Yichun Zheng

Expression of androgen and oestrogen receptors and its prognostic significance in urothelial neoplasm of the urinary bladder

Whats known on the subject? and What does the study add?

Androgen receptor signals regulate UDP‐glucuronosyltransferases in the urinary bladder: A potential mechanism of androgen‐induced bladder carcinogenesis

UDP‐glucuronosyltransferases (UGTs), major phase II drug metabolism enzymes, play an important role in urinary bladder cancer initiation by detoxifying carcinogens. We aimed to determine if androgens regulate UGT expression via the androgen receptor (AR) pathway in the bladder. Real‐time reverse transcription‐polymerase chain reaction and Western blot analyses were used to assess UGT1A levels in the normal urothelium SVHUC cell line stably expressed with AR and in bladder tissues from AR knockout (ARKO) and castrated male mice. Immunohistochemistry was also performed in radical cystectomy specimens. Dihydrotestosterone (DHT) treatment in SVHUC‐AR reduced mRNA expression of all the UGT1A subtypes (19–75% decrease), and hydroxyflutamide antagonized the DHT effects. In contrast, DHT showed only marginal effects on UGT1A expression in SVHUC‐Vector. Of note were higher expression levels of UGT1As in SVHUC‐Vector than in SVHUC‐AR. In ARKO mice, all the Ugt1a subtypes were up‐regulated, compared to wild‐type littermates. In wild‐type male mice, castration increased the expression of Ugt1a8, Ugt1a9, and Ugt1a10. Additionally, wild‐type female mice had higher levels of Ugt1a than wild‐type males. Immunohistochemical studies showed strong (3+) UGT1A staining in 11/24 (46%) cancer tissues, which was significantly lower than in corresponding benign tissues [17/18 (94%) cases (P = 0.0009)]. These results suggest that androgen‐mediated AR signals promote bladder carcinogenesis by down‐regulating the expression of UGTs in the bladder.

Nuclear Hormone Receptor Signals as New Therapeutic Targets for Urothelial Carcinoma

Unlike prostate and breast cancers, urothelial carcinoma of the urinary bladder is not yet considered as an endocrine-related neoplasm, and hormonal therapy for bladder cancer remains experimental. Nonetheless, there is increasing evidence indicating that nuclear hormone receptor signals are implicated in the development and progression of bladder cancer. Androgen-mediated androgen receptor (AR) signals have been convincingly shown to induce bladder tumorigenesis. Androgens also promote the growth of AR-positive bladder cancer cells, although it is controversial whether AR plays a dominant role in bladder cancer progression. Both stimulatory and inhibitory functions of estrogen receptor signals in bladder cancer have been reported. Various studies have also demonstrated the involvement of other nuclear receptors, including progesterone receptor, glucocorticoid receptor, vitamin D receptor, and retinoid receptors, as well as some orphan receptors, in bladder cancer. This review summarizes and discusses available data suggesting the modulation of bladder carcinogenesis and cancer progression via nuclear hormone receptor signaling pathways. These pathways have the potential to be an extremely important area of bladder cancer research, leading to the development of effective chemopreventive/therapeutic approaches, using hormonal manipulation. Considerable uncertainty remains regarding the selection of patients who are likely to benefit from hormonal therapy and optimal options for the treatment.

Epidermal growth factor induces bladder cancer cell proliferation through activation of the androgen receptor

Androgen receptor (AR) signals have been suggested to contribute to bladder tumorigenesis and cancer progression. Activation of epidermal growth factor receptor (EGFR) also leads to stimulation of bladder tumor growth. However, crosstalk between AR and EGFR pathways in bladder cancer remains uncharacterized. We have recently shown that androgens activate the EGFR pathway in bladder cancer cells. The purpose of this study was to investigate the effects of EGF on AR activity in bladder cancer. EGF increased AR transcriptional activity by 1.2-, 1.9- and 2.0-fold in UMUC3, 5637-AR and J82-AR cell lines, respectively, over mock treatment and a specific EGFR inhibitor, PD168393, antagonized the EGF effect. Combined treatment of EGF and dihydrotestosterone (DHT) further induced AR transactivation while an AR antagonist, hydroxyflutamide (HF), abolished the effect of not only DHT but also EGF. In growth assays, EGF alone/DHT alone/EGF+DHT increased cell numbers by 16/12/19%, 6/14/18% and 30/12/38% in UMUC3-control-shRNA, 5637-AR and J82-AR, respectively, whereas the effects of EGF were marginal or less significant in UMUC3-AR-shRNA (8%) or AR-negative 5637-V (<1%) and J82-V (17%) cells. HF treatment at least partially counteracted the EGF effect on the growth of AR-positive cells. Western blotting demonstrated that EGF, especially in the presence of DHT, upregulated the expression of the p160 coactivator TIF2 and HF again blocked this stimulation. Co-immunoprecipitation revealed the association between AR and estrogen receptor (ER)-β or Src in UMUC3 cells and stronger associations with EGF treatment, implying the involvement of the AR/ER/Src complex in EGF-increased AR transactivation and cell growth. Current results, thus, suggest that EGF promotes bladder cancer cell proliferation via modulation of AR signals. Taken together with our previous findings, crosstalk between EGFR and AR pathways can play an important role in the progression of bladder cancer.

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.

Contrary Regulation of Bladder Cancer Cell Proliferation and Invasion by Dexamethasone-Mediated Glucocorticoid Receptor Signals

In patients with advanced bladder cancer, glucocorticoids are frequently given to reduce acute toxicity, particularly hyperemesis, during chemotherapy, as well as to improve cachectic conditions. However, it remains unclear whether glucocorticoids directly affect the development and progression of bladder cancer through the glucocorticoid receptor pathway. Glucocorticoid receptor expression was first investigated in human bladder cancer lines and tissue microarrays. Then, the effects of dexamethasone on glucocorticoid receptor transcription, cell proliferation, apoptosis/cell cycle, and invasion were examined in bladder cancer lines. Finally, mouse xenograft models for bladder cancer were used to assess the efficacy of dexamethasone on tumor progression. All the cell lines and tissues examined were found to express glucocorticoid receptor. Dexamethasone increased glucocorticoid receptor–mediated reporter activity and cell proliferation, and inhibited apoptosis in the presence or absence of cisplatin. In contrast, dexamethasone suppressed cell invasion, the expression of its related genes [MMP-2/MMP-9, interleukin (IL)-6, VEGF], and the activity of MMP-2/MMP-9, and also induced mesenchymal-to-epithelial transition. In addition, dexamethasone increased IκBα protein levels and cytosolic accumulation of NF-κB. In xenograft-bearing mice, dexamethasone slightly augmented the growth of the inoculated tumors but completely prevented the development of bloody ascites, suggestive of peritoneal dissemination of tumor cells, and actual metastasis. In all these assays, dexamethasone effects were abolished by a glucocorticoid receptor antagonist or glucocorticoid receptor knockdown via RNA interference. Thus, glucocorticoid receptor activation resulted in promotion of cell proliferation via inhibiting apoptosis yet repression of cell invasion and metastasis. These results may provide a basis of developing improved chemotherapy regimens, including or excluding glucocorticoid receptor agonists/antagonists, for urothelial carcinoma. Mol Cancer Ther; 11(12); 2621–32. ©2012 AACR.

Expression of UDP‐glucuronosyltransferase 1A in bladder cancer: Association with prognosis and regulation by estrogen

Although UDP‐glucuronosyltransferase 1A (UGT1A) plays an important role in preventing bladder cancer initiation by detoxifying carcinogenic compounds, its contribution to bladder cancer progression is poorly understood. We immunohistochemically stained for UGT1A in bladder specimens. UGT1A was positive in 130/145 (90%; 28 [19%] weak, 53 [37%] moderate, and 49 [34%] strong) urothelial neoplasms, which was significantly weaker than in matched non‐neoplastic urothelial tissues (100/101 [99%]; 2 [2%] weak, 17 [17%] moderate, and 81 [80%] strong). Fifty (98%) of 51 low‐grade/79 (99%) of 80 non‐muscle‐invasive tumors were immunoreactive to UGT1A, whereas 80 (85%) of 94 high‐grade/51 (78%) of 65 muscle‐invasive tumors were UGT1A‐positive. Kaplan‐Meier analysis showed strong associations between lower UGT1A expression versus the risk of recurrence in high‐grade non‐muscle‐invasive tumors (P = 0.038) or disease‐specific mortality in muscle‐invasive tumors (P = 0.016). Multivariate analysis further revealed UGT1A loss as an independent prognosticator for disease‐specific mortality in patients with muscle‐invasive tumor (P = 0.010). Additionally, the expression of UGT1A was positively and negatively correlated with those of estrogen receptor‐α and estrogen receptor‐β, respectively. We then assessed UGT1A/Ugt1a levels in human cell lines/mouse tissues. 17β‐Estradiol increased and decreased UGT1A expression in normal urothelium and bladder cancer lines, respectively, and an anti‐estrogen abolished these effects. Ovariectomy in mice resulted in down‐regulation of Ugt1a subtypes. These results suggest the involvement of UGT1A in not only bladder carcinogenesis but tumor progression. Moreover, UGT1A is likely regulated by estrogens in non‐neoplastic urothelium versus bladder tumor in opposite manners, which could be underlying mechanisms of gender‐specific differences in bladder cancer incidence and progression.

Expression of semenogelins I and II and its prognostic significance in human prostate cancer

Little is known about the role of semenogelins, seminal plasma proteins that play critical roles in semen clotting and subsequent liquefaction in the presence of zinc and prostate‐specific antigen, in human malignancies.

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.

Seminal plasma proteins in prostatic carcinoma: increased nuclear semenogelin I expression is a predictor of biochemical recurrence after radical prostatectomy

Semenogelins and eppin are seminal plasma proteins that form a complex and inhibit sperm motility. However, the role of these proteins in prostate cancer is poorly understood. We immunohistochemically stained for semenogelins I and II and eppin in 291 radical prostatectomy specimens. We then evaluated the association between their expressions in nuclei, cytoplasms, or intraluminal secretions of benign/high-grade prostatic intraepithelial neoplasia/carcinoma cells and clinicopathologic profile available for our patient cohort. Stains were positive in 32%/77%/84% (nuclear semenogelin I), 87%/94%/84% (nuclear semenogelin II), 56%/64%/37% (nuclear eppin), 7%/15%/11% (cytoplasmic semenogelin I), 6%/11%/9% (cytoplasmic semenogelin II), 68%/74%/95% (cytoplasmic eppin), 97%/98%/13% (secreted semenogelin I), 98%/97%/11% (secreted semenogelin II), and 97%/98%/48% (secreted eppin) of benign/prostatic intraepithelial neoplasia/carcinoma, respectively. The levels of nuclear semenogelin I/cytoplasmic eppin were significantly higher in carcinoma than in benign (P<.001/P<.001) or prostatic intraepithelial neoplasia (P<.001/P<.001) and in prostatic intraepithelial neoplasia than in benign (P<.001/P=.006). Significantly higher nuclear semenogelin II expression was found in prostatic intraepithelial neoplasia than in benign (P<.001) or carcinoma (P<.001). Significantly lower nuclear eppin expression was seen in carcinoma than in benign (P<.001) or prostatic intraepithelial neoplasia (P<.001). Secreted semenogelin I, secreted semenogelin II, and secreted eppin were all significantly lower in carcinoma than in benign (P<.001) or prostatic intraepithelial neoplasia (P<.001). There were no statistically significant correlations between each stain and clinicopathologic features except significantly lower nuclear eppin expression in Gleason score 8 or higher tumors. Kaplan-Meier and log-rank tests further revealed that patients with nuclear semenogelin I-positive tumor had a significantly higher risk for biochemical recurrence (P=.046). Multivariate Cox model showed a trend toward significance (P=.093) in nuclear semenogelin I positivity as an independent predictor for recurrence. These results suggest that nuclear semenogelin I expression could be a reliable prognosticator in men who undergo radical prostatectomy.