Taichi Mizushima

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.

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.

Role of the androgen receptor in urothelial cancer

Men have had a substantially higher risk of developing bladder cancer than women. This has prompted research on androgen-mediated androgen receptor (AR) signaling in urothelial cancer. Indeed, increasing preclinical evidence indicates that AR activation correlates with the promotion of urothelial carcinogenesis and tumor outgrowth. In this article, we summarize and discuss available data suggesting the involvement of androgens and the AR pathway in the development and progression of urothelial cancer. Although precise mechanisms for the functions of AR and related signals in urothelial cells remain far from being fully understood, current observations may offer effective chemopreventive and therapeutic approaches for urothelial cancer. Clinical application of various anti-AR therapies available for AR-dependent prostate cancer to urothelial cancer patients is thus anticipated.

GATA3 immunohistochemistry in urothelial carcinoma of the upper urinary tract as a urothelial marker and a prognosticator

Immunohistochemistry of a transcription factor, GATA3, has been widely used as a promising urothelial marker in diagnostic surgical pathology practice. However, the expression status of GATA3 in upper urinary tract urothelial carcinomas (UUTUCs) and its prognostic significance have not been fully investigated. We immunohistochemically stained for GATA3 in 99 UUTUC samples and paired nonneoplastic urothelial tissues. GATA3 was positive in 51 (51.5%; 32 [32.3%] weak, 11 [11.1%] moderate, 8 [8.1%] strong) of 99 UUTUCs, which was significantly lower than in benign urothelium (79 [96.3%] of 82; 33 [40.2%] weak, 35 [42.7%] moderate, 11 [13.4%] strong; P<.001). However, there were no statistically significant associations between GATA3 expression and tumor grade, pT stage, lymph node involvement, or distant metastasis. Meanwhile, the rate of GATA3 positivity was significantly higher (P=.004) in ureteral tumors (66.0%) than in renal pelvic tumors (35.6%). Kaplan-Meier and log-rank tests revealed that GATA3 negativity was significantly associated with lower recurrence-free survival (P=.037 for all cases, P=.026 for muscle-invasive tumors) and cancer-specific survival (P=.007 for all cases, P=.012 for muscle-invasive tumors, P=.035 for cases with adjuvant chemotherapy) rates. Multivariate analysis further identified strong correlations of GATA3 expression with tumor progression (all cases: hazard ratio [HR], 0.479 [95% confidence interval {CI},0.229-1.003; P=.051]; muscle-invasive tumors: HR, 0.387 [95% CI, 0.166-0.903; P=.028) or cancer-specific mortality (all cases: HR, 0.354 [95% CI, 0.135-0.925; P=.034]; muscle-invasive tumors: HR, 0.402 [95% CI, 0.149-1.086; P=.072]). Thus, compared with nonneoplastic urothelium, a significant decrease in the expression of GATA3 in UUTUC was seen. Moreover, loss of GATA3 expression was found to be an independent predictor of poor patient outcomes. Of note was that only roughly half of high-grade and/or muscle-invasive UUTUCs were immunoreactive for GATA3.

NFATc1 Expression as a Prognosticator in Urothelial Carcinoma of the Upper Urinary Tract

We recently found that NFATc1, a member of the NFAT family and a key regulator of the immune response, could induce bladder carcinogenesis and cancer progression. In this study, we immunohistochemically stained for NFATc1 in upper urinary tract urothelial carcinoma (UUTUC) specimens and paired nonneoplastic urothelial tissues. NFATc1 was positive in 51 [52%; 40 (40%) weak (1+), 9 (9%) moderate (2+), and 2 (2%) strong (3+)] of 99 UUTUCs, which was significantly higher than in benign urothelium [30 (36%) of 83; 28 (34%) weak and 2 (2%) moderate] (0 vs 1+/2+/3+, P = .038; 0/1+ vs 2+/3+, P = .023). There were no significant associations between NFATc1 expression pattern and tumor grade or pT stage. However, the positive rates of NFATc1 expression tended to be higher in renal pelvic tumors (60%) than in ureteral tumors (42%; P = .080) as well as in pN+ tumors (75%) than in pN0 tumors (49%; P = .089). Kaplan-Meier and log-rank tests revealed that moderate (2+) to strong (3+) NFATc1 expression correlated with lower progression-free survival (P = .032) and cancer-specific survival (P = .005) rates in the 99 cases. Patients with high (2+/3+) NFATc1 muscle-invasive tumor (n = 9) also had a significantly higher risk of cancer-specific mortality (P = .021) compared to those with low (0/1+) NFATc1 muscle-invasive tumor (n = 53). Thus, compared with nonneoplastic urothelium, a significant increase in the expression of NFATc1 in UUTUC was seen, implying the involvement of NFATc1 signals in the development of UUTUC. The current results further suggest that NFATc1 overexpression serves as a predictor of poor prognosis in patients with UUTUC.

Prostaglandin receptors induce urothelial tumourigenesis as well as bladder cancer progression and cisplatin resistance presumably via modulating PTEN expression

Background:We investigated the role of prostaglandin receptors (e.g. prostaglandin E2 receptor 2 (EP2), EP4) and the efficacy of celecoxib in urothelial tumourigenesis and cancer progression.Methods:We performed immunohistochemistry in bladder cancer (BC) tissue microarrays, in vitro transformation assay in a normal urothelial SVHUC line, and western blot/reverse transcription–polymerase chain reaction/cell growth assays in BC lines.Results:EP2/EP4 expression was elevated in BCs compared with non-neoplastic urothelial tissues and in BCs from those who were resistant to cisplatin-based neoadjuvant chemotherapy. Strong positivity of EP2/EP4 in non-muscle-invasive tumours or positivity of EP2/EP4 in muscle-invasive tumours strongly correlated with disease progression or disease-specific mortality, respectively. In SVHUC cells, exposure to a chemical carcinogen 3-methylcholanthrene considerably increased and decreased the expression of EP2/EP4 and phosphatase and tensin homologue (PTEN), respectively. Treatment with selective EP2/EP4 antagonist or celecoxib also resulted in prevention in 3-methylcholanthrene-induced neoplastic transformation of SVHUC cells. In BC lines, EP2/EP4 antagonists and celecoxib effectively inhibited cell viability and migration, as well as augmented PTEN expression. Furthermore, these drugs enhanced the cytotoxic activity of cisplatin in BC cells. EP2/EP4 and PTEN were also elevated and reduced, respectively, in cisplatin-resistant BC sublines.Conclusions:EP2/EP4 activation correlates with induction of urothelial cancer initiation and outgrowth, as well as chemoresistance, presumably via downregulating PTEN expression.

Androgen receptor activation: A prospective therapeutic target for bladder cancer?

ABSTRACT Introduction: Patients with non-muscle-invasive or muscle-invasive bladder cancer undergoing surgery and currently available conventional therapy remain having a high risk of tumor recurrence or progression, respectively. Novel targeted molecular therapy is therefore expected to improve patient outcomes. Meanwhile, substantially higher incidence of bladder cancer in men has prompted research on androgen-mediated androgen receptor (AR) signaling in this malignancy. Indeed, preclinical evidence has suggested that AR signaling plays an important role in urothelial carcinogenesis and tumor outgrowth as well as resistance to some of the currently available conventional non-surgical therapies. Areas covered: We summarize and discuss available data suggesting the involvement of AR and its potential downstream targets in the development and progression of bladder cancer. Associations between AR signaling and sensitivity to cisplatin/doxorubicin or bacillus Calmette-Guérin treatment are also reviewed. Expert opinion: AR activation is likely to correlate with the promotion of urothelial carcinogenesis and cancer outgrowth as well as resistance to conventional therapies. Molecular therapy targeting the AR may thus provide effective chemopreventive and therapeutic approaches for urothelial cancer. Accordingly, bladder cancer can now be considered as an endocrine-related neoplasm. Clinical application of various anti-AR therapies available for AR-dependent prostate cancer to bladder cancer patients is anticipated.

Nuclear Factor-κB Promotes Urothelial Tumorigenesis and Cancer Progression via Cooperation with Androgen Receptor Signaling

We investigated the role of NF-κB in the development and progression of urothelial cancer as well as cross-talk between NF-κB and androgen receptor (AR) signals in urothelial cells. Immunohistochemistry in surgical specimens showed that the expression levels of NF-κB/p65 (P = 0.015)/phospho-NF-κB/p65 (P < 0.001) were significantly elevated in bladder tumors, compared with those in nonneoplastic urothelial tissues. The rates of phospho-NF-κB/p65 positivity were also significantly higher in high-grade (P = 0.015)/muscle-invasive (P = 0.033) tumors than in lower grade/non–muscle-invasive tumors. Additionally, patients with phospho-NF-κB/p65-positive muscle-invasive bladder cancer had significantly higher risks of disease progression (P < 0.001) and cancer-specific mortality (P = 0.002). In immortalized human normal urothelial SVHUC cells stably expressing AR, NF-κB activators and inhibitors accelerated and prevented, respectively, their neoplastic transformation induced by a chemical carcinogen 3-methylcholanthrene. Bladder tumors were identified in 56% (mock), 89% (betulinic acid), and 22% (parthenolide) of N-butyl-N-(4-hydroxybutyl)nitrosamine-treated male C57BL/6 mice at 22 weeks of age. NF-κB activators and inhibitors also significantly induced and reduced, respectively, cell proliferation/migration/invasion of AR-positive bladder cancer lines, but not AR-knockdown or AR-negative lines, and their growth in xenograft-bearing mice. In both nonneoplastic and neoplastic urothelial cells, NF-κB activators/inhibitors upregulated/downregulated, respectively, AR expression, whereas AR overexpression was associated with increases in the expression levels of NF-κB/p65 and phospho-NF-κB/p65. Thus, NF-κB appeared to be activated in bladder cancer, which was associated with tumor progression. NF-κB activators/inhibitors were also found to modulate tumorigenesis and tumor outgrowth in AR-activated urothelial cells. Accordingly, NF-κB inhibition, together with AR inactivation, has the potential of being an effective chemopreventive and/or therapeutic approach for urothelial carcinoma. Mol Cancer Ther; 17(6); 1303–14. ©2018 AACR.

The interaction between androgen receptor and semenogelin I: a synthetic LxxLL peptide antagonist inhibits the growth of prostate cancer cells

Background:We previously demonstrated that a seminal plasma protein, semenogelin I (SgI), functioned as an androgen receptor (AR) coactivator. Meanwhile, several short sequence motifs in AR coregulators, such as LxxLL (L=leucine), have been shown to mediate specific interactions with AR.Methods:We investigated the role of the LxxLL motif within SgI in the interactions with AR and cell growth in prostate cancer lines in vitro.Results:A full-length SgI with mutations in the motif (i.e., LxxAA; A=alanine) failed to significantly increase cell proliferation/migration as well as androgen-mediated AR transcription. Co-immunoprecipitation showed no physical interactions between AR and the mutant SgI. In addition, transfection of an 18-amino acid peptide of SgI containing LxxLL, but not LxxAA, resulted in considerable reduction in cell growth and prostate-specific antigen expression in LNCaP and C4-2 lines.Conclusions:The LxxLL motif of SgI could be a novel therapeutic target for both androgen-sensitive and castration-resistant prostate cancers.

ZKSCAN3 expression in urothelial carcinoma of the upper urinary tract and its impact on patient outcomes

We recently found that a zinc-finger transcription factor, ZKSCAN3, could induce the growth of bladder cancer cells. In this study, we immunohistochemically stained for ZKSCAN3 in upper urinary tract urothelial carcinoma (UUTUC) specimens. ZKSCAN3 was positive in 52 [42.4%; 26 (26.3%) weak (1+), 13 (13.1%) moderate (2+), and 3 (3.0%) strong (3+)] of 99 UUTUCs, which was significantly (P<0.001) lower than in paired normal-appearing urothelial tissues [70 (86.4%) of 81; 17 (21.0%) weak, 36 (44.4%) moderate, and 17 (21.0%) strong]. There were no statistically significant associations between ZKSCAN3 expression pattern and tumor grade, pT/pN stage, or distant metastasis. However, the positive rate of ZKSCAN3 expression was significantly (P<0.001) higher in ureteral tumors (54.0%) than in renal pelvic tumors (26.7%). Kaplan-Meier and log-rank tests revealed that the levels of ZKSCAN expression did not considerably correlate with progressionfree or cancer-specific survival in the entire cohort of the patients. Meanwhile, strong (3+) ZKSCAN3 expression in muscle-invasive tumor was correlated with the risk of disease progression (P=0.001) and cancer-specific mortality (P=0.069), while only one patient had a ZKSCAN3(3+) muscle-invasive tumor. Thus, in contrast to the observations in bladder specimens, the decreased expression of ZKSCAN3 in UUTUC, compared with non-neoplastic urothelium, was seen. Moreover, the current results failed to provide conclusive evidence suggesting the prognostic value of ZKSCAN3 expression in patients with UUTUC.