The Expression and Actions of Androgen Receptor in Upper Urinary Tract
Urothelial Carcinoma (UUTUC) Tissues and Primary Cultured Cells
Chih-Rong Shyr*,¶, Chi-Cheng Chen*, Teng-Fu Hsieh*, Chao-Hsiang Chang, Wen-Lung Ma, Shuyuan Yeh, Edward Messing, Tsung-Heng Li, Fu-Yin Li, Chawnshang Chang
C.-R. Shyr W.-L. Ma T.-H. Li F.-Y. Li C.-C. Chen C.-H. Chang T.-F. Hsieh and C. Chang Sex Hormone Research Center, China Medical University/Hospital, Taichung 404, Taiwan
C.-C. Chen and C.-H. Chang
Department of Urology, China Medical University Hospital, Taichung 404, Taiwan C.-R. Shyr
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan
T.-F. Hsieh
Division of Urology, Department of Surgery, Buddhist Tzu Chi General Hospital, Taichung Branch, Taichung 427, Taiwan
S. Yeh E Messing and C. Chang
George Whipple Lab for Cancer Research, Departments of Pathology and Urology, University of Rochester Medical Center, Rochester, New York
W.-L. Ma, T.-F. Hsieh, and C. Chang
Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan
¶ Corresponding authors
Abstract.
Purpose
Sex hormone receptors, androgen receptor (AR) and estrogen receptors (ERs) including both ER and ER, mediate the actions of sex hormones. In this study, we aimed to evaluate sex hormone receptors expression in upper urinary tract urothelial carcinomas (UUTUCs) of ureter and renal pelvis with different tumor stages and grades as well as their possible roles in tumor progression.
Methods
Immunohistochemistry was used to assay the expression of AR and ERs in primary UUTUCs. XTT viability test was applied to evaluate cell responses for anticancer drug treatment. Wound healing assay was performed to determine cell migration abilities. Results
AR and ERβ immunoreactivities were observed in both UUTUCs, but ERα was not detected in either UUTUCs. In UUTUC of ureter specimens, higher AR expression was found in superficial or lower grade tumors. In contrast, little difference of ERβ expression was found in superficial vs muscle-invasive tumor stages or low grades vs high grades in UUTUCs of ureter specimens. Furthermore in primary cultured cells from UUTUC specimens, the addition of functional AR reduced cell chemosensitivity, but increased cell migration.
Conclusions
These results provide the first data showing the expression patterns of sex hormone receptors in both renal pelvis and ureter UUTUCs. From results, we concluded there is a positive correlation for higher AR expression found in superficial or low grade UUTUCs of ureter and identified the functional roles of AR in UUTUC progression.
Keywords: upper urinary tract urothelial carcinoma; sex hormone; androgen receptor (AR); estrogen receptors (ERs)
Introduction
The urinary collecting system from renal pelvis, ureter, bladder to urethra is lined with urothelial cells, so urothelial carcinomas of renal pelvis, ureter, and bladder share similar morphological patterns and development . Upper urinary tract urothelial carcinomas (UUTUCs) are relatively uncommon tumors, accounting for about 5% of all urothelial tumors and 5–10% of all renal tumors, but cause severe morbidity and mortality . UUTUCs are located more commonly in the renal pelvis than in the ureter with a ratio of 3:1 . The incidence of bilateral UUTUCs ranges from 2% to 8% . The development of UUTUC after primary diagnosis of bladder cancer is a rare event, occurring in only 2–4% of patients with bladder cancer , but the development of secondary bladder cancer after primary UUTUC is more frequent with a risk of 20–50% . In addition, UUTUCs are more invasive and poorly differentiated than bladder cancer . Furthermore, microsatellite alterations in UUTUCs differ from bladder cancer . These findings suggest UUTUCs and bladder cancer may have some variations in term of cancer initiation and progression.
UUTUCs afflict more men than women with a male-to-female ratio of 3:2 for tumors in the renal pelvis and 2:1 for a ureteral location . However, disease specific annual mortality was greater in women than in men . Postmenopausal women, compared with premenopausal women, were shown to have an increased risk for developing urothelial bladder cancer, suggesting menopausal status and age at menopause may affect the risk of bladder cancer among women . But how the gender difference affects the progression and prognosis of UUTUCs is not clearly understood.
