Author's Accepted Manuscript Increased Expression of Androgen Receptor mRNA in Human Renal Cell Carcinoma Cells is Associated with Poor Prognosis in Patients with Localized Renal Cell Carcinoma Yun-Sok Ha , Geun Taek Lee , Parth Modi , Joseph Kwon , Hanjong Ahn , WunJae Kim , Isaac Yi Kim PII: DOI: Reference:
S0022-5347(15)03375-3 10.1016/j.juro.2015.03.078 JURO 12467
To appear in: The Journal of Urology Accepted Date: 11 March 2015 Please cite this article as: Ha YS, Lee GT, Modi P, Kwon J, Ahn H, Kim WJ, Kim IY, Increased Expression of Androgen Receptor mRNA in Human Renal Cell Carcinoma Cells is Associated with Poor Prognosis in Patients with Localized Renal Cell Carcinoma, The Journal of Urology® (2015), doi: 10.1016/j.juro.2015.03.078. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.
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Increased Expression of Androgen Receptor mRNA in Human Renal Cell Carcinoma Cells is Associated with Poor Prognosis in Patients with Localized Renal Cell
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Carcinoma
Yun-Sok Ha1, 2, Geun Taek Lee1, Parth Modi1, Joseph Kwon1, Hanjong Ahn3, Wun-Jae Kim4,
Section of Urologic Oncology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ
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1
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and Isaac Yi Kim1
08903, USA; 2Department of Urology, Kyungbuk National University, Daegu, Korea; 3
Department of Urology, Asan Medical Center, University of Ulsan College of Medicine,
Seoul, Korea; 4Department of Urology, Chungbuk National University College of Medicine,
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Cheongju, Korea.
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Address correspondence to:
Isaac Yi Kim, M.D., Ph.D.
Section of Urologic Oncology Rutgers Cancer Institute of New Jersey Rutgers, The State University of New Jersey 195 Little Albany Street 1
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New Brunswick, NJ 08903, USA Phone: 732 235 2043
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Fax: 732 235 6596
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E-mail: [email protected]
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Keywords: Renal cell carcinoma, Androgen receptor, Prognosis, Enzalutamide
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ABSTRACT PURPOSE: The role of AR in RCC is not well understood. In this study, the correlation
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between the AR mRNA expression and clinicopathologic features in patients with localized RCC was investigated. Additionally, human RCC cell lines were examined for the presence
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and effect of AR.
MATERIALS AND METHODS: AR mRNA expression was evaluated by qPCR in 115
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tumor samples from patients with primary pathologic stage T1 or T2 (pT1/pT2) RCC and 57 specimens of corresponding normal kidney tissue. RT-PCR and Western blot were used to examine the expression of AR in human RCC cell lines. The effects on cellular proliferation
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were investigated after activating and blocking androgen signaling in tissue culture. RESULTS: AR mRNA expression levels were significantly higher in patients with pT2
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tumors than in those with pT1 tumors (P = 0.011). Kaplan-Meier estimates revealed
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significant differences in the time to progression and cancer specific survival between lowand high-AR mRNA expression groups, regardless of gender. Multivariate Cox regression analysis demonstrated that the level of AR expression is an independent predictor of cancerspecific survival (HR, 15.546; 95% CI, 1.320–183.131; P = 0.029). In tissue culture, treatment with DHT caused proliferation in AR-positive cell lines while enzalutamide resulted in a reduction in cell viability in a dose-dependent manner. 3
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CONCLUSIONS: In patients with localized RCC, AR mRNA expression level is associated with prognosis. In addition, cell culture data suggests that enzalutamide may have an effect in
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limiting the growth of AR-positive RCC.
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Introduction RCC is the most frequent and lethal malignant tumor of the kidney in adults.1 Approximately
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20-30% of patients have metastatic disease at diagnosis, and 20-40% of the patients who undergo nephrectomy for clinically localized RCC relapse at distant sites.2 The prognosis for patients with mRCC is extremely poor.3 Although targeted therapies have revolutionized the
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treatment of mRCC, the lack of curative potential, the chronic nature of the treatment, and its
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side effects are clear limitations.4 Therefore, it is crucial to improve our understanding of RCC and attempt to identify new targets of intervention in patients with RCC. The AR mediates the effects of androgens in a variety of reproductive and non-
conditions.5,
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reproductive tissues in both males and females under physiological and pathophysiological The transcriptional activation function of AR is associated with human
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carcinogenesis, most notably prostate cancer.7 Previously, it has been reported that AR expression is found in 14.8–42% of RCCs.8, 9 Noh et al. recently reported that patients with
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clear cell RCC expressing AR had higher nuclear grade, shorter OS, RFS, and CSS.10 Contrary to these findings, however, Zhu et al. reported that AR inversely correlates with pathologic stage and Fuhrman's grade.11 Similarly, a study by Langner et al. revealed that AR expression predicted favorable prognosis in RCC.8 Collectively, these conflicting reports have not been able to definitively characterize the role of AR in RCC. 5
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Since previous studies on AR in RCC exclusively used immunohistochemistry, we investigated AR mRNA expression as a prognostic marker in tissue samples from patients
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with localized RCC. Additionally, the biological role of AR was studied in human RCC cell
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lines in vitro.
Patients and tumor samples
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Materials and Methods
After Institutional Review Board approval (IRB #2006-01-001), we obtained tissue samples from 115 patients treated with radical nephrectomy or partial nephrectomy for localized RCC
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between April 1996 and December 2010 at the Chungbuk National University Hospital, Cheongju, Korea. Matched normal-appearing kidney tissues were available in 57 patients.
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The samples were re-examined by a pathologist to confirm the presence of tumor or normal
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tissue. All tumors were macrodissected within 15 min of surgical resection, snap frozen in liquid nitrogen, and stored at –80 °C until use. Each cancer specimen was confirmed by analyzing adjacent tissue with fresh frozen sections. Staging was based on the American Joint Committee on Cancer classification12 and histological differentiation was graded according to the Fuhrman’s nuclear grading system.13 All patients were evaluated postoperatively every 3 months for the first 2 years, every 6 months for the next 2 years, and yearly thereafter. The 6
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definition of disease progression was local recurrence, lymph node metastasis, or distant metastasis by CT scan and/or bone scan.
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RNA Extraction and Construction of cDNA RNA was isolated from tissues with 1 ml of TRIzol (Invitrogen,Carlsbad, Calif., USA) using the manufacturer’s recommended protocol. The quality and integrity of the RNA were
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confirmed by agarose gel electrophoresis and ethidium bromide staining, followed by visual
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inspection under ultraviolet light. cDNA was prepared from 1 µg of total RNA using a FirstStrand cDNA Synthesis Kit (Amersham Biosciences Europe GmbH, Freiburg, Germany). Real-time PCR
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Real-time PCR amplification was performed using a Rotor Gene 6000 instrument (Corbett Research, Mortlake, Australia) to quantify the expression of AR. Real-time PCR assays were
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carried out in micro-reaction tubes (Corbett Research, Mortlake, Australia) using SYBR
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Premix EX Taq (TAKARA BIO INC., Otsu, Japan). All samples were run in triplicate. GAPDH was used as the endogenous RNA reference gene.
Cell culture and transfection
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LNCAP, A498, HT891.T, CAKI-1, and CAKI-2 cell lines were purchased from the American Type Collection Tissue Collection (Manassas, VA). For transient transfection, cells
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were grown to 80% confluence and Lipofectamine 2000 (Invitrogen) was used.
RT-PCR
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Total RNA was isolated from cells using TRIzol reagent (Invitrogen) and ImProm-II Reverse
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Transcription System (Promega, WI) was used for reverse transcription. The primers for AR are forward (5’- TGC TCC GCT GAC CTT AAA GA -3’) and reverse (5’- CTC TCG CCT TCT AGC CCT TT -3’) and for β-actin are forward (5′-GGCATCGTGATCGACTTC-3′) and
Western blot analysis
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reverse (5′-GCTGGAAGGTGGACAGCG-3′).
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Whole-cell lysates were prepared using lysis buffer (Cell Signaling) and were centrifuged at
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14,000 x g for 10 min. Fifty µg of protein was loaded onto SDS-polyacrylamide gel and transferred onto nitrocellulose membranes for immunoblotting analysis. To standardize protein levels, the blots were reprobed with β-actin antibody. Commercially available antibodies against AR (Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (SigmaAldrich) were used.
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Knock down of AR
Five shRNAs were obtained from Sigma-Aldrich and the level of expression of the target
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gene was measured using real-time PCR.
Cell proliferation assay
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For cell counts, cells were plated at 50,000 cells/well in 24-well culture and allowed to
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adhere for 24 h. Then the cells were transfected with AR expression constructs or treated by DHT or enzalutamide (Selleckchem, Houston, TX). Three days after treatment, cells were detached and counted using a hemocytometer.
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Statistics
A repeated ANOVA with post hoc tests using the Bonferroni correction and Kruskal-Wallis
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methods were used to calculate the statistical significances for in vitro cell proliferation. To normalize the highly skewed distribution of AR mRNA expression, the data were examined
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as the natural log and subsequently back transformed for the model results.14 Results were expressed as a geometric mean (anti-log CI).14 The paired t test was used to calculate the significance of differences between data from tumor tissues and matched normal tissues. To compare gene expression levels among the groups, the two-sample t test or ANOVA was performed. ROC curves were used to determine the optimal cutoff point for the mRNA level 9
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that yielded the highest combined sensitivity and specificity for progression (sensitivity, 63.6%; specificity 72.1%) and cancer specific survival (sensitivity, 66.7%; specificity 91.7%).
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Using these values, patients were classified into high or low AR expression groups. The Kaplan-Meier method was used to estimate the time to progression, and differences were assessed using log-rank statistics. The prognostic value of AR for CSS was analyzed using
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multivariate Cox proportional hazard regression models. Statistical analysis was performed
considered statistically significant.
Baseline characteristics
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Results
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using the SPSS 12.0 software (SPSS Inc., Chicago, IL), and a P value of < 0.05 was
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The mean age of the subjects was 57.4 years (range, 21–83 years). The mean postsurgical
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follow-up period was 58.2 months (range, 3.5–156.6 months). Males and females accounted for 86 (74.8%) and 29 (25.2%), respectively. Sixty-three patients (54.8%) received an open radical (N=59) or partial (N=4) nephrectomy. Laparoscopic surgery was performed in 52 patients (45.2%), including 49 patients treated with radical nephrectomy and 3 with partial nephrectomy. Histologic subtypes of the main component were clear cell in 98 (85.2%),
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papillary in 13 (11.3%), and chromophobe in 4 (3.5%) cases. Eleven patients underwent immune-chemotherapy or targeted therapy due to progression.
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Expression of AR in tumor and surrounding matched normal-appearing tissues
The AR mRNA expression in 57 RCC specimens was compared to that in matched normal-
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appearing tissues. AR mRNA expression was not significantly different between tumor tissue
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(0.240 x 103 copies/µl, 0.184–0.314) and normal tissue (0.257 x 103 copies/µl, 0.209–0.317) (p = 0.684) (Table 1 and Fig 1). Subgroup analyses stratified by stage and grade, showed no significant differences in AR mRNA expression between normal and tumor tissues (Fig 1). In contrast, AR mRNA levels were more frequently elevated in pT2 tumors when compared to
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those in pT1 tissues (Table 1).
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Western blot in human RCC and surrounding normal-appearing tissues
To correlate the expression levels of AR mRNA and protein in human RCC tissues, we
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performed a Western blot analysis (Fig 2A). The corresponding quantitative RT-PCR result is shown in Fig 2B. Compared to the human prostate cancer cell line LNCaP, RCC tissues expressed significantly lower levels of AR protein and mRNA. In kidney tissues, there were no significant differences in AR expression in malignant and matched normal tissues. However, the protein expression of AR in RCC was higher in T2 than T1 disease. 11
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Correlation of AR expression with clinical parameters in localized RCC
Table 1 summarizes the correlation between AR mRNA expression and clinicopathological
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parameters in patients with RCC. Pathologic stage T2 tumors showed significantly higher AR mRNA expression compared to T1 (P= 0.011). The expression levels of AR mRNA in
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localized RCC were significantly higher among patients who experienced progression and cancer specific deaths (P= 0.021 and P= 0.001, respectively). There was no significant
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difference in AR mRNA expression levels between males and females.
Prognostic value of the AR mRNA expression level for progression and cancer specific
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death
Kaplan-Meier estimates revealed that significant differences in the time to progression
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between low- and high-expression groups (cut off value, 0.229 x103 copies/µl) [log-rank test, P= 0.040; Fig. 3(A)]. When stratified by gender, AR expression was marginally associated
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with progression free survival in both genders. In terms of cancer specific death (cut off value, 0.776 x 103 copies/µl), the patients with higher AR expression had shorter cancer-specific survival than those with lower AR expression [log-rank test, P= 0.040; Fig. 3(B)]. These significant findings were also found in male and female patients by subgroup analyses.
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Multivariate Cox regression analysis revealed that the level of AR expression (HR, 15.546; 95% CI, 1.320-183.131; P = 0.029) was an independent predictor of progression (Table 2).
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DHT effect on proliferation of human RCC cell lines
To determine the effect of DHT, cell numbers were determined after treating human RCC
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cell lines with increasing concentrations of DHT for 3 days. The results shown in Fig. 4(A)
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reveal that DHT enhanced the proliferation of CAKI2 cells in a dose-dependent manner. The human prostate cancer cell line LNCAP was used as a positive control.
Expression of AR in RCC cell lines
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To investigate whether AR is expressed in RCC cell lines, AR mRNA and protein expression were assessed. RT-PCR analyses demonstrated AR mRNA in CAKI-2 and HS891.T cells
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[Fig 4(B)]. Subsequently, Western blot confirmed AR protein expression in CAKI-2. Although the 110 kDa AR protein was not detected in HS891.T, a slightly lower molecular
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weight protein band was present. Collectively, these results suggest that HS891.T cells express an AR splice variant.
Overexpression of AR enhances RCC cell proliferation
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As A498 and CAKI-1 had undetectable AR expression [Fig 4(B)], these two cell lines were selected for overexpression experiments. Following transfection with AR, proliferation of
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both A498 and CAKI-1 significantly increased with DHT treatment in a concentrationdependent manner (Fig 5).
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Effect of anti-androgens
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Because treatment with DHT enhanced the growth potential of RCC in AR positive cell lines, the effect of the antiandrogen MDV3100 (enzalutamide) was investigated (Fig 6). Cell proliferation was significantly suppressed by MDV3100 in CAKI-2 cells in a concentrationdependent manner. Interestingly, MDV3100 also blocked the proliferation of AR-negative
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HT891.T, suggesting that the therapeutic effect of MDV3100 involves both AR-dependent and –independent pathways.
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AR knockdown attenuates the effect of MDV3100 in CAKI-2 cells
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Given the results showing that MDV3100 blocked the proliferation of CAKI-2 and HS891.T, we performed similar experiments after knocking down AR with shRNAs. The AR shRNA transfected cells showed an mRNA expression of AR that was weaker than control cells in CAKI2 [Fig 7(A)] and HS891.T [Fig 7(B)]. When CAKI2 and HS891.T were transfected with AR shRNA, MDV3100’s inhibitory effect was no longer observed. 14
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Discussion
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In the present study, we have demonstrated that the high AR mRNA level correlates with poor prognosis in RCC regardless of sex. In tissue culture, DHT stimulated the proliferation of AR-positive cells in a concentration dependent manner. More importantly,
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the antiandrogen MDV3100 inhibited the proliferation of some human RCC cell lines. Taken
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together, the current findings confirm the importance of AR signaling pathway during RCC progression and the potential therapeutic role of AR antagonists in RCC. The “kidney” nature of RCC is characterized by a well-developed secretory apparatus
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with high expression of membrane proteins specialized for elimination of xenobiotics.15 This attribute appears to explain the resistance of RCC to chemotherapeutic agents.16 Due to the
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gender difference in tumor incidence,17 many investigators have been interested in hormonal receptors in RCC.8, 11, 18, 19 However, previous research examining the significance of AR in
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RCC has been contradictory, and consistent findings have not been reported.
The largest study performing immunohistochemical staining of AR was reported
by Langner et al. in 2004.8 In this study, AR expression was detected in 27 of 182 (15%) of RCC cases and was significantly associated with lower pathological stage and grade as well as improved progression free survival. Zhu et al. also used IHC in tissue samples from 136 15
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associated with lower pathological stage and Fuhrman grade. Interestingly, Zhu et al also investigated the transactivation function of AR in two cell lines and found that AR, while present, had little function. In contrast with these reports, a recent study by Noh et al.
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demonstrated worse OS with AR positive clear cell RCC.10 The present study also confirms
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these findings as increased AR mRNA was associated with higher stage tumors and shorter cancer-specific survival. The reason for the disparity in the results of these studies is not known, but may be associated with methodological differences. Immunohistochemical
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staining is a semi-quantitative method and can be influenced by the observer’s subjective view. However, qPCR analysis, such as those used in the present study, are quantitative
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methods with improved accuracy and sensitivity. The mechanism of AR involvement in carcinogenesis is poorly understood.
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Studies have suggested that AR inhibits the tumor suppressor P53 in hepatocelluar carcinoma and activates G protein signaling cascades in ovarian cancer.20, 21 Further evidence for a role of AR in other malignancies has been reported: AR expression is decreased after chemotherapy in ovarian cancer and increased AR expression has been associated with decreased responsiveness and increased migration of tumor cells in upper urinary tract 16
ACCEPTED MANUSCRIPT urothelial cancer.22, 23 However, the exact role of AR in RCC is still unclear. Further research that can identify the distinctive pathways of AR in RCC cells is needed.
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Hormonal therapy has been studied as a therapy for renal malignancy since the 1940s.24 Despite the poor response rates, flutamide, progestin, medroxyprogesterone, megesterol, and tamoxifen have been used as primary or adjuvant treatment modalities in
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selected patients by some investigators.18, 25-28 Enzalutamide (MDV3100, XTANDI®) is a
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novel AR antagonist that overcomes resistance to conventional anti-androgens by inhibiting nuclear localization and chromatin binding of AR.29 Our data demonstrates that enzalutamide decreases cell proliferation rates in some RCC cell lines. Whether enzalutamide is more
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effective in RCC than previous generation of AR antagonists remains to be investigated via a
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It should be noted that enzalutamide also inhibited the proliferation of HS891.T cells that lack the full length AR protein. Although the precise mechanism for this
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observation remains unclear, it is likely that HS891.T may be expressing alternative forms of AR. Consistent with this concept, a lower molecular weight protein band was detected on Western blot and knockdown of AR reversed the inhibitory effect of HS891.T. Indeed, Lu et al. recently demonstrated that AR splice variants can bind to DNA independent of full-length AR in the absence of androgen and modulate a unique set of genes that is not regulated by 17
ACCEPTED MANUSCRIPT full-length AR.30 Notwithstanding, it is entirely possible that enzalutamide has a cytotoxic effect that is independent of AR. Additional studies are underway to verify this concept.
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Despite the clinical implications, the limitation of the present study is that Fuhrman grade did not predict cancer-specific death. This observation likely reflects the relatively restricted distribution of our patient population. Specifically, we only analyzed patients with
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T1 and T2 disease. Therefore the number of Fuhrman grade 4 was very small (N= 4). Given
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this, the precise role of AR in RCC patients with higher grade and stage remains uncertain.
Conclusions
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The present findings suggest the important role of AR in RCC. More importantly, tissue culture studies have demonstrated that the AR signaling pathway can be exploited for therapy
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in RCC patients. Future translational investigation will include in vivo studies to confirm the
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efficacy of AR blockade in mice bearing RCC cells with or without AR expression.
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ACKNOWLEGMENTS
This work is supported by a grant from the National Cancer Institute (P30CA072720). This
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work is also supported in part by generous grants from the Tanzman Foundation and by Basic Science Research Program through the National Research Foundation of Korea (NRF)
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funded by the Ministry of Education Science and Technology (2008-0062611) and supported by a grant from the Next-Generation BioGreen 21 Program (No.PJ009621), Rural
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Development Administration, Republic of Korea.
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Figure legends
Fig 1. AR expression in 57 RCC specimens was compared to that in matched normal-
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appearing tissues. There were no significant differences in AR expression overall (A), T1 patients (B), T2 patients (C), patients with Fuhrman grade 1-2 (D) and those with Fuhrman
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grade 3-4 (E).
Fig 2. Representative Western blot (A) and quantitative RT-PCR (B) of AR in RCC and
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normal kidney tissues. Western blot analysis of protein expression was used to confirm the gene expression data obtained by qPCR. Lane 1, LNCaP; Lane 2, normal tissue of T1 patient; Lane 3, tumor of T1 patient; Lane 4, normal tissue of T2 patient; Lane 5, tumor of T2 patient.
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Fig 3. Kaplan-Meier curves estimates the progression (A) and cancer specific death (B) according to AR mRNA expression levels.
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Fig 4. Androgens and human RCC cell lines. (A) Effect of DHT on human RCC cells. DHT
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increased cellular proliferation of CAKI2 in a concentration-dependent manner. LNCAP was used as a positive control. (B) AR mRNA (RT-PCR) and protein expression were detected only in CAKI2. In HS891.T, AR mRNA but not full length AR protein was detectable. Rather, a lower molecular weight protein was observed in HS891.T, suggesting the presence of an AR splice variant. Again, LNCAP was used as a positive control. Fig 5. Overexpression of AR. When AR was transfected into AR-negative A498 and CAKI1 25
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cells, DHT increased cellular proliferation in a concentration-dependent manner. Fig 6. Enzalutamide and cellular proliferation. There were significant decreases in cellular
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proliferation rates in a concentration-dependent manner in CAKI2 (AR-positive) and HS891T (AR-negative). Enzalutamide had no obvious effect on A498 and CAKI1 cells.
Fig 7. AR knockdown using shRNA. Transfection with shRNA knocked down AR mRNA
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expression significantly in both CAKI2 (A) and HS891.T (B). When AR was knocked down,
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no significant effect of MDV3100 was seen in CAKI2 and HS891.T cells.
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Table 1. AR mRNA expression stratified by clinicopathological parameters mRNA expression of AR (
103 P
copies/µl)
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Parameters (N)
0.684a
Paired tissue samples Donor-matched adjacent
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0.257 (0.209–0.317)
Tumor tissue (57) Gender
Female (29)
0.240 (0.184–0.314)
0.245 (0.183–0.329)
Pathologic T stage
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pT1 (94)
0.737 b
0.262 (0.213–0.322)
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Male (86)
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normal tissue (57)
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pT2 (21)
0.011 b
0.220 (0.191–0.253) 0.523 (0.276–0.992) 0.135C
Nuclear grade 1 (26)
0.193 (0.148–0.253)
2 (52)
0.296 (0.226–0.387)
3 (33)
0.241 (0.172–0.340)
4 (4)
0.464 (0.089–2.420) 1
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0.021 b
Progression 0.242 (0.204–0.286)
Yes (11)
0.471 (0.218–1.015)
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No (104)
0.001 b
Cancer specific death
Yes (6)
0.471 (0.218–1.015)
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0.242 (0.204–0.286)
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Paired t test; b2-samples t test; cANOVA; AR, androgen receptor
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a
No (109)
2
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Table 2. Cox regression analysis for Cancer specific death
Variables HR (95% CI)
Mutivariate analysis
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Univariate analysis P
HR (95% CI)
P
0.906
1.112 (0.125-9.901)
0.924
0.755
0.932 (0.046-18.856)
0.963
1.004 (0.934-1.080)
Sex (male vs. female)
0.705 (0.079-6.335)
T Stage (T1 vs. T2)
14.352 (1.586-129.863)
0.018
5.684 (0.441-73317)
0.183
Fuhrman grade (1-2 vs. 3-4)
1.445 (0.239-8.728)
0.688
0.850 (0.105-6.915)
0.879
AR expression (low vs. high)
26.529 (2.885-243-983)
0.004
15.546 (1.320-183.131)
0.029
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Age
1
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Fig. 1 (C)
(D)
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(B)
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(A)
(E)
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(A)
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Fig. 2
Full length AR (110 kd)
3
4
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2
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10
5
1
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Level (Natural Log)
(B)
Relative AR mRNA
LNCaP
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β-actin
0
1
2
3
4
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Total
Male
Female
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Male
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Total
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(B)
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Fig. 3 (A)
Female
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Fig. 4 control
50
A498
HS891.T
CAKI1
CAKI2
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LNCAP
β-actin
10n
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100
AR mRNA
1n
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150
0
(B)
0.1n
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Cell growth (%)
(A)
AR Protein β-actin
LNCAP A498 HS891.T CAKI1 CAKI2
Wild type Splice variant
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Fig. 5
180
P< 0.001
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P< 0.001
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140 120 100
60
20 0
2 days 4 days 6 days A498
AR_DHT1N AR_DHT10N
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40
PCDNA3_DHT1N PCDNA3_DHT10N
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80
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Proliferation rate (%)
160
2 days 4 days 6 days CAKI1
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MDV_1uM
140 P= 0.020
100
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40 20 0
P= 0.004
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60
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Cell Count (% control)
120
80
P= 0.095
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P= 0.561
MDV_25uM
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Control
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Fig. 6
A498
HT891.T
CAKI1
CAKI2
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Fig. 7 (A) Caki2
100
120 100 80 60 40 20 0
60 40
sh-AR
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Con
HS891.T
MDV_25uM
Control
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150
50 0 Con
sh-AR
sh-AR
MDV_1uM
MDV_25uM Cell counts (%
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100
Scramble
HS891.T
200 control)
20
(B)
MDV_1uM
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80
0
AR mRNA
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Control
Cell counts (% control)
120
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AR mRNA expression (%…
Caki2
100 0 Con
sh-AR
ACCEPTED MANUSCRIPT Abbreviations RCC, Renal cell carcinoma mRCC, metastatic RCC
RFS, recurrence-free survival CSS, cancer specific survival GAPDH, Glyceraldehyde-3-phosphate dehydrogenase
qPCR, quantitative real-time PCR DHT, dihydrotestosterone CI, confidence interval HR, hazard ratio
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RT-PCR, Reverse transcriptase PCR
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OS, overall survival
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AR, androgen receptor
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ROC, Receiver operating characteristics
1
S0022-5347(15)03375-3 10.1016/j.juro.2015.03.078 JURO 12467
To appear in: The Journal of Urology Accepted Date: 11 March 2015 Please cite this article as: Ha YS, Lee GT, Modi P, Kwon J, Ahn H, Kim WJ, Kim IY, Increased Expression of Androgen Receptor mRNA in Human Renal Cell Carcinoma Cells is Associated with Poor Prognosis in Patients with Localized Renal Cell Carcinoma, The Journal of Urology® (2015), doi: 10.1016/j.juro.2015.03.078. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.
Embargo Policy All article content is under embargo until uncorrected proof of the article becomes available online. We will provide journalists and editors with full-text copies of the articles in question prior to the embargo date so that stories can be adequately researched and written. The standard embargo time is 12:01 AM ET on that date. Questions regarding embargo should be directed to [email protected].
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Increased Expression of Androgen Receptor mRNA in Human Renal Cell Carcinoma Cells is Associated with Poor Prognosis in Patients with Localized Renal Cell
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Carcinoma
Yun-Sok Ha1, 2, Geun Taek Lee1, Parth Modi1, Joseph Kwon1, Hanjong Ahn3, Wun-Jae Kim4,
Section of Urologic Oncology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ
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1
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and Isaac Yi Kim1
08903, USA; 2Department of Urology, Kyungbuk National University, Daegu, Korea; 3
Department of Urology, Asan Medical Center, University of Ulsan College of Medicine,
Seoul, Korea; 4Department of Urology, Chungbuk National University College of Medicine,
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Cheongju, Korea.
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Address correspondence to:
Isaac Yi Kim, M.D., Ph.D.
Section of Urologic Oncology Rutgers Cancer Institute of New Jersey Rutgers, The State University of New Jersey 195 Little Albany Street 1
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New Brunswick, NJ 08903, USA Phone: 732 235 2043
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Fax: 732 235 6596
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E-mail: [email protected]
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Keywords: Renal cell carcinoma, Androgen receptor, Prognosis, Enzalutamide
2
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ABSTRACT PURPOSE: The role of AR in RCC is not well understood. In this study, the correlation
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between the AR mRNA expression and clinicopathologic features in patients with localized RCC was investigated. Additionally, human RCC cell lines were examined for the presence
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and effect of AR.
MATERIALS AND METHODS: AR mRNA expression was evaluated by qPCR in 115
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tumor samples from patients with primary pathologic stage T1 or T2 (pT1/pT2) RCC and 57 specimens of corresponding normal kidney tissue. RT-PCR and Western blot were used to examine the expression of AR in human RCC cell lines. The effects on cellular proliferation
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were investigated after activating and blocking androgen signaling in tissue culture. RESULTS: AR mRNA expression levels were significantly higher in patients with pT2
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tumors than in those with pT1 tumors (P = 0.011). Kaplan-Meier estimates revealed
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significant differences in the time to progression and cancer specific survival between lowand high-AR mRNA expression groups, regardless of gender. Multivariate Cox regression analysis demonstrated that the level of AR expression is an independent predictor of cancerspecific survival (HR, 15.546; 95% CI, 1.320–183.131; P = 0.029). In tissue culture, treatment with DHT caused proliferation in AR-positive cell lines while enzalutamide resulted in a reduction in cell viability in a dose-dependent manner. 3
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CONCLUSIONS: In patients with localized RCC, AR mRNA expression level is associated with prognosis. In addition, cell culture data suggests that enzalutamide may have an effect in
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limiting the growth of AR-positive RCC.
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Introduction RCC is the most frequent and lethal malignant tumor of the kidney in adults.1 Approximately
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20-30% of patients have metastatic disease at diagnosis, and 20-40% of the patients who undergo nephrectomy for clinically localized RCC relapse at distant sites.2 The prognosis for patients with mRCC is extremely poor.3 Although targeted therapies have revolutionized the
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treatment of mRCC, the lack of curative potential, the chronic nature of the treatment, and its
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side effects are clear limitations.4 Therefore, it is crucial to improve our understanding of RCC and attempt to identify new targets of intervention in patients with RCC. The AR mediates the effects of androgens in a variety of reproductive and non-
conditions.5,
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reproductive tissues in both males and females under physiological and pathophysiological The transcriptional activation function of AR is associated with human
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carcinogenesis, most notably prostate cancer.7 Previously, it has been reported that AR expression is found in 14.8–42% of RCCs.8, 9 Noh et al. recently reported that patients with
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clear cell RCC expressing AR had higher nuclear grade, shorter OS, RFS, and CSS.10 Contrary to these findings, however, Zhu et al. reported that AR inversely correlates with pathologic stage and Fuhrman's grade.11 Similarly, a study by Langner et al. revealed that AR expression predicted favorable prognosis in RCC.8 Collectively, these conflicting reports have not been able to definitively characterize the role of AR in RCC. 5
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Since previous studies on AR in RCC exclusively used immunohistochemistry, we investigated AR mRNA expression as a prognostic marker in tissue samples from patients
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with localized RCC. Additionally, the biological role of AR was studied in human RCC cell
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lines in vitro.
Patients and tumor samples
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Materials and Methods
After Institutional Review Board approval (IRB #2006-01-001), we obtained tissue samples from 115 patients treated with radical nephrectomy or partial nephrectomy for localized RCC
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between April 1996 and December 2010 at the Chungbuk National University Hospital, Cheongju, Korea. Matched normal-appearing kidney tissues were available in 57 patients.
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The samples were re-examined by a pathologist to confirm the presence of tumor or normal
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tissue. All tumors were macrodissected within 15 min of surgical resection, snap frozen in liquid nitrogen, and stored at –80 °C until use. Each cancer specimen was confirmed by analyzing adjacent tissue with fresh frozen sections. Staging was based on the American Joint Committee on Cancer classification12 and histological differentiation was graded according to the Fuhrman’s nuclear grading system.13 All patients were evaluated postoperatively every 3 months for the first 2 years, every 6 months for the next 2 years, and yearly thereafter. The 6
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definition of disease progression was local recurrence, lymph node metastasis, or distant metastasis by CT scan and/or bone scan.
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RNA Extraction and Construction of cDNA RNA was isolated from tissues with 1 ml of TRIzol (Invitrogen,Carlsbad, Calif., USA) using the manufacturer’s recommended protocol. The quality and integrity of the RNA were
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confirmed by agarose gel electrophoresis and ethidium bromide staining, followed by visual
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inspection under ultraviolet light. cDNA was prepared from 1 µg of total RNA using a FirstStrand cDNA Synthesis Kit (Amersham Biosciences Europe GmbH, Freiburg, Germany). Real-time PCR
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Real-time PCR amplification was performed using a Rotor Gene 6000 instrument (Corbett Research, Mortlake, Australia) to quantify the expression of AR. Real-time PCR assays were
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carried out in micro-reaction tubes (Corbett Research, Mortlake, Australia) using SYBR
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Premix EX Taq (TAKARA BIO INC., Otsu, Japan). All samples were run in triplicate. GAPDH was used as the endogenous RNA reference gene.
Cell culture and transfection
7
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LNCAP, A498, HT891.T, CAKI-1, and CAKI-2 cell lines were purchased from the American Type Collection Tissue Collection (Manassas, VA). For transient transfection, cells
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were grown to 80% confluence and Lipofectamine 2000 (Invitrogen) was used.
RT-PCR
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Total RNA was isolated from cells using TRIzol reagent (Invitrogen) and ImProm-II Reverse
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Transcription System (Promega, WI) was used for reverse transcription. The primers for AR are forward (5’- TGC TCC GCT GAC CTT AAA GA -3’) and reverse (5’- CTC TCG CCT TCT AGC CCT TT -3’) and for β-actin are forward (5′-GGCATCGTGATCGACTTC-3′) and
Western blot analysis
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reverse (5′-GCTGGAAGGTGGACAGCG-3′).
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Whole-cell lysates were prepared using lysis buffer (Cell Signaling) and were centrifuged at
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14,000 x g for 10 min. Fifty µg of protein was loaded onto SDS-polyacrylamide gel and transferred onto nitrocellulose membranes for immunoblotting analysis. To standardize protein levels, the blots were reprobed with β-actin antibody. Commercially available antibodies against AR (Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (SigmaAldrich) were used.
8
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Knock down of AR
Five shRNAs were obtained from Sigma-Aldrich and the level of expression of the target
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gene was measured using real-time PCR.
Cell proliferation assay
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For cell counts, cells were plated at 50,000 cells/well in 24-well culture and allowed to
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adhere for 24 h. Then the cells were transfected with AR expression constructs or treated by DHT or enzalutamide (Selleckchem, Houston, TX). Three days after treatment, cells were detached and counted using a hemocytometer.
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Statistics
A repeated ANOVA with post hoc tests using the Bonferroni correction and Kruskal-Wallis
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methods were used to calculate the statistical significances for in vitro cell proliferation. To normalize the highly skewed distribution of AR mRNA expression, the data were examined
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as the natural log and subsequently back transformed for the model results.14 Results were expressed as a geometric mean (anti-log CI).14 The paired t test was used to calculate the significance of differences between data from tumor tissues and matched normal tissues. To compare gene expression levels among the groups, the two-sample t test or ANOVA was performed. ROC curves were used to determine the optimal cutoff point for the mRNA level 9
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that yielded the highest combined sensitivity and specificity for progression (sensitivity, 63.6%; specificity 72.1%) and cancer specific survival (sensitivity, 66.7%; specificity 91.7%).
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Using these values, patients were classified into high or low AR expression groups. The Kaplan-Meier method was used to estimate the time to progression, and differences were assessed using log-rank statistics. The prognostic value of AR for CSS was analyzed using
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multivariate Cox proportional hazard regression models. Statistical analysis was performed
considered statistically significant.
Baseline characteristics
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Results
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using the SPSS 12.0 software (SPSS Inc., Chicago, IL), and a P value of < 0.05 was
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The mean age of the subjects was 57.4 years (range, 21–83 years). The mean postsurgical
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follow-up period was 58.2 months (range, 3.5–156.6 months). Males and females accounted for 86 (74.8%) and 29 (25.2%), respectively. Sixty-three patients (54.8%) received an open radical (N=59) or partial (N=4) nephrectomy. Laparoscopic surgery was performed in 52 patients (45.2%), including 49 patients treated with radical nephrectomy and 3 with partial nephrectomy. Histologic subtypes of the main component were clear cell in 98 (85.2%),
10
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papillary in 13 (11.3%), and chromophobe in 4 (3.5%) cases. Eleven patients underwent immune-chemotherapy or targeted therapy due to progression.
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Expression of AR in tumor and surrounding matched normal-appearing tissues
The AR mRNA expression in 57 RCC specimens was compared to that in matched normal-
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appearing tissues. AR mRNA expression was not significantly different between tumor tissue
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(0.240 x 103 copies/µl, 0.184–0.314) and normal tissue (0.257 x 103 copies/µl, 0.209–0.317) (p = 0.684) (Table 1 and Fig 1). Subgroup analyses stratified by stage and grade, showed no significant differences in AR mRNA expression between normal and tumor tissues (Fig 1). In contrast, AR mRNA levels were more frequently elevated in pT2 tumors when compared to
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those in pT1 tissues (Table 1).
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Western blot in human RCC and surrounding normal-appearing tissues
To correlate the expression levels of AR mRNA and protein in human RCC tissues, we
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performed a Western blot analysis (Fig 2A). The corresponding quantitative RT-PCR result is shown in Fig 2B. Compared to the human prostate cancer cell line LNCaP, RCC tissues expressed significantly lower levels of AR protein and mRNA. In kidney tissues, there were no significant differences in AR expression in malignant and matched normal tissues. However, the protein expression of AR in RCC was higher in T2 than T1 disease. 11
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Correlation of AR expression with clinical parameters in localized RCC
Table 1 summarizes the correlation between AR mRNA expression and clinicopathological
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parameters in patients with RCC. Pathologic stage T2 tumors showed significantly higher AR mRNA expression compared to T1 (P= 0.011). The expression levels of AR mRNA in
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localized RCC were significantly higher among patients who experienced progression and cancer specific deaths (P= 0.021 and P= 0.001, respectively). There was no significant
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difference in AR mRNA expression levels between males and females.
Prognostic value of the AR mRNA expression level for progression and cancer specific
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death
Kaplan-Meier estimates revealed that significant differences in the time to progression
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between low- and high-expression groups (cut off value, 0.229 x103 copies/µl) [log-rank test, P= 0.040; Fig. 3(A)]. When stratified by gender, AR expression was marginally associated
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with progression free survival in both genders. In terms of cancer specific death (cut off value, 0.776 x 103 copies/µl), the patients with higher AR expression had shorter cancer-specific survival than those with lower AR expression [log-rank test, P= 0.040; Fig. 3(B)]. These significant findings were also found in male and female patients by subgroup analyses.
12
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Multivariate Cox regression analysis revealed that the level of AR expression (HR, 15.546; 95% CI, 1.320-183.131; P = 0.029) was an independent predictor of progression (Table 2).
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DHT effect on proliferation of human RCC cell lines
To determine the effect of DHT, cell numbers were determined after treating human RCC
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cell lines with increasing concentrations of DHT for 3 days. The results shown in Fig. 4(A)
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reveal that DHT enhanced the proliferation of CAKI2 cells in a dose-dependent manner. The human prostate cancer cell line LNCAP was used as a positive control.
Expression of AR in RCC cell lines
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To investigate whether AR is expressed in RCC cell lines, AR mRNA and protein expression were assessed. RT-PCR analyses demonstrated AR mRNA in CAKI-2 and HS891.T cells
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[Fig 4(B)]. Subsequently, Western blot confirmed AR protein expression in CAKI-2. Although the 110 kDa AR protein was not detected in HS891.T, a slightly lower molecular
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weight protein band was present. Collectively, these results suggest that HS891.T cells express an AR splice variant.
Overexpression of AR enhances RCC cell proliferation
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As A498 and CAKI-1 had undetectable AR expression [Fig 4(B)], these two cell lines were selected for overexpression experiments. Following transfection with AR, proliferation of
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both A498 and CAKI-1 significantly increased with DHT treatment in a concentrationdependent manner (Fig 5).
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Effect of anti-androgens
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Because treatment with DHT enhanced the growth potential of RCC in AR positive cell lines, the effect of the antiandrogen MDV3100 (enzalutamide) was investigated (Fig 6). Cell proliferation was significantly suppressed by MDV3100 in CAKI-2 cells in a concentrationdependent manner. Interestingly, MDV3100 also blocked the proliferation of AR-negative
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HT891.T, suggesting that the therapeutic effect of MDV3100 involves both AR-dependent and –independent pathways.
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AR knockdown attenuates the effect of MDV3100 in CAKI-2 cells
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Given the results showing that MDV3100 blocked the proliferation of CAKI-2 and HS891.T, we performed similar experiments after knocking down AR with shRNAs. The AR shRNA transfected cells showed an mRNA expression of AR that was weaker than control cells in CAKI2 [Fig 7(A)] and HS891.T [Fig 7(B)]. When CAKI2 and HS891.T were transfected with AR shRNA, MDV3100’s inhibitory effect was no longer observed. 14
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Discussion
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In the present study, we have demonstrated that the high AR mRNA level correlates with poor prognosis in RCC regardless of sex. In tissue culture, DHT stimulated the proliferation of AR-positive cells in a concentration dependent manner. More importantly,
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the antiandrogen MDV3100 inhibited the proliferation of some human RCC cell lines. Taken
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together, the current findings confirm the importance of AR signaling pathway during RCC progression and the potential therapeutic role of AR antagonists in RCC. The “kidney” nature of RCC is characterized by a well-developed secretory apparatus
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with high expression of membrane proteins specialized for elimination of xenobiotics.15 This attribute appears to explain the resistance of RCC to chemotherapeutic agents.16 Due to the
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gender difference in tumor incidence,17 many investigators have been interested in hormonal receptors in RCC.8, 11, 18, 19 However, previous research examining the significance of AR in
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RCC has been contradictory, and consistent findings have not been reported.
The largest study performing immunohistochemical staining of AR was reported
by Langner et al. in 2004.8 In this study, AR expression was detected in 27 of 182 (15%) of RCC cases and was significantly associated with lower pathological stage and grade as well as improved progression free survival. Zhu et al. also used IHC in tissue samples from 136 15
ACCEPTED MANUSCRIPT patients with RCC.11 They demonstrated that the expression of AR was more common in normal kidney than in RCC tissue. Furthermore, in their study, the presence of AR was
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associated with lower pathological stage and Fuhrman grade. Interestingly, Zhu et al also investigated the transactivation function of AR in two cell lines and found that AR, while present, had little function. In contrast with these reports, a recent study by Noh et al.
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demonstrated worse OS with AR positive clear cell RCC.10 The present study also confirms
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these findings as increased AR mRNA was associated with higher stage tumors and shorter cancer-specific survival. The reason for the disparity in the results of these studies is not known, but may be associated with methodological differences. Immunohistochemical
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staining is a semi-quantitative method and can be influenced by the observer’s subjective view. However, qPCR analysis, such as those used in the present study, are quantitative
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methods with improved accuracy and sensitivity. The mechanism of AR involvement in carcinogenesis is poorly understood.
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Studies have suggested that AR inhibits the tumor suppressor P53 in hepatocelluar carcinoma and activates G protein signaling cascades in ovarian cancer.20, 21 Further evidence for a role of AR in other malignancies has been reported: AR expression is decreased after chemotherapy in ovarian cancer and increased AR expression has been associated with decreased responsiveness and increased migration of tumor cells in upper urinary tract 16
ACCEPTED MANUSCRIPT urothelial cancer.22, 23 However, the exact role of AR in RCC is still unclear. Further research that can identify the distinctive pathways of AR in RCC cells is needed.
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Hormonal therapy has been studied as a therapy for renal malignancy since the 1940s.24 Despite the poor response rates, flutamide, progestin, medroxyprogesterone, megesterol, and tamoxifen have been used as primary or adjuvant treatment modalities in
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selected patients by some investigators.18, 25-28 Enzalutamide (MDV3100, XTANDI®) is a
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novel AR antagonist that overcomes resistance to conventional anti-androgens by inhibiting nuclear localization and chromatin binding of AR.29 Our data demonstrates that enzalutamide decreases cell proliferation rates in some RCC cell lines. Whether enzalutamide is more
clinical trial.
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effective in RCC than previous generation of AR antagonists remains to be investigated via a
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It should be noted that enzalutamide also inhibited the proliferation of HS891.T cells that lack the full length AR protein. Although the precise mechanism for this
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observation remains unclear, it is likely that HS891.T may be expressing alternative forms of AR. Consistent with this concept, a lower molecular weight protein band was detected on Western blot and knockdown of AR reversed the inhibitory effect of HS891.T. Indeed, Lu et al. recently demonstrated that AR splice variants can bind to DNA independent of full-length AR in the absence of androgen and modulate a unique set of genes that is not regulated by 17
ACCEPTED MANUSCRIPT full-length AR.30 Notwithstanding, it is entirely possible that enzalutamide has a cytotoxic effect that is independent of AR. Additional studies are underway to verify this concept.
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Despite the clinical implications, the limitation of the present study is that Fuhrman grade did not predict cancer-specific death. This observation likely reflects the relatively restricted distribution of our patient population. Specifically, we only analyzed patients with
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T1 and T2 disease. Therefore the number of Fuhrman grade 4 was very small (N= 4). Given
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this, the precise role of AR in RCC patients with higher grade and stage remains uncertain.
Conclusions
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The present findings suggest the important role of AR in RCC. More importantly, tissue culture studies have demonstrated that the AR signaling pathway can be exploited for therapy
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in RCC patients. Future translational investigation will include in vivo studies to confirm the
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efficacy of AR blockade in mice bearing RCC cells with or without AR expression.
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ACKNOWLEGMENTS
This work is supported by a grant from the National Cancer Institute (P30CA072720). This
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work is also supported in part by generous grants from the Tanzman Foundation and by Basic Science Research Program through the National Research Foundation of Korea (NRF)
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funded by the Ministry of Education Science and Technology (2008-0062611) and supported by a grant from the Next-Generation BioGreen 21 Program (No.PJ009621), Rural
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Development Administration, Republic of Korea.
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Figure legends
Fig 1. AR expression in 57 RCC specimens was compared to that in matched normal-
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appearing tissues. There were no significant differences in AR expression overall (A), T1 patients (B), T2 patients (C), patients with Fuhrman grade 1-2 (D) and those with Fuhrman
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grade 3-4 (E).
Fig 2. Representative Western blot (A) and quantitative RT-PCR (B) of AR in RCC and
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normal kidney tissues. Western blot analysis of protein expression was used to confirm the gene expression data obtained by qPCR. Lane 1, LNCaP; Lane 2, normal tissue of T1 patient; Lane 3, tumor of T1 patient; Lane 4, normal tissue of T2 patient; Lane 5, tumor of T2 patient.
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Fig 3. Kaplan-Meier curves estimates the progression (A) and cancer specific death (B) according to AR mRNA expression levels.
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Fig 4. Androgens and human RCC cell lines. (A) Effect of DHT on human RCC cells. DHT
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increased cellular proliferation of CAKI2 in a concentration-dependent manner. LNCAP was used as a positive control. (B) AR mRNA (RT-PCR) and protein expression were detected only in CAKI2. In HS891.T, AR mRNA but not full length AR protein was detectable. Rather, a lower molecular weight protein was observed in HS891.T, suggesting the presence of an AR splice variant. Again, LNCAP was used as a positive control. Fig 5. Overexpression of AR. When AR was transfected into AR-negative A498 and CAKI1 25
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cells, DHT increased cellular proliferation in a concentration-dependent manner. Fig 6. Enzalutamide and cellular proliferation. There were significant decreases in cellular
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proliferation rates in a concentration-dependent manner in CAKI2 (AR-positive) and HS891T (AR-negative). Enzalutamide had no obvious effect on A498 and CAKI1 cells.
Fig 7. AR knockdown using shRNA. Transfection with shRNA knocked down AR mRNA
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expression significantly in both CAKI2 (A) and HS891.T (B). When AR was knocked down,
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no significant effect of MDV3100 was seen in CAKI2 and HS891.T cells.
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Table 1. AR mRNA expression stratified by clinicopathological parameters mRNA expression of AR (
103 P
copies/µl)
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Parameters (N)
0.684a
Paired tissue samples Donor-matched adjacent
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0.257 (0.209–0.317)
Tumor tissue (57) Gender
Female (29)
0.240 (0.184–0.314)
0.245 (0.183–0.329)
Pathologic T stage
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pT1 (94)
0.737 b
0.262 (0.213–0.322)
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Male (86)
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normal tissue (57)
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pT2 (21)
0.011 b
0.220 (0.191–0.253) 0.523 (0.276–0.992) 0.135C
Nuclear grade 1 (26)
0.193 (0.148–0.253)
2 (52)
0.296 (0.226–0.387)
3 (33)
0.241 (0.172–0.340)
4 (4)
0.464 (0.089–2.420) 1
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0.021 b
Progression 0.242 (0.204–0.286)
Yes (11)
0.471 (0.218–1.015)
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No (104)
0.001 b
Cancer specific death
Yes (6)
0.471 (0.218–1.015)
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0.242 (0.204–0.286)
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Paired t test; b2-samples t test; cANOVA; AR, androgen receptor
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a
No (109)
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Table 2. Cox regression analysis for Cancer specific death
Variables HR (95% CI)
Mutivariate analysis
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Univariate analysis P
HR (95% CI)
P
0.906
1.112 (0.125-9.901)
0.924
0.755
0.932 (0.046-18.856)
0.963
1.004 (0.934-1.080)
Sex (male vs. female)
0.705 (0.079-6.335)
T Stage (T1 vs. T2)
14.352 (1.586-129.863)
0.018
5.684 (0.441-73317)
0.183
Fuhrman grade (1-2 vs. 3-4)
1.445 (0.239-8.728)
0.688
0.850 (0.105-6.915)
0.879
AR expression (low vs. high)
26.529 (2.885-243-983)
0.004
15.546 (1.320-183.131)
0.029
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Age
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Fig. 1 (C)
(D)
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(B)
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(A)
(E)
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(A)
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Fig. 2
Full length AR (110 kd)
3
4
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10
5
1
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Level (Natural Log)
(B)
Relative AR mRNA
LNCaP
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β-actin
0
1
2
3
4
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Total
Male
Female
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Male
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Total
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(B)
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Fig. 3 (A)
Female
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Fig. 4 control
50
A498
HS891.T
CAKI1
CAKI2
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LNCAP
β-actin
10n
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100
AR mRNA
1n
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150
0
(B)
0.1n
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Cell growth (%)
(A)
AR Protein β-actin
LNCAP A498 HS891.T CAKI1 CAKI2
Wild type Splice variant
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Fig. 5
180
P< 0.001
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P< 0.001
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140 120 100
60
20 0
2 days 4 days 6 days A498
AR_DHT1N AR_DHT10N
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40
PCDNA3_DHT1N PCDNA3_DHT10N
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80
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Proliferation rate (%)
160
2 days 4 days 6 days CAKI1
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MDV_1uM
140 P= 0.020
100
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40 20 0
P= 0.004
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Cell Count (% control)
120
80
P= 0.095
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P= 0.561
MDV_25uM
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Control
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Fig. 6
A498
HT891.T
CAKI1
CAKI2
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Fig. 7 (A) Caki2
100
120 100 80 60 40 20 0
60 40
sh-AR
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Con
HS891.T
MDV_25uM
Control
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150
50 0 Con
sh-AR
sh-AR
MDV_1uM
MDV_25uM Cell counts (%
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100
Scramble
HS891.T
200 control)
20
(B)
MDV_1uM
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80
0
AR mRNA
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Control
Cell counts (% control)
120
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AR mRNA expression (%…
Caki2
100 0 Con
sh-AR
ACCEPTED MANUSCRIPT Abbreviations RCC, Renal cell carcinoma mRCC, metastatic RCC
RFS, recurrence-free survival CSS, cancer specific survival GAPDH, Glyceraldehyde-3-phosphate dehydrogenase
qPCR, quantitative real-time PCR DHT, dihydrotestosterone CI, confidence interval HR, hazard ratio
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RT-PCR, Reverse transcriptase PCR
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OS, overall survival
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AR, androgen receptor
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ROC, Receiver operating characteristics
1
