Development Of Urothelial Cancer Research Articles

MP06-14 ROLE OF LATROPHILINS AS KEY DOWNSTREAM EFFECTORS OF THE ANDROGEN RECEPTOR IN MODULATING UROTHELIAL TUMORIGENESIS

You have accessJournal of UrologyCME1 May 2022MP06-14 ROLE OF LATROPHILINS AS KEY DOWNSTREAM EFFECTORS OF THE ANDROGEN RECEPTOR IN MODULATING UROTHELIAL TUMORIGENESIS Takuro Goto, Yuki Teramoto, Masato Yasui, Guiyang Jiang, Yujiro Nagata, George Netto, and Hiroshi Miyamoto Takuro GotoTakuro Goto More articles by this author , Yuki TeramotoYuki Teramoto More articles by this author , Masato YasuiMasato Yasui More articles by this author , Guiyang JiangGuiyang Jiang More articles by this author , Yujiro NagataYujiro Nagata More articles by this author , George NettoGeorge Netto More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We and others have indicated that androgen receptor (AR) signaling plays a critical role in urothelial cancer outgrowth. Meanwhile, our DNA microarray data suggest that latrophilin-3 (LPHN3), one of latrophilins (LPHNs) that are a group of the G-protein-coupled receptor where a spider venom latrotoxin (LTX) is known to bind, represents a downstream target of AR in bladder cancer cells. The present study aimed to determine the functional role of LPHNs, including LPHN1, LPHN2, and LPHN3, in the development of urothelial cancer. METHODS: We compared the levels of LPHN expression in 83 bladder tumors vs. paired normal bladder tissues (by immunohistochemistry) as well as in human normal urothelial SVHUC sublines stably expressing wild-type AR vs. vector only (by western blot). We then assessed the effects of ligand (e.g. LTX, FLRT3) treatment, as well as silencing of each LPHN via shRNA virus infection, on the neoplastic transformation of SVHUC-derived cells induced by a chemical carcinogen 3-methylcholanthrene (MCA). Bladder tumor development was also compared in C57BL/6 mice treated with a carcinogen BBN and LTX/FLRT3. RESULTS: The rates of immunoreactivity for LPHN1 (87% vs. 69%, P=0.012), LPHN2 (55% vs. 31%, P=0.002), and LPHN3 (51% vs. 37%, P=0.087) were significantly higher in bladder tumors than in non-neoplastic urothelial tissues. Moreover, LPHN3 positivity in non-muscle-invasive tumors was associated with the risk of disease recurrence after transurethral surgery (P=0.051). The expression levels of each LPHN were also higher in SVHUC-AR cells than in AR-negative SVHUC-vector cells, and androgen treatment in SVHUC-AR further increased them. Chromatin immunoprecipitation assay then revealed the binding of AR to the promoter region of each LPHN in SVHUC-derived cells. Meanwhile, induction in LPHN expression by LTX or FLRT3 treatment was seen in SVHUC cells. The in vitro transformation assay showed that LTX and FLRT3 induced the MCA-mediated oncogenic activity particularly in SVHUC-vector cells, while knockdown of LPHN1, LPHN2, or LPHN3 resulted in similar inhibition in that of MCA-SVHUC-AR cells. LTX (P=0.002) and FLRT3 (P=0.001) also accelerated the development of bladder tumors in BBN-treated female mice. CONCLUSIONS: These findings suggest that LPHNs, which are activated in bladder tumors as key downstream effectors of the AR, promote urothelial tumorigenesis. Accordingly, LPHN inhibition, along with AR inactivation, has the potential of being an effective chemopreventive approach for urothelial cancer. Source of Funding: Ferring Research Institute © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e82 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takuro Goto More articles by this author Yuki Teramoto More articles by this author Masato Yasui More articles by this author Guiyang Jiang More articles by this author Yujiro Nagata More articles by this author George Netto More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement PDF DownloadLoading ...