Sex steroid hormones and their receptors are proposed to play a major role in the UUTUCs rate and their development and prognosis . The actions of sex hormones on target organs are mediated by their cognate receptors, androgen receptor (AR),
estrogen receptors (ERs) both ERand ER, in target cells to regulate development, reproduction, metabolism, and homeostasis . These receptors were shown to be expressed in bladder , although physiological functions of androgens and estrogens in the bladder are unclear. Currently the expression pattern of AR and ERs in UUTUCs is not well characterized. One study showed that AR expressed more in stage II than in stage I of UUTUCs and the cytosolic location of AR expression was correlated to the cytosolic expression of activin A, which is correlated with longer disease-free intervals . Although several studies examined the AR expression pattern in human bladder cancers, the prognostic significance of AR expression is conflicting with the report showing that the loss of AR expression was associated with invasive bladder cancer or has no association . However, ERβ, but not ERwas shown to express in the normal urothelium of the rat urinary bladder as the main subtype of ER expressed , but no reports on the expression pattern of ERs on UUTUCs have yet been published. Therefore, sex hormone receptors have been identified in patients with localized and advanced transitional cell carcinoma of the bladder and correlated with clinico-pathological features. But, the expression of sex hormone receptors and their roles in UUTUCs are still unclear. Hence, we aimed to investigate and analyze the expression and action of AR and ERs (bothand β) in neoplastic urothelial specimens of UUTUCs. Primary cultured tumor cells represent the heterogeneity and diversity of the multicellular tumors, and primary cultured tumor cells from different tumors have been used to study the development of tumors . Tumor cells from patients also provide a model to assay diagnosis-specific activity of cytotoxic drugs, in which cells are used in ex vivo assays to correlate the cytotoxic drug activity patterns observed in fresh human tumor cells ex vivo and the drug response in the clinic . Therefore, primary cultured cells of tumors obtained from individuals after open surgery or endoscopic resections have a
great diagnostic value to an individual, which is the goal of personalized medicine to find an optimal care and treatment for an individual. Therefore, we also examined the expression of sex hormone receptor in primary cultured tumor cells and the effect of AR on the chemosensitivity and migration of primary cultured cells.
Materials and Methods
Patients
The clinical data of 91 patients (collected continuously from 2010-2011 in our hospital) with UUTUCs treated at our institution and their specimens obtained from nephroureterectomy (NU) were collected for this study. Institutional Review Board approval was obtained for the study. After histopathologic evaluation, the tumors were staged according to the American Joint Committee on Cancer/International Union Against Cancer TNM classification. Superficial tumors included tumors confined to the urothelium or extending into the lamina propria only (pTa, carcinoma in situ [CIS], and pT1), and muscle-invasive tumors were characterized by tumor involvement of the muscularis propria or beyond (pT2, pT3, and pT4). Tumors were graded using the 2004 World Health Organization/International Society of Urological Pathology classification as papillary urothelial neoplasm of low malignant potential, low-grade urothelial carcinoma, and high-grade urothelial carcinoma. Of the 91 patients, 37 were men and 54 were female. Because in Taiwan, UUTUCs occur in females more than males , we have collected more female samples than male samples. Eight cases with simultaneous ureteral and renal pelvic tumors were excluded from further analysis. All the tumors were primary and one patient had received neoadjuvant therapy prior to tissue collection.
UUTUC cancer cell lines and Primary culture of UUTUC tumors
The UUTUC cell line, BFTC 909 cells (from a UUTUCs of renal pelvis patient) and SV-HUC cells (ureter cells immortalized by SV40, from an 11-year-old male accident victim) , obtained from ATCC, were cultured in Dulbecco's modified Eagle's medium and Ham's F-12 medium respectively, containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C in an atmosphere of 5% CO2.
To obtain primary culture of UUTUC tumor cells from patient specimens, the upper tract urothelium tumor tissues, which were later confirmed histologically as urothelial carcinomas, were collected immediately after surgical resection in the operating room and were rinsed with HBSS (GIBCO). After washing, the tumor tissues were minced into small pieces and digested in HBSS buffer containing collagenase type I incubated at 37°C, 5% CO2 for 2 h. The cells were filtered through a 70 μm nylon cell strainer (SPL) and spun down to collect the cell pellets. The cells were suspended in DMEM with 10% FBS and cultured in an incubator at 37°C, 5% CO2 and 95% humidified air environment. Differential trypsinization method was then repeatedly used to remove stromal fibroblasts until 99% of the cells exhibited epithelial cell morphology. The cell type was examined by E-cadherin (an epithelial cell marker) immunofluorescent staining to confirm their epithelial origin.
Transduction of AR-expressing lentiviral vectors
To overexpress AR in primary cultured cells, a recombinant lentiviral vector containing wild-type AR (pWPI hAR) and a control lentiviral vector expressing the enhanced green fluorescent protein (pWPI) were used to exogenously express AR. Lentiviral pWPI-AR/pWPI-control with pMD2.G packaging and psPAX2 envelope plasmids (lentivirus:packaging:envelopeZ 2:1:1) were co-transfected into 293T cells. After 48 h transfection, the target cells were cultured in the presence of viral supernatant containing 8 mg/ml polybrene (Millipore) for 6 h.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue blocks from each tumor were selected that contained tumor and nontumorous urothelium whenever possible. Sections were cut,
deparaffinized, and rehydrated in a graded series of ethanol. Tissue localization of the AR protein was performed on paraffin tissue sections using a mouse monoclonal anti-AR antibody (NCL-AR-318, Novocastra), of the ERprotein using a mouse monoclonal anti-ERantibody (NCL-ER-6F11, Novocastra), and of the ERprotein using a mouse monoclonal anti-ERantibody (NCL-ER-beta, Novocastra). The staining was performed on the automated immunostainer Leica BOND-MAX (Leica Microsystems). The sections were counterstained with hematoxylin, dehydrated, and mounted. Incubation without the primary antibody was used as a negative control. Formalin-fixed paraffin-embedded tissue sections with benign prostate obtained from radical prostatectomy were used as a positive control. The negative controls showed an absence of staining. The staining results of receptor expression were determined independently by two pathologists. Positive staining was given if more than 10% of cells were stained. The protein expression was tested for correlation to the tumor stage and grade.
XTT Assay
Cells infected with pWPI or pWPI hAR by lentiviral system (1 × 105 in 100 μl culture medium per well) were seeded in 96-well plates for 24 h, and then treated with Doxorubicin at various concentration for 24 h. To measure cell viability using cell proliferation kit (XTT) (Roche), 50 μl XTT labeling mixture per well was added and reacted for 3 h. The spectrophotometric absorbance of the sample was measured using a microplate reader. The wavelength to measure absorbance of the formazan product was 490 nm, and the reference wavelength was 690 nm.
Cells infected with pWPI or pWPI hAR by lentiviral system were plated into six-well tissue culture dishes and grown to confluence. Cell monolayers were then wounded by sterile pipette tips (1 mm) that generated a gap. Wounded monolayers were then washed 3 times with PBS to remove cell debris and incubated in 10% FBS medium in the presence of dihydrotestosterone (DHT) (10 nM) for 24 h to allow cells to migrate. Photos of the cells were then taken via a microscope equipped with a camera. The relative migration folds were determined by the area covered by migrating cells of test cells in the wounded area compared to that of control cells, analyzed with NIS-Elements BR (Nikon) software.
Dual-luciferase reporter assay
Primary cultured UUTUC 7630 hAR cells were co-transfected with ARE-Luc, and pRL-TK plasmids using Lipofectamine 2000 reagent (Invitrogen). After 24 h, the cells were treated with 10 nM DHT for 24 h and then lysed. The firefly and renilla luciferase activities were analyzed using the dual luciferase assay system (Promega). Firefly luciferase activity was normalized to the respective renilla luciferase activity. The luciferase activity of the ethanol treated cells was set as one.
Quantitative real-time PCR
Total RNA was isolated by TRIzol method (Invitrogen) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using BluePrint RT Reagent Kit (Takara). The primer sequences were as follows: human AR: forward: 5′-TGTCCATCTTGTCGTCTTC-3′ and reverse: 5′- CTC TCC TTC CTC CTG TAG-3′, and human β-actin: forward: 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′ and reverse : 5′-CAG CGG AAC CGC TCAT TGC CAA TGG-3′. Quantitative RT-PCR was performed
using Bio-Rad Real-time thermal cycler CFX96 and SYBR Premix. The relative mRNA expression level was normalized to β-actin (as an internal control) and determined by the 2−ΔΔCT method.
Statistical analysis
Statistical analyses were performed with SPSS 11.5 package program for Windows (SPSS Inc., Chicago, IL). Nominal data comparisons were tested by Pearson's χ2 test or student t test when appropriate. A P value < 0.05 was considered statistically significant, and all P values were two-sided.
Results
Immunohistochemical staining of AR, ER
and ER
proteins in UUTUCs of renalpelvis and ureter
We evaluated 83 specimens, consisting of 43 primary urothelial tumors of renal pelvis, and 40 primary urothelial tumors of ureter. To determine the roles of sex hormone receptors in UUTUCs progression, we performed IHC to detect where and how receptors express in the UUTUCs. Fig. 1a shows both nuclear and cytoplasmic staining in UUTUC tissue sections, but only nuclear reactivity was assessed because sex hormone receptors exert their transcription activity in the nucleus and the nuclear translocation from cytoplasm to nucleus is mediated by sex hormones. AR and ER immunoreactivities were observed in both UUTUCs, but little ER was detected in either of the sites. The expression of AR and ER was mainly localized to the nuclei of cancer cells.
The AR and ERβ expression status in UUTUC tumor specimens by IHC
Because AR and ER immunoreactivities were observed in both types of UUTUCs, we analyzed the correlation of their expression to ages, tumor locations, grades, and stages. As Table 1 shows, there was no statistically significant difference in AR and ER expression pattern at various ages. AR protein was shown to appear more in UUTUCs of ureters than renal pelvises (p<0.01). However, this phenomenon was not demonstrated in ERexpression.
To further investigate the significance of higher expression of AR in UUTUCs of ureter, we separated UUTUCs into two types. When analyzing only UUTUCs of ureter specimens (Table 2a), a significant difference in AR expression was observed between the superficial and muscle-invasive tumor stages (P < 0.01) as well as low and high
grades (P < 0.01). High AR expression percentage was associated with superficial and low grade tumors. However, ERβ expression was similar between superficial and muscle-invasive tumor stages as well as low and high grades.
However, when analyzing UUTUCs of pelvis specimens (Table 2b), the percentage of tumors that expressed the AR or ERβ showed no significant difference between superficial and muscle-invasive tumors stages. And both AR and ERβ expression were similar between low and high grades.
And the expression levels of AR (p=0.748) and ERp=0.887) were not significantly different between male and female (data not shown).
Detections of AR, ERα, and ERβ expression status in UUTUC cancer primary culture
The protein expression of sex hormone receptors was also determined by Western blot analyses in different UUTUC cells: BFTC-909 cells as well as 3 primary cultured cancer cells from UUTUCs of renal pelvis patients (#7630 from a high grade and pTaN0M0 tumor of a 64 year-old female patient; #8808 from a high grade and pT4N0M0 tumor of a 62 year-old female patient; and #7475from a high grade and pT1N0M0 tumor of a 59 year-old male patient). As shown in Fig. 1b, no detectable AR proteins were seen in any UUTUC cells. ER was seen in all cells and #7630 had highest expression, but #8808 and #7475 cells had low expression. ER protein expression was detected in lysates from all cells. Greater expression of ERwas detected in the 3 primary cultured tumor cells. The relative AR mRNA expression was also examined by Q-RT-PCR (Fig. 1c) and the result showed that AR mRNA expression was much lower in UUTUC cells than prostate cancer cells (LNCaP cells).
The effect of AR on the chemosensitivity of UUTUC cells
To prove that different sex hormone receptor expression level has impacts on UUTUC tumor cells, we tested whether the presence of AR might affect the cytotoxic effect of doxorubicin on UUTUC cells. Doxorubicin has been used as an anticancer drug in the adjuvant chemotherapy of UUTUCs to improve the overall mortality rate associated with UUTUCs by targeting the remaining cancer cells. We used the primary cultured tumor cells (#7630) and infected cells with lentiviral system carrying pWPI hAR or control pWPI parental vectors and treated with doxorubicin. With exogenously expressed AR via a lentiviral vector in primary cultured UUTUC cells (Fig. 2a), the AR was shown to have a transcriptional activity in primary cultured cells in the presence of DHT, demonstrated by the reporter gene assay (Fig. 2b). The growth of cells infected with pWPI hAR cells was not different than cells infected with pWPI in the presence or absence of DHT (Fig. 2c). However the cells had less cell death caused by doxorubicin compared to cells infected with pWPI control vector (Fig. 2d), suggesting that AR expressed in UUTUCs cells might be able to protect cells from being killed by doxorubicin and it was not due to the cell growth difference. We also measured the cell migration ability and found the overexpression of AR in primary cultured UUTUC cells increased the cell mobility (Fig. 2e). The results indicate the presence of AR influences the behaviors of UUTUC cells.
Discussion
Smoking, occupational carcinogens, and analgesic abuse are the risk factors for UUTUCs. In addition, gender is also a risk factor for UUTUCs, but the sex difference effects on the incidence and prognosis of UUTUCs are not clearly understood . We investigated the potential relationship between sex hormones and urothelial carcinoma development by determining whether the status of sex hormone receptors influences clinicopathological features of UUTUC patients. In this study, we found that AR and ER immunoreactivities were observed in both UUTUC sites, but ER was not detected in either of the sites. And the levels of AR and ERβ expression in UUTUC were similar in both sexes. However the expression of sex hormone receptors had different expression profiles in UUTUC cancer and primary cultured tumor cells.
When analyzing UUTUCs of pelvis specimens, the results showed that the percentage of tumors that expressed the AR or ERβ was not significantly different between superficial and muscle-invasive tumors stages (Table 2b). And both AR and ERβ expression were similar between low and high grades. But, we found a significant difference in AR expression was observed between the superficial and muscle-invasive tumor stages (P < 0.01) as well as low and high grades (P < 0.01) in only UUTUCs of ureter specimens, indicating that high AR expression percentage was associated with superficial and low grade tumors (Table 2a). In bladder cancer, Boorjian et al. reported that 75% of superficial tumors expressed the AR compared with 21.4% of invasive tumors, suggesting that the loss of AR expression is associated with invasive bladder cancer . Laor et al. also reported high-grade tumors had a lower AR content than low-grade tumors . Another study also pointed out that AR expression was significantly lower in T2-tumors than in Ta-tumors and T1-tumors . Our finding of the AR expression in UUTUCs of ureter was consistent with these results. The expression pattern of ERin
UUTUCs was similar to that in bladder cancer, showing that no significant relationship was found between ER expression levels in tumor grades or stages . However, others have demonstrated that ER expression was found higher in invasive high-grade bladder tumors . Therefore, the significance of ER expression in urothelial tumors (bladder or UUTUC) needs further clarified.
Sex steroid hormone receptors were also shown to have differential expression on UUTUC cells and primary cultured tumor cells (Fig. 1b). The expression profiles of receptors were different between tumor tissue sections from patients and cultured cells. In cultured cells, AR protein was barely detected, but ER reappeared after cell culture, suggesting the receptor expression level changes as the tumor microenvironment changes. Since the tumor tissues consist of different clones of cells with different receptor expression profiles, in primary culture, certain populations of cells expand in specific conditions. For example, human breast epithelial cells derived from reduction mammoplasty were shown to form type I estrogen receptor positive and type II estrogen receptor negative cells during the primary culture . The primary cultured tumor cells with AR receptor overexpression had lower responses to the antitumor drug, doxorubicin, but higher mobility (Fig. 2), indicating that the receptor expression status is a deciding factor to affect the efficacy of chemotherapeutic agents and tumor progression. The expression pattern of receptors on primary cultured tumor cells isolated from an individual may be a promising marker to evaluate the progression of UUTUCs. Moreover, primary cultured tumor cells isolated from a specific individual provide a good model to test the efficacy of chemotherapeutic agents for a specific individual to achieve the benefits of a personalized medicine.
The expression variations of receptors may be due to the developmental process of the urogenital system and de-differentiation of carcinogenesis, in which gene
expressions are turned on and off . The urothelium is derived primarily from the urogenital sinus during embryogenesis, which in males also gives rise to the prostate, regulated by AR in urogenital mesenchymal cells, but without functional AR, the urogenital epithelium develops into urothelium of UUT, bladder, and urethras, thus indicating the important role of AR in both normal development and carcinogenesis of urogenital organs including UUT, bladder, and urethra . Indeed, when bladder epithelium is induced by the urogenital sinus mesenchyme, the cells undergo prostatic differentiation possibly due to increased Nkx3.1 expression, which is androgen inducible . The role of ERs are not clearly defined, but the discovery of high expression of ER in recent studies suggests that ER may play a role in urothelium carcinogenesis .
The pathophysiologic significance of sex hormone receptor expression in UUTUCs may be linked to de-differentiation of neoplastic cells, which may alter local sex hormonal levels, resulting in the different expression levels of receptors in different individual tumors. Nevertheless, with the presence of AR and ER in UUTUC tumors, together with the decreased AR expression noted in high-grade and high-stage disease, AR and ER are potential players in the hormonal regulation during the development and progression of UUTUCs. Understanding the roles of sex hormone receptors in UUTUCs development may help to design a novel therapeutic approach combining hormonal therapy and chemotherapy to have improved survival and/or organ sparing and preservation of function for cancer patients.
In conclusion, comparing the expression of sex hormone receptors in UUTUC specimens, we found higher expression of AR, but not ERs, in superficial stages and low grades of UUTUCs of ureter. Furthermore, in primary cultured UUTUC cells, the overexpression of AR reduced cell chemosensitivity, but increased cell migration. These
findings suggest that AR may contribute to the UUTUCs tumor progression and the efficacy of treatment.
Acknowledgments. We thank Karen Wolf at University of Rochester for kindly editing the manuscript. This study was supported by the grant (DMR-101-053) from China Medical University Hospital, Taichung, Taiwan. This study was also supported in part by Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004).
Disclosures: None of the contributing authors have any conflict of interest, including specific financial interests or relationships and affiliations relevant to the subject matter or materials discussed in the manuscript.
References
1. Kakizoe, T.: Development and progression of urothelial carcinoma.
Cancer Sci. 97, 821-828 (2006)
2. Kirkali, Z., Tuzel, E.: Transitional cell carcinoma of the ureter and renal
pelvis. Critical Reviews in Oncology Hematology. 47, 155-169 (2003)
3. Oosterlinck, W., Solsona, E., van der Meijden, A.P., Sylvester, R., Bohle,
A., et al.: EAU guidelines on diagnosis and treatment of upper urinary
tract transitional cell carcinoma. Eur Urol. 46, 147-154 (2004)
4. Krogh, J., Kvist, E., Rye, B.: Transitional cell carcinoma of the upper
urinary tract: prognostic variables and post-operative recurrences. Br J
Urol. 67, 32-36 (1991)
5. Lehmann, J., Suttmann, H., Kovac, I., Hack, M., Kamradt, J., et al.:
Transitional cell carcinoma of the ureter: prognostic factors influencing
progression and survival. Eur Urol. 51, 1281-1288 (2007)
6. Sanderson, K.M., Cai, J., Miranda, G., Skinner, D.G., Stein, J.P.: Upper
cell carcinoma of the bladder: an analysis of 1,069 patients with
10-year followup. J Urol. 177, 2088-2094 (2007)
7. Hall, M.C., Womack, S., Sagalowsky, A.I., Carmody, T., Erickstad, M.D.,
et al.: Prognostic factors, recurrence, and survival in transitional cell
carcinoma of the upper urinary tract: a 30-year experience in 252
patients. Urology. 52, 594-601 (1998)
8. Kang, C.H., Yu, T.J., Hsieh, H.H., Yang, J.W., Shu, K., et al.: The
development of bladder tumors and contralateral upper urinary tract
tumors after primary transitional cell carcinoma of the upper urinary
tract. Cancer. 98, 1620-1626 (2003)
9. Zigeuner, R.E., Hutterer, G., Chromecki, T., Rehak, P., Langner, C.:
Bladder tumour development after urothelial carcinoma of the upper
urinary tract is related to primary tumour location. BJU Int. 98,
1181-1186 (2006)
10. Catto, J.W., Yates, D.R., Rehman, I., Azzouzi, A.R., Patterson, J., et al.:
Behavior of urothelial carcinoma with respect to anatomical location. J
11. Catto, J.W., Azzouzi, A.R., Amira, N., Rehman, I., Feeley, K.M., et al.:
Distinct patterns of microsatellite instability are seen in tumours of the
urinary tract. Oncogene. 22, 8699-8706 (2003)
12. Zigeuner, R., Pummer, K.: Urothelial carcinoma of the upper urinary
tract: surgical approach and prognostic factors. Eur Urol. 53, 720-731
(2008)
13. Munoz, J.J., Ellison, L.M.: Upper tract urothelial neoplasms: incidence
and survival during the last 2 decades. J Urol. 164, 1523-1525 (2000)
14. McGrath, M., Michaud, D.S., De Vivo, I.: Hormonal and reproductive
factors and the risk of bladder cancer in women. Am J Epidemiol. 163,
236-244 (2006)
15. Fajkovic, H., Halpern, J.A., Cha, E.K., Bahadori, A., Chromecki, T.F., et
al.: Impact of gender on bladder cancer incidence, staging, and
prognosis. World J Urol. 29, 457-463 (2011)
16. Mangelsdorf, D., Thummel, C., Beato, M., Herrlich, P., Schutz, G., et al.:
The nuclear receptor superfamily: the second decade. Cell. 83,
17. Pelletier, G.: Localization of androgen and estrogen receptors in rat
and primate tissues. Histol Histopathol. 15, 1261-1270 (2000)
18. Rau, K.M., Chen, Y.J., Sun, M.T., Kang, H.Y.: Prognostic effects and
regulation of activin A, maspin, and the androgen receptor in upper
urinary tract urothelial carcinoma. Anticancer Res. 31, 1713-1720
(2011)
19. Tuygun, C., Kankaya, D., Imamoglu, A., Sertcelik, A., Zengin, K., et al.:
Sex-specific hormone receptors in urothelial carcinomas of the human
urinary bladder: a comparative analysis of clinicopathological features
and survival outcomes according to receptor expression. Urol Oncol.
29, 43-51 (2011)
20. Boorjian, S., Ugras, S., Mongan, N.P., Gudas, L.J., You, X., et al.:
Androgen receptor expression is inversely correlated with pathologic
tumor stage in bladder cancer. Urology. 64, 383-388 (2004)
21. Mir, C., Shariat, S.F., van der Kwast, T.H., Ashfaq, R., Lotan, Y., et al.:
Loss of androgen receptor expression is not associated with
22. Makela, S., Strauss, L., Kuiper, G., Valve, E., Salmi, S., et al.:
Differential expression of estrogen receptors alpha and beta in adult
rat accessory sex glands and lower urinary tract. Mol Cell Endocrinol.
170, 219-229 (2000)
23. Buhler, P., Wolf, P., Katzenwadel, A., Schultze-Seemann, W.,
Wetterauer, U., et al.: Primary prostate cancer cultures are models for
androgen-independent transit amplifying cells. Oncol Rep. 23, 465-470
(2010)
24. Fridborg, H., Jonsson, E., Nygren, P., Larsson, R.: Relationship between
diagnosis-specific activity of cytotoxic drugs in fresh human tumour
cells ex vivo and in the clinic. Eur J Cancer. 35, 424-432 (1999)
25. Yang, M.H., Chen, K.K., Yen, C.C., Wang, W.S., Chang, Y.H., et al.:
Unusually high incidence of upper urinary tract urothelial carcinoma in
Taiwan. Urology. 59, 681-687 (2002)
26. Tzeng, C.C., Liu, H.S., Li, C., Jin, Y.T., Chen, R.M., et al.:
Characterization of two urothelium cancer cell lines derived from a
blackfoot disease endemic area in Taiwan. Anticancer Res. 16,
27. Reznikoff, C.A., Loretz, L.J., Christian, B.J., Wu, S.Q., Meisner, L.F.:
Neoplastic transformation of SV40-immortalized human urinary tract
epithelial cells by in vitro exposure to 3-methylcholanthrene.
Carcinogenesis. 9, 1427-1436 (1988)
28. Ackland, S.P., Ratain, M.J., Vogelzang, N.J., Choi, K.E., Ruane, M., et al.:
Pharmacokinetics and pharmacodynamics of long-term
continuous-infusion doxorubicin. Clin Pharmacol Ther. 45, 340-347 (1989)
29. Laor, E., Schiffman, Z.J., Braunstein, J.D., Reid, R.E., Tolia, B.M., et al.:
Androgen receptors in bladder tumors. Urology. 25, 161-163 (1985)
30. Shen, S.S., Smith, C.L., Hsieh, J.T., Yu, J., Kim, I.Y., et al.: Expression of
estrogen receptors-alpha and -beta in bladder cancer cell lines and
human bladder tumor tissue. Cancer. 106, 2610-2616 (2006)
31. Miyamoto, H., Yao, J.L., Chaux, A., Zheng, Y., Hsu, I., et al.: Expression
of androgen and oestrogen receptors and its prognostic significance in
urothelial neoplasm of the urinary bladder. BJU Int. 109, 1716-1726
32. Kang, K.S., Morita, I., Cruz, A., Jeon, Y.J., Trosko, J.E., et al.: Expression
of estrogen receptors in a normal human breast epithelial cell type
with luminal and stem cell characteristics and its neoplastically
transformed cell lines. Carcinogenesis. 18, 251-257 (1997)
33. Cunha, G.R., Ricke, W., Thomson, A., Marker, P.C., Risbridger, G., et al.:
Hormonal, cellular, and molecular regulation of normal and neoplastic
prostatic development. J Steroid Biochem Mol Biol. 92, 221-236 (2004)
34. Bhatia-Gaur, R., Donjacour, A.A., Sciavolino, P.J., Kim, M., Desai, N., et
al.: Roles for Nkx3.1 in prostate development and cancer. Genes Dev.
Figure Captions
Fig.1 (a) Immunohistochemical staining of AR, ERand ERproteins in UUTUC of renal pelvis and ureter. Magnification: 100x (upper panel) and 400x (lower panel). (b) Western blot analysis of AR, ER, and ERin UUTUC cells: BFTC 909, SV-HUC, primary cultured cells: #7630, #8808, and #7475. LNCaP cells (a prostate cancer cell line) were used as a reference. (c) Q-RT-PCR of AR mRNA expression in UUTUC cells.
Fig. 2 The effect of AR on the cytotoxic effect of doxorubicin in primary cultured UUTUC cells. Primary cultured UUTUC cells (#7630) were infected with pWPI or pWPI hAR by lentiviral system to overexpress AR. (a) Western blotting of AR expression. (b) The transcritonal activity of AR in 7630 cells infected with pWPI hAR. The cells were co-transfected with ARE-Luc and RL-TK plasmids and treated with 10 nM dihydrotestosterone (DHT). The luciferase activity of vehicle (ethanol) treatment cells was set as one. (c) The growth curve of 7630 cells infected with pWPI or pWPI hAR by lentiviral system. Cells were seeded into 96 well plates in the presence of absence of DHT (10nM) treatment for 8, 24 and 48h and measured the cell viability by colorimetric XTT assay. (d) The cytotoxic effect of doxorubicin on primary cultured tumor cells. After lentiviral infection, Primary cultured tumor cells: 7630-pWPI and 7630-pWPIhAR cells were treated with doxorubicin (Dox) (40uM) in the presence or absence of dihydrotestosterone (DHT) (10nM) for 24 h. Ethanol (Etoh) was used as vehicle control. The cell viability was determined by colorimetric XTT assay. The value from the cells without Dox treatment was set as 100 %. (e) Wound healing cell migration assay on primary cultured UUTUC cells (#7630) infected with pWPI or pWPI hAR by lentiviral system to overexpress AR. Relative cell migration is expressed as fold increase compared with control cells. Values represent mean ± SD of three separate experiments. * P<0.05 is considered significant, VS control 7630-pWPI cells
