a promising target to suppress tumor growth. Several studies demonstrated that alpha1-adrenoceptor (α1-AR) antagonists, therapeutic drugs for benign prostatic hyperplasia, have growth inhibitory effects on human prostate cancer (PCa) cells through induction of apoptosis or G1 cell-cycle arrest. However, their direct actions on stromal cells surrounding cancer cells have not yet been elucidated. In this study, we investigated the effects of subtype-selective α1-AR antagonists (naftopidil, tamsulosin, and silodosin) on prostate tumor growth with a focus on the role of stroma, using commercially available fibroblast cells (PrSC). Tumorigenic studies in vivo showed significant reductions in tumor growth when E9 cells (an androgen low-sensitive LNCaP subline) grafted with PrSC were treated with naftopidil. In in vitro analyses, naftopidil and silodosin showed antiproliferative effects on PCa cells regardless of androgen sensitivity and α1-AR subtype expression. In PrSC, a strong growth inhibitory effect was observed with naftopidil but not silodosin. Flow cytometric analysis revealed that naftopidil, but not silodosin, induced G1 cell-cycle arrest in both PCa cells and PrSC. In naftopidil-treated PrSC, total interleukin-6 protein was significantly reduced with increased suppression of cell proliferation. Silodosin induced weak early apoptosis only in PCa cells. These findings demonstrated that naftopidil strongly suppressed cell proliferation of stromal cells, resulting in decreased tumorigenic soluble factor, suggesting that naftopidil might be effective in preventing stromal support of tumor cells. Cancer Prev Res; 4(1); 87–96. ©2011 AACR.

Evidence that androgen-independent stromal growth factor signals promote androgen-insensitive prostate cancer cell growth in vivo

Activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. To discover new therapeutic targets for hormone-refractory prostate tumor growth under androgen ablation therapy, androgen-sensitive LNCaP cells and the derived sublines, E9 (androgen-low-sensitive), and AIDL (androgen-insensitive), were recombined with androgen-dependent embryonic rat urogenital sinus mesenchyme (UGM). Tumors of E9 + UGM and AIDL + UGM were approximately three times as large as those of LNCaP + UGM. Tumors grown in castrated hosts exhibited reduced growth as compared with those in intact hosts. However, in castrated hosts, E9 + UGM and AIDL + UGM tumors were still approximately twice as large as those of LNCaP + UGM. Cell proliferation in tumors of E9 + UGM and AIDL + UGM grown in castrated host, was significantly higher than that in tumors of LNCaP + UGM. In vitro, expression of fibroblast growth factor (FGF)-2 and IGF-I, but not FGF-7 mRNA, was significantly reduced in UGM under androgen starvation. In cell culture, E9 cells were responsive to FGF-2 and FGF-7 stimulation, while AIDL responded to FGF-7 and IGF-1. Expression of FGFR1 and FGFR2 was considerably higher in E9 than those in LNCaP, similarly expression of FGFR2 and IGF-IR were elevated in AIDL. These data suggest that activation of prostate cancer cell growth through growth factor receptor expression may result in the activity of otherwise androgen-independent stromal growth factor signals such as FGF-7 under conditions of androgen ablation.