Urine phthalate metabolites are associated with urothelial cancer in chronic kidney disease patients

BackgroundDi(2-ethylhexyl) phthalate (DEHP) is one of the most widely used phthalates and is associated with breast cancer. Ths association between DEHP and other types of cancer is not clear. DEHP may increase matrix metalloproteinase-9 that is critical for the development of urothelial cancer (UC). We examined the association between urinary phthalate metabolites and UC. CKD patients were selected as a control group because CKD patients are more at risk of UC than the general population. MethodsIn this cross-sectional study, we measured seven urinary phthalate metabolites that are abundant and can be measured using HPLC-MS/MS in Taiwan CKD patients between Jul 2013 and Dec 2015. MiBP (a urinary metabolite of Dibutyl phthalates[DBP]) and MEHHP (a urinary metabolite of DEHP) were described because they are the most abundant phthalate metabolites. The association of phthalate (log-transformed) and UC were analyzed using logistic regression with adjustments for age, gender, renal function, use of traditional Chinese medicine, toxins (dye, organic solvent), and non-steroidal anti-inflammatory drugs. ResultsWe measured the urinary MEHHP and MiBP of 496 patients (224 UC and 272 CKD patients). The urinary MEHHP was associated with UC but MiBP was not. Medical history including the use of non-steroid anti-inflammatory drugs, exposure to environmental toxins (dye, paint, and organic solvent), and the use of traditional Chinese medicine was independently associated with UC. The adjusted odds ratio of MEHHP was 1.42 (95% confidence interval: 1.21–1.68). ConclusionPhthalate urinary metabolite(MEHHP) may be associated with UC in CKD patients and the association is independent of well-known risk factors of UC.

1136 - Does double J stenting during transurethral resection of a bladder tumor increase the risk for metachronous upper urinary tract urothelial cancer development?

1136 - Does double J stenting during transurethral resection of a bladder tumor increase the risk for metachronous upper urinary tract urothelial cancer development?

Specificity of the Metallothionein-1 Response by Cadmium-Exposed Normal Human Urothelial Cells.

Occupational and environmental exposure to cadmium is associated with the development of urothelial cancer. The metallothionein (MT) family of genes encodes proteins that sequester metal ions and modulate physiological processes, including zinc homeostasis. Little is known about the selectivity of expression of the different MT isoforms. Here, we examined the effect of cadmium exposure on MT gene and isoform expression by normal human urothelial (NHU) cell cultures. Baseline and cadmium-induced MT gene expression was characterized by next-generation sequencing and RT-PCR; protein expression was assessed by Western blotting using isoform-specific antibodies. Expression of the zinc transporter-1 (SLC30A1) gene was also assessed. NHU cells displayed transcription of MT-2A, but neither MT-3 nor MT-4 genes. Most striking was a highly inducer-specific expression of MT-1 genes, with cadmium inducing transcription of MT-1A, MT-1G, MT-1H, and MT-1M. Whereas MT-1G was also induced by zinc and nickel ions and MT-1H by iron, both MT-1A and MT-1M were highly cadmium-specific, which was confirmed for protein using isoform-specific antibodies. Protein but not transcript endured post-exposure, probably reflecting sequestration. SLC30A1 transcription was also affected by cadmium ion exposure, potentially reflecting perturbation of intracellular zinc homeostasis. We conclude that human urothelium displays a highly inductive profile of MT-1 gene expression, with two isoforms identified as highly specific to cadmium, providing candidate transcript and long-lived protein biomarkers of cadmium exposure.

Clinical and molecular characterization of urothelial cancer developing after pelvic radiation.

518 Background: The development of urothelial cancer (UC) after pelvic radiotherapy (RT) is an increasingly recognized phenomenon. Numerous studies show a relationship between ionizing radiation and urothelial dysplasia and DNA damage. It is not known if UC that develops after pelvic RT is associated with a distinct molecular signature, which could have treatment implications for this subset of UC. Methods: The University of North Carolina (UNC) Genitourinary Oncology Database was queried for cases of UC between 01/2012 and 06/2016 in patients with prior pelvic RT. Demographics, clinical stage, and details of prior RT were collected. Patients with available archival FFPE tumor tissue underwent next generation targeted exome sequencing per the UNCSeq pipeline. Somatic mutations and copy number alterations were analyzed. Total mutation burden (TMB) (mutations per total Megabase region of 30X coverage, mut/Mb) and insertion-to-deletion (indel) ratio was calculated. Wilcoxon rank sum was used to compare TMB and indel ratio of RT-associated cases with all cases of UC sequenced at UNC. Results: 38 patients with UC developing after prior pelvic RT were identified. 68% had muscle-invasive UC. Median age was 76. Prior RT was for the purpose of prostate cancer treatment in all patients. 26% had received external beam radiotherapy, 29% brachytherapy, and 16% both (29% unknown). Median time from RT to diagnosis of UC was 8 years. Ten patients with muscle-invasive UC had tumors available for sequencing. The most commonly identified somatic alterations were TP53 in 60%, RB1 40%, CREBBP 30%, ELF3 30%, HLA-DRB1 30%, KDM6A 30%, and MLL2 30%, including co-occurrence of TP53 and RB1 mutations in 30% of patients. The median TMB was 12.1 mut/Mb, significantly lower than the median TMB of 25.6 mut/Mb in all cases of UC sequenced at UNC (p = 0.006). The indel ratio was not different between the two groups (p = 0.11). Conclusions: UC that develops after pelvic RT is a frequent entity that occurs years after RT. Tumors of patients who received prior RT had a lower overall TMB than other UC tumors, suggesting that RT-induced UC may be a different entity than carcinogen-induced UC. Further studies should expand our analysis to confirm these findings.

Influence of diabetes on the risk of urothelial cancer according to body mass index: a 10-year nationwide population-based observational study.

To examine the association between obesity and urothelial cancer, we used a representative data from the National Health Insurance System (NHIS). Participants included 826,170 men aged 20 years and older who experienced a health examination at least one time between 2004 and 2008. The study thus excluded people aged <20 years and women. We used a multivariate adjusted Cox regression analysis to examine the association between urothelial cancer and body mass index (BMI) via a hazard ratio (HR) and 95% confidence interval (CI).The age- or multivariable-adjusted HR for urothelial cancer was stratified by BMI. Men with a higher BMI were more likely to acquire urothelial cancer independent of variables. In the population with diabetes, there showed a considerable, increasing trend in the risk of urothelial cancer in the overweight and obesity group, compared to the group with the same BMI but without diabetes. This population-based study showed evidence of an association between obesity and the development of urothelial cancer, where the presence of diabetes increased the risk of urothelial cancer. Additionally, the higher the BMI, the higher the risk for urothelial cancer.

Effects of Aspirin, Nonsteroidal Anti-inflammatory Drugs, Statin, and COX2 Inhibitor on the Developments of Urological Malignancies: A Population-Based Study with 10-Year Follow-up Data in Korea.

PurposeThe purpose of this study was to determine the impact of aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs), statin, and cyclooxygenase 2 (COX-2) inhibitor on the development of kidney, prostate, and urothelial cancers by analyzing the Korean National Health Insurance Service–National Sample Cohort (NHIS-NSC) database.Materials and MethodsAmong a representative sample cohort of 1,025,340 participants in NHIS-NSC database in 2002, we extracted data of 799,850 individuals who visited the hospital more than once, and finally included 321,122 individuals aged 40 and older. Following a 1-year washout period between 2002 and 2003, we analyzed 143,870 (male), 320,861 and 320,613 individuals for evaluating the risk of prostate cancer, kidney cancer and urothelial cancer developments, respectively, during 10-year follow-up periods between 2004 and 2013. The medication group consisted of patients prescribed these drugs more than 60% of the time in 2003. To adjustfor various parameters of the patients, a multivariate Cox regression model was adopted.ResultsDuring 10-year follow-up periods between 2004 and 2013, 9,627 (6.7%), 1,107 (0.4%), and 2,121 (0.7%) patients were diagnosed with prostate cancer, kidney cancer, and urothelial cancer, respectively. Notably, multivariate analyses revealed that NSAIDs significantly increased the risk of prostate cancer (hazard ratio [HR], 1.35). Also, it was found that aspirin (HR, 1.28) and statin (HR, 1.55) elevated the risk of kidney cancer. No drugs were associated with the risk of urothelial cancer.ConclusionIn sum, our study provides the valuable information for the impact of aspirin, NSAID, statin, and COX-2 inhibitor on the risk of prostate, kidney, and urothelial cancer development and its survival outcomes.

Bladder cancer and urothelial impairment: the role of TRPV1 as potential drug target.

Urothelium, in addition to its primary function of barrier, is now understood to act as a complex system of cell communication that exhibits specialized sensory properties in the regulation of physiological or pathological stimuli. Furthermore, it has been hypothesized that bladder inflammation and neoplastic cell growth, the two most representative pathological conditions of the lower urinary tract, may arise from a primary defective urothelial lining. Transient receptor potential vanilloid channel 1 (TRPV1), a receptor widely distributed in lower urinary tract structures and involved in the physiological micturition reflex, was described to have a pathophysiological role in inflammatory conditions and in the genesis and development of urothelial cancer. In our opinion new compounds, such as curcumin, the major component of turmeric Curcuma longa, reported to potentiate the effects of the chemotherapeutic agents used in the management of recurrent urothelial cancer in vitro and also identified as one of several compounds to own the vanillyl structure required to work like a TRPV1 agonist, could be thought as complementary in the clinical management of both the recurrences and the inflammatory effects caused by the endoscopic resection or intravesical chemotherapy administration or could be combined with adjuvant agents to potentiate their antitumoral effect.

Loss of Sh3gl2/Endophilin A1 Is a Common Event in Urothelial Carcinoma that Promotes Malignant Behavior

Urothelial carcinoma (UC) causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying urothelial cancer development and tumor progression are still largely unknown. Using informatics analysis, we identified Sh3gl2 (endophilin A1) as a bladder urothelium-enriched transcript. The gene encoding Sh3gl2 is located on chromosome 9p, a region frequently altered in UC. Sh3gl2 is known to regulate endocytosis of receptor tyrosine kinases implicated in oncogenesis, such as the epidermal growth factor receptor (EGFR) and c-Met. However, its role in UC pathogenesis is unknown. Informatics analysis of expression profiles as well as immunohistochemical staining of tissue microarrays revealed Sh3gl2 expression to be decreased in UC specimens compared to nontumor tissues. Loss of Sh3gl2 was associated with increasing tumor grade and with muscle invasion, which is a reliable predictor of metastatic disease and cancer-derived mortality. Sh3gl2 expression was undetectable in 19 of 20 human UC cell lines but preserved in the low-grade cell line RT4. Stable silencing of Sh3gl2 in RT4 cells by RNA interference 1) enhanced proliferation and colony formation in vitro, 2) inhibited EGF-induced EGFR internalization and increased EGFR activation, 3) stimulated phosphorylation of Src family kinases and STAT3, and 4) promoted growth of RT4 xenografts in subrenal capsule tissue recombination experiments. Conversely, forced re-expression of Sh3gl2 in T24 cells and silenced RT4 clones attenuated oncogenic behaviors, including growth and migration. Together, these findings identify loss of Sh3gl2 as a frequent event in UC development that promotes disease progression.

Abstract 4066: Loss of Sh3gl2/endophilin A1 is an early event in urothelial carcinoma that regulates malignant behavior.

Abstract Urothelial carcinoma (UC) causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying urothelial cancer development and tumor progression are still largely unknown. Using informatics analysis we identified Sh3gl2 (endophilin A1) as a transcript and protein highly enriched in bladder epithelium (urothelium). The gene encoding Sh3gl2 is located on chromosome 9p, a genomic region frequently altered in UC. Sh3gl2 is known to regulate endocytosis of receptor tyrosine kinases implicated in oncogenesis, such as the epidermal growth factor receptor (EGFR) and c-Met. However, its role in UC pathogenesis is unknown. Informatics analysis of expression profiles in Oncomine™, as well as immunohistochemical staining of tissue microarrays revealed Sh3gl2 expression to be decreased in UC specimens compared to non-tumor tissues. Loss of Sh3gl2 was associated with increasing tumor grade and with muscle invasion, a reliable predictor of metastatic disease and cancer-derived mortality. Sh3gl2 expression was undetectable in 15 of 16 human UC cell lines, but preserved in the low grade, well- differentiated cell line RT4. In UC cell lines lacking expression of Sh3gl2, treatment with the demethylating agent 5-aza-deoxycytidine led to upregulation of Sh3gl2 mRNA levels. Methylation-specific restriction analysis and PCR of genomic DNA revealed hypermethylation of a CpG island in the Sh3gl2 promoter in cells lacking Sh3gl2 expression, but no promoter methylation in RT4 cells that express Sh3gl2, suggesting that lack of Sh3gl2 expression results, at least in part, from methylation-induced gene silencing. Stable knockdown of Sh3gl2 in RT4 cells by RNA interference (i) enhanced proliferation and colony formation in vitro; (ii) inhibited EGF-induced EGFR internalization and increased EGFR phosphorylation; (iii) stimulated phosphorylation of Src family kinases and STAT3; (iv) altered sensitivity to chemotherapeutic agents, including the dual-specificity EGFR/ERBB2 inhibitor lapatinib; and (v) promoted formation of RT4 xenografts in subrenal capsule tissue recombination experiments. Together, these findings identify loss of Sh3gl2 as an early event in UC development that promotes disease progression. Sh3gl2 status in UC biopsies may provide a potential marker of risk for UC progression or recurrence and may enable determination of which patients require more aggressive intervention in the form of surgery or chemotherapy in order to improve prognosis. Citation Format: Shyama Majumdar, Edward Gong, Dolores Di Vizio, Jonathan Dreyfuss, David DeGraff, Martin Hager, Peter Park, Joaquim Bellmunt, Robert Matusik, Jonathan Rosenberg, Rosalyn Adam. Loss of Sh3gl2/endophilin A1 is an early event in urothelial carcinoma that regulates malignant behavior. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2013-4066

ACB‐PCR measurement of H‐ras codon 61 CAA→CTA mutation provides an early indication of aristolochic acid I carcinogenic effect in tumor target tissues

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen, which induces forestomach and kidney tumors in mice and is associated with development of urothelial cancer in humans. This study sought to gain mechanistic insight into AAI-induced carcinogenesis through analysis of a tumor-relevant endpoint. Female Hupki mice were treated daily with 5 mg AAI/kg body weight by gavage for 3, 12, or 21 days. Histopathology and DNA adduct analysis confirmed kidney and forestomach as target tissues for AAI-induced toxicity. H-ras codon 61 CAA→CTA mutations were measured in mouse kidney and forestomach, as well as liver and glandular stomach (nontarget organs) by allele-specific competitive blocker-PCR (ACB-PCR), because A→T transversion is the predominant mutation induced by AA and this particular mutation was found previously in AA-induced rodent forestomach tumors. Treatment-related differences were observed, with the H-ras mutant fraction (MF) of mouse kidney and forestomach exposed to 5 mg AAI/kg body weight for 21 days significantly higher than that of vehicle-treated controls (Fisher's exact test, P < 0.05). Statistically significant correlations between dA-AAI adduct levels (measured previously in the same animals) and induced H-ras MFs were evident in forestomach of mice treated for 21 days (linear regression, P < 0.05). The significant increase in H-ras MF in kidney and forestomach, along with the correlation between DNA adducts, histopathology, and oncogene mutation, provide definitive evidence that AA induces tumors through a directly mutagenic mode of action. Thus, measurement of tumor-associated mutations is a useful tool for elucidating the mechanisms underlying the tissue specificity of carcinogenesis.

Aristolochic acid-induced carcinogenesis examined by ACB-PCR quantification of H-Ras and K-Ras mutant fraction

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen associated with the development of urothelial cancer in humans. AA induces forestomach, kidney and urothelial tract tumours in rats and mice. This study was conducted to characterise AA's carcinogenic mechanism of action and compare allele-specific competitive blocker-polymerase chain reaction (ACB-PCR)-based early detection of carcinogenic effect using two different tumour-relevant endpoints. H-Ras codon 61 CAA→CTA mutation was analysed because it is found in rodent forestomach tumours and A:T→T:A transversion is the predominant mutational specificity induced by AA. K-Ras codon 12 GGT→GAT mutation was analysed because it is a common spontaneous mutation present in various rodent tissues and may be a useful generic biomarker for carcinogenic effect. DNA samples from Big Blue rats treated with 0, 0.1, 1.0 or 10.0 mg AA/kg body weight (bw) by gavage, 5 days/week for 12 weeks were used in ACB-PCR in order to examine the induction of the two specific mutations. A significant dose-dependent induction of H-Ras mutant fraction (MF) was observed in liver and kidney. Statistically significant correlations were observed between AA-induced DNA adduct levels or cII mutant frequencies (previously measured in the same rats) and H-Ras MF measurements. No correlation between AA dose and K-Ras MF was found in liver or kidney, although there was a significant induction of K-Ras mutation in kidneys exposed to 0.1 mg/kg bw AA relative to controls. Thus, the data establish a straightforward dose-related increase in H-Ras MF due to fixation of AA-induced DNA adducts, whereas the common spontaneous K-Ras mutation showed a non-monotonic dose-response, consistent with loss of non-targeted mutation at cytotoxic doses.

Loss of p53 and Acquisition of Angiogenic MicroRNA Profile Are Insufficient to Facilitate Progression of Bladder Urothelial Carcinoma in Situ to Invasive Carcinoma

Activation of oncogenes or inactivation of tumor suppressors in urothelium is considered critical for development of urothelial cancer. Here we report cloning of the urothelium-specific promoter uroplakin-II (UPK II) and generation of transgenic mice in which expression of SV40 large T antigen is driven by UPK II promoter. Inactivation of tumor suppressor p53 and pRb in urothelium by SV40 T antigen resulted in urothelial carcinoma, resembling human high-grade carcinoma in situ. Specific deletion of p53 in urothelial cells using the newly generated UPK II-Cre mice results in normal bladders without any evidence of cancer. The high-grade carcinoma in situ in the UPK II-SV40 mice is associated with significant activation of angiogenic signals consisting of hypoxia-inducible factor-1α (HIF-1α) and VEGF and a down-regulation of thrombospondin-1. Interestingly, such pro-angiogenic activity was not associated with progression to invasive cancer. Analysis of bladder-associated microRNAs in carcinoma in situ lesions reveals a pro-angiogenic profile, with specific overexpression of miR-18a and miR-19a and down-regulation of miR-107. A group of microRNAs (miRs) identified as associated with invasive human urothelial cancer remained unchanged in this mouse model. Collectively, our results support the notion that activation of angiogenesis and loss of p53 are not sufficient for progression to invasive cancer. Our studies identify a new mouse model for bladder cancer that can be used to study factors that determine progression to an invasive phenotype of bladder cancer.

Personalized therapy for urothelial cancer: review of the clinical evidence.

Despite a detailed understanding of the molecular aberrations driving the development of urothelial cancers, this knowledge has not translated into advances for the treatment of this disease. Urothelial cancers are chemosensitive, and platinum-based combination chemotherapy remains the standard of care for advanced disease, as well as neoadjuvant and adjuvant therapy for locally advanced disease. However, nearly half of patients who undergo resection of locally advanced urothelial cancer will relapse and eventually develop platinum-resistant disease. Clinical trials of targeted agents against angiogenesis and growth factors, as well as novel chemotheraputics, have generally been unsuccessful in urothelial cancers. Improvements in the theraputic arsenal for urothelial cancer depend upon identification of new targets and strategies to overcome platinum resistance.

Abstract 1837: Exposure to Aristolochic Acid is associated with endemic (Balkan) nephropathy

Abstract Endemic (Balkan) Nephropathy (EN) is a rare, chronic tubulointerstitial nephritis that gradually progresses to end state renal disease. EN is also associated with development of urothelial cancer. EN has been the subject of many epidemiologic investigations during the past 50 years and is now thought to occur more broadly than just the endemic Balkan regions. Numerous hypotheses have been investigated but no causal factor has been identified. Exposure to Aristolochic Acid (AA) was proposed 30 years ago as a potential cause of EN, but was dismissed. The most likely route of human exposure to AA is via ingestion of home-baked bread prepared from flour contaminated by seeds of Aristolochia clematitis. Methods: Studies are underway in Croatia in collaboration with US investigators to evaluate this hypothesis. Data for this study comes from a population-based survey conducted in 2005 that includes 1081 subjects from 3 endemic and 1 nonendemic village. Logistic regression was used to evaluate the association between exposure to AA and EN. Results: Exposure to Aristolochic Acid (AA) from bread consumption is associated with a nearly 2 fold increase in risk of EN (OR=1.7; 95%CI: 1.07,2.98), growing wheat for flour is associated with a 3 fold increase risk (OR=3.5; 1.27, 10.1)), and recalling the AA containing weed in the wheat field was also significant (OR=2.4; 1.32, 4.62). Adjustment for covariates reduced the significance, but the elevated risk was still observed. A larger population-based assessment of several new villages in Croatia was recently completed. Conclusion: Understanding the etiology of this debilitating disease is the first step in developing an effective prevention strategy. Next steps will include evaluating specific genetic components and their interactions with exposure to AA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1837.

Evolution on experimental animal model for upper urothelium carcinogenesis

No optimal, well designed and reproducible animal model for upper urothelial carcinogenesis exists. This study characterized the histopathological features on top of immunolocalization of alpha-dystroglycans (alpha-DG) and matrix metalloproteinase (MMP-9) and cell turn-over in the upper urinary tract using a novel experimental model. Seventy-five female Fischer 344 rats were divided into three groups: the control group received a 0.30-ml dose of 0.9% physiological saline; the MNU group (chemical carcinogen N-methyl-N-nitrosourea) received 0.30 ml of MNU; and the MNU-citrate group received 0.30 ml of MNU plus sodium citrate, every one intravesically every other week for a total of 4 doses. After 15 weeks, bladder, ureters and renal pelvis were collected for morphological and molecular analysis. Associated management with MNU and sodium citrate was able to lead to 100% of both urinary bladder and upper urinary tract tumors, being the high-grade noninvasive papillary urothelial carcinoma the most frequent lesion. The upper urothelium showed reduced alpha-DG and increased MMP-9 and Ki-67 immunoreactivities in the MNU-citrate group in relation to the other groups. MNU group presented no upper urothelium tumor and 100% bladder tumor. This is a relevant evolution on experimental animal model for upper urinary tract carcinogenesis field. MMP-dependent disruption of the DG complex plays an important role in urothelial tumor carcinogenesis and showed the model applicability and significance. MNU-citrate model could contribute to a better understanding of human upper urothelial cancer development as well as to its local treatment strategies in a near future.

NUCLEAR EXPRESSION OF ARYL HYDROCARBON RECEPTOR PREDICTS DISEASE SPECIFIC SURVIVAL OF UROTHELIAL CARCINOMA OF THE UPPER URINARY TRACT

199 NUCLEAR EXPRESSION OF ARYL HYDROCARBON RECEPTOR PREDICTS DISEASE SPECIFIC SURVIVAL OF UROTHELIAL CARCINOMA OF THE UPPER URINARY TRACT Masaru Ishida*, Eiji Kikuchi, Shuji Mikami, Takeo Kosaka, Akira Miyajima, Makio Mukai, Yasunori Okada, Mototsugu Oya. Tokyo, Japan. INTRODUCTION AND OBJECTIVE: Cigarette smoke contributes to the development of urothelial cancer (UC). The aryl hydrocarbon receptor (AhR), which is the receptor for xenobiotics such as cigarette smoke, induces the CYP1 family, which becomes involved in tumor initiation and progression. AhR overexpression is observed in a few cancers, such as lung and pancreatic cancer, but expression of AhR in UCs, including bladder cancer, has not been investigated. We detected

DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by 32P-postlabeling and mutant frequency (MF) was determined using the λ Select- cII Mutation Detection System. Three major adducts (7-[deoxyadenosin- N 6-yl]-aristolactam I, 7-[deoxyadenosin- N 6-yl]-aristolactam II and 7-[deoxyguanosin- N 2-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10 8 nucleotides in liver and 95–4598 adducts/10 8 nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 × 10 −6 in liver compared with the MFs of 78–1319 × 10 −6 that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.

High throughput comparative genomic hybridization array analysis of multifocal urothelial cancers

The purpose of this study was to examine genetic alterations occur during synchronous or metachronous multifocal development of urothelial cancers on the whole genome using a comparative genomic hybridization (CGH) array. We used 10 tumor pairs (2 tumors for each patient), in which we had previously defined a clonal relationship by microsatellite analysis. For CGH array analysis, Vysis GenoSensor Array 300 kit was used. An unsupervised hierarchical cluster analysis revealed that the tumors from one patient were clustered together independent of the tumors of all other patients. On the other hand, many genetic divergences among multifocal urothelial cancers were newly found by a CGH array analysis. The concordant genetic alteration patterns of the chromosomal arm in tumor pairs were most frequently observed in 9p, 9q, 8p, 7p, 7q and 11q, while discordant patterns were most frequently found in 15q, 20q, 2q, 10p and 11q. Investigation using a CGH array showed that genetically stable multifocal tumors were less frequent, and that a large percentage of urothelial cancers accumulate genetic alterations during multifocal development by clonal evolution. We might have to consider these genetic accumulations during multifocal development when designing strategies for prevention and detection of recurrent multifocal urothelial cancers. CGH array can be a powerful tool for genetic analysis of multifocal urothelial cancer.

Mutations induced by carcinogenic doses of aristolochic acid in kidney of Big Blue transgenic rats

Aristolochic acid (AA) is present in at least 65 different kinds of plants, many of which are used as herbal folk remedies. AA is considered one of the most potent plant carcinogens in humans and animals. It has been associated with the development of urothelial cancers in humans, and kidney and forestomach tumors in rats. In the present study, we used the Big Blue transgenic rat model to evaluate the mutagenicity of AA in kidney of rats and to define the mechanism of action for the tumor induction by AA. Groups of six male Big Blue transgenic rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight 5 times a week for 12 weeks, a treatment protocol that resulted in tumors in kidneys and other tissues. The animals were sacrificed 1 day after the final treatment and the kidneys were isolated for assays to determine the mutant frequencies (MFs) and types of mutations induced by AA in the transgenic cII gene. AA treatment resulted in a strong linear relationship between MF inductions and treatment dose ( R 2 = 0.998). The cII MFs were 29 ± 6 × 10 −6, 78 ± 21 × 10 −6, 242 ± 104 × 10 −6 and 1319 ± 360 × 10 −6 in the control, low, medium and high dose treatment groups, respectively ( p < 0.001 for all pair wise comparisons among the four treatment groups). These MFs correlated strongly with tumor incidences induced by the different doses of AA ( Mengs et al., 1982). Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutational spectra in the AA-treated and control rats ( p < 0.05). A:T → T:A transversion was the predominant type of mutation in the AA-treated rats whereas G:C → A:T transition was the main type of mutations in the control rats. These results suggest that AA induces kidney tumors in rats though a mutagenic mechanism of action.