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PD38-10 BLADDER CANCER EXOSOMES FROM HIGH-GRADE MUSCLE INVASIVE BLADDER CANCER CONTAIN LONG NON-CODING RNA AND MESSENGER RNA
THE JOURNAL OF UROLOGYâ
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PD38-09 STEROID SULFATASE PREDICTS THE PROGRESSION OF BLADDER CANCER Minoru Kato*, Min Gi, Taro Iguchi, Satoshi Tamada, Hideki Wanibuchi, Tatsuya Nakatani, Osaka, Japan INTRODUCTION AND OBJECTIVES: Androgen signaling is recently suggested to be involved in the growth of bladder cancer. We have previously found that steroid sulfatase (STS) is highly expressed in muscle invasive bladder cancer. STS converts dehydroepiandrosterone sulfate to dehydroepiandrosterone, and is considered one of the key enzymes in androgen signaling pathway. However, the role of STS in bladder cancer has not been elucidated. The purpose of the present study is to determine the clinical and functional significance of STS in bladder cancer. METHODS: STS expression was measured in 170 bladder cancer tissues by immunohistochemistry. Recurrence free survival (RFS) and cancer specific survival (CSS) were evaluated. The functional role of STS on cell proliferation, migration and invasion capacity was evaluated using bladder cancer cell line (TCCSUP and T24) by loss-of-function assay. RESULTS: STS was highly