Eiji Kashiwagi

ELK1 is up-regulated by androgen in bladder cancer cells and promotes tumor progression

Little is known about biological significance of ELK1, a transcriptional factor that activates downstream targets including c-fos proto-oncogene, in bladder cancer. Recent preclinical evidence also suggests the involvement of androgen receptor (AR) signaling in bladder cancer progression. In this study, we aim to investigate the functions of ELK1 in bladder cancer growth and their regulation by AR signals. Immunohistochemistry in bladder tumor specimens showed that the levels of phospho-ELK1 (p-ELK1) expression were significantly elevated in urothelial neoplasms, compared with non-neoplastic urothelium tissues, and were also correlated with AR positivity. Patients with p-ELK1-positive non-muscle-invasive and muscle-invasive tumors had significantly higher risks for tumor recurrence and progression, respectively. In AR-positive bladder cancer cell lines, dihydrotestosterone treatment increased ELK1 expression (mRNA, protein) and its nuclear translocation, ELK1 transcriptional activity, and c-fos expression, which was restored by an anti-androgen hydroxyflutamide. ELK1 silencing via short hairpin RNA (shRNA) resulted in decreases in cell viability/colony formation, and cell migration/invasion as well as an increase in apoptosis. Importantly, ELK1 appears to require activated AR to regulate bladder cancer cell proliferation, but not cell migration. Androgen also failed to significantly induce AR transactivation in ELK1-knockdown cells. In accordance with our in vitro findings, ELK1-shRNA expression considerably retarded tumor formation as well as its growth in xenograft-bearing male mice. Our results suggest that ELK1 plays an important role in bladder tumorigenesis and cancer progression, which is further induced by AR activation. Accordingly, ELK1 inhibition, together with AR inactivation, has the potential of being a therapeutic approach for bladder cancer.

The role of NFATc1 in prostate cancer progression: Cyclosporine A and tacrolimus inhibit cell proliferation, migration, and invasion

The functional role of nuclear factor of activated T‐cells (NFAT), a well‐characterized regulator of the immune response, in prostate cancer progression remains largely unknown. We aim to investigate biological significance of NFATc1, a NFAT isoform shown to function as an oncogene in a sarcoma model, in human prostate cancer.

Loss of GATA3 in bladder cancer promotes cell migration and invasion.

The transcription factor GATA3 is known as a breast tumor suppressor as well as a urothelial marker, and its loss is often seen in high-grade invasive bladder cancer. Nonetheless, GATA3 functions in bladder cancer cells remain largely unknown. In this study, we assessed the effects of GATA3 silencing via RNA interference on cell migration, invasion, and proliferation of bladder cancer. GATA3 expression was downregulated in all four bladder cancer lines examined, compared with a non-neoplastic urothelial line SVHUC. Knockdown of GATA3 in the bladder cancer lines (5637, TCC-SUP, J82) resulted in promotion of cell migration and invasion as well as increases in the expression of their related molecules, such as vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9, and the activity of MMP-2 and MMP-9. GATA3 loss was also associated with an increasing level of a mesenchymal marker N-cadherin and a decreasing level of an epithelial marker β-catenin. Consistent with these findings, enforced expression of GATA3 in UMUC3 inhibited cell migration and invasion. However, GATA3 showed marginal effects on bladder cancer cell viability and the expression of cell cycle- or apoptosis-related molecules. Additionally, in contrast to bladder cancer lines, no significant effects of GATA3 silencing on cell migration were seen in SVHUC. These findings suggest that GATA3 plays an important role in the prevention of bladder cancer progression and metastasis by inhibiting cell migration and invasion as well as epithelial-to-mesenchymal transition.

Enzalutamide inhibits androgen receptor-positive bladder cancer cell growth.

PURPOSE Emerging preclinical evidence suggests that androgen-mediated androgen receptor (AR) signals promote bladder cancer progression. However, little is known about the efficacy of an AR signaling inhibitor, enzalutamide, in the growth of bladder cancer cells. In this study, we compared the effects of enzalutamide and 2 other classic antiandrogens, flutamide and bicalutamide, on androgen-induced bladder cancer cell proliferation, migration, and invasion as well as tumor growth in vivo. METHODS Thiazolyl blue cell viability assay, flow cytometry, scratch wound-healing assay, transwell invasion assay, real-time polymerase chain reaction, and reporter gene assay were performed in AR-positive (e.g., UMUC3, TCCSUP, and 647V-AR) and AR-negative (e.g., UMUC3-AR-short hairpin RNA [shRNA], TCCSUP-AR-shRNA, 647V) bladder cancer lines treated with dihydrotestosterone and each AR antagonist. We also used a mouse xenograft model for bladder cancer. RESULTS Dihydrotestosterone increased bladder cancer cell proliferation, migration, and invasion indicating that endogenous or exogenous AR was functional. Enzalutamide, hydroxyflutamide, and bicalutamide showed similar inhibitory effects, without significant agonist activity, on androgen-mediated cell viability/apoptosis, cell migration, and cell invasion in AR-positive lines. No significant effects of dihydrotestosterone as well as AR antagonists on the growth of AR-negative cells were seen. Correspondingly, in UMUC3 cells, these AR antagonists down-regulated androgen-induced expression of AR, matrix metalloproteinase-2, and interleukin-6. Androgen-enhanced AR-mediated transcriptional activity was also blocked by each AR antagonist exhibiting insignificant agonist activity. In UMUC3 xenograft-bearing mice, oral gavage treatment with each antiandrogen retarded tumor growth, and only enzalutamide demonstrated a statistically significant suppression compared with mock treatment. CONCLUSIONS Our current data support recent observations indicating the involvement of the AR pathway in bladder cancer growth and further suggest that AR antagonists, including enzalutamide, are of therapeutic benefit in AR-positive bladder cancer.

Androgen receptor activity modulates responses to cisplatin treatment in bladder cancer

Cisplatin (CDDP)-based combination chemotherapy remains the mainstream treatment for advanced bladder cancer. However, its efficacy is often limited due to the development of resistance for which underlying mechanisms are poorly understood. Meanwhile, emerging evidence has indicated the involvement of androgen-mediated androgen receptor (AR) signals in bladder cancer progression. In this study, we aimed to investigate whether AR signals have an impact on sensitivity to CDDP in bladder cancer cells. UMUC3-control-short hairpin RNA (shRNA) cells with endogenous AR and AR-negative 647V/5637 cells stably expressing AR were significantly more resistant to CDDP treatment at its pharmacological concentrations, compared with UMUC3-AR-shRNA and 647V-vector/5637-vector control cells, respectively. A synthetic androgen R1881 significantly reduced CDDP sensitivity in UMUC3, 647V-AR, or 5637-AR cells, and the addition of an anti-androgen hydroxyflutamide inhibited the effect of R1881. In these AR-positive cells, R1881 treatment also induced the expression levels of NF-κB, which is known to involve CDDP resistance, and its phosphorylated form, as well as nuclear translocation of NF-κB. In CDDP-resistant bladder cancer sublines established following long-term culture with CDDP, the expression levels of AR as well as NF-κB and phospho-NF-κB were considerably elevated, compared with respective control sublines. In bladder cancer specimens, there was a strong trend to correlate between AR positivity and chemoresistance. These results suggest that AR activation correlates with CDDP resistance presumably via modulating NF-κB activity in bladder cancer cells. Targeting AR during chemotherapy may thus be a useful strategy to overcome CDDP resistance in patients with AR-positive bladder cancer.

Cyclosporine A and tacrolimus inhibit urothelial tumorigenesis

The functional role of nuclear factor of activated T‐cells (NFAT), while it has been extensively investigated in the immune system, remains uncertain in bladder cancer development. We here aim to assess the effects of cyclosporine A (CsA) and tacrolimus (FK506), immunosuppressants known to specifically inactivate the NFAT pathway in immune cells, on neoplastic transformation of urothelial cells. Immunohistochemistry revealed that the expression levels of NFATc1, a NFAT isoform shown to function as an oncogene in a sarcoma model, were elevated in urothelial neoplasms, compared with non‐neoplastic urothelial tissues, and in low‐grade and high‐grade papillary urothelial carcinomas, compared with papillary urothelial neoplasms of low malignant potential. In an immortalized normal urothelial cell line SVHUC, CsA and FK506 reduced NFATc1 expression, NFAT transcriptional activity, and the expression of c‐myc, a downstream target of NFATc1 signals. Treatment with CsA or FK506 in the SVHUC cells undergoing neoplastic transformation induced by exposure to a chemical carcinogen 3‐methylcholanthrene resulted in strong inhibition in colony formation in vitro as well as tumor formation in NOD‐SCID mice. CsA and FK506 were additionally found to up‐regulate the expression of several molecules that play a protective role in bladder tumorigenesis, including p53, p21, and p27, and down‐regulate that of oncogenic genes, such as cyclin D1, cyclin D3, and cyclin E, in SVHUC cells with the carcinogen challenge. Thus, CsA and FK506 likely inhibit urothelial tumorigenesis. These findings offer a potential chemopreventive approach for urothelial tumors using NFAT inhibitors.

Expression of steroid hormone receptors and its prognostic significance in urothelial carcinoma of the upper urinary tract

ABSTRACT To assess the expression status of steroid hormone receptors in upper urinary tract urothelial carcinoma (UUTUC), we immunohistochemically stained for androgen receptor (AR), estrogen receptor-α (ERα), ERβ, glucocorticoid receptor (GR), and progesterone receptor (PR) in 99 UUTUC specimens and paired non-neoplastic urothelial tissues. AR/ERα/ERβ/GR/PR was positive in 20%/18%/62%/63%/16% of tumors, which was significantly lower (except PR) than in benign urothelial tissues [57% (P < 0.001)/40% (P = 0.001)/85% (P = 0.001)/84% (P = 0.002)/13% (P = 0.489)]. There were no significant associations between each receptor expression pattern and histopathological characteristic of the tumors including tumor grade/stage. Kaplan-Meier and log-rank tests revealed no significant prognostic value of each receptor expression in these 99 patients. However, patients with UUTUC positive for either ERα or PR had a significantly higher risk of disease-specific mortality (P = 0.025), compared with those with UUTUC negative for both. PR positivity alone in pT3 or pT4 tumors was also strongly associated with the risk of disease-specific mortality (P = 0.040). Multivariate analysis further identified the expression of ERα and/or PR as a strong predictor for disease-specific mortality in the entire cohort of the patients (hazard ratio, 2.434; P = 0.037). Thus, in accordance with previous observations in bladder specimens, significant decreases in the expression of AR/ERα/ERβ/GR in UUTUC, compared with that in non-neoplastic urothelium, were observed. Meanwhile, the negativity of both ERα and PR in UUTUC as well as the negativity of PR alone in deeply invasive tumor was suggested to serve as a prognosticator.

Differential regulation of bladder cancer growth by various glucocorticoids: corticosterone and prednisone inhibit cell invasion without promoting cell proliferation or reducing cisplatin cytotoxicity

PurposeA synthetic glucocorticoid, dexamethasone, was recently shown to inhibit bladder cancer cell invasion and metastasis through the glucocorticoid receptor (GR) pathway but increased cell proliferation via inhibiting apoptosis particularly induced by cisplatin. Therefore, comedication with dexamethasone in bladder cancer patients may lead to unfavorable outcomes such as chemoresistance. We here look for any glucocorticoids with inhibitory effects on tumor cell invasion yet inhibitory or at least no stimulatory effects on cell viability.MethodsThe effects of 10 glucocorticoids on cell viability were first assessed in three bladder cancer lines. Selected compounds were further assessed for their ability in cell viability and apoptosis, with or without cisplatin, as well as in cell invasion.ResultsMost of the compounds (hydrocortisone, betamethasone, flumethasone, triamcinolone, budesonide, fluticasone propionate, and fludrocortisone acetate) increased GR-positive cell growth, which was similar to or even stronger than the effect of dexamethasone. Nonetheless, two glucocorticoids (corticosterone, prednisone) showed only marginal effects on cell growth of all the lines tested. They did not significantly reduce the effects of cisplatin on cell proliferation or cisplatin-induced apoptosis. Conversely, corticosterone, prednisone, and dexamethasone similarly inhibited cell invasion and expression of related genes, including MMP-9, VEGF, and IL-6, in GR-positive lines.ConclusionCorticosterone and prednisone are suggested to have the potential of being harmless, in contrast to dexamethasone, without promoting cell proliferation or inhibiting cytotoxic activity of cisplatin, yet beneficial to bladder cancer patients via suppressing tumor invasion. Our results are thus useful in improving chemotherapy regimens, including optimal glucocorticoids, for urothelial carcinoma.

ZKSCAN3 promotes bladder cancer cell proliferation, migration, and invasion

The expression status of ZKSCAN3, a zinc-finger transcription factor containing KRAB and SCAN domains, as well as its biological significance, in human bladder cancer remains largely unknown. In the current study, we aimed to determine the functional role of ZKSCAN3 in bladder cancer progression. Immunohistochemistry in tissue specimens detected ZKSCAN3 signals in 138 (93.2%) of 148 urothelial neoplasms, which was significantly higher than in non-neoplastic urothelial tissues [76 (84.4%) of 90; P=0.044]. Correspondingly, the levels of ZKSCAN3 gene were significantly elevated in bladder tumors, compared with those in adjacent normal-appearing bladder mucosae (P=0.008). In a validation set of tissue microarray, significantly higher ZKSCAN3 expression was observed in high-grade and/or muscle-invasive urothelial carcinomas than in low-grade and/or non-muscle-invasive tumors. Two bladder cancer cell lines, UMUC3 and 647V, were found to strongly express ZKSCAN3 protein/mRNA, whereas its expression in 5637 bladder cancer and SVHUC normal urothelium cell lines was very weak. ZKSCAN3 silencing via its short hairpin RNA (shRNA) in UMUC3 and 647V resulted in significant decreases in cell viability/colony formation, cell migration/invasion, and the expression of matrix metalloproteinase (MMP)-2/MMP-9 and oncogenes c-myc/FGFR3, as well as significant increases in apoptosis and the expression of tumor suppressor genes p53/PTEN. ZKSCAN3 overexpression in 5637 also induced cell growth and migration. In addition, ZKSCAN3-shRNA expression considerably retarded tumor formation as well as its subsequent growth in xenograft-bearing mice. These results suggest that ZKSCAN3 plays an important role in bladder cancer outgrowth. Thus, ZKSCAN3 inhibition has the potential of being a therapeutic approach for bladder cancer.

Silodosin inhibits prostate cancer cell growth via ELK1 inactivation and enhances the cytotoxic activity of gemcitabine

Biological significance of ELK1, a transcriptional factor whose phosphorylation is necessary for c‐fos proto‐oncogene activation, in prostate cancer remains far from fully understood. In this study, we aim to investigate the role of ELK1 in tumor growth as well as the efficacy of a selective α1A‐adrenergic blocker, silodosin, in ELK1 activity in prostate cancer cells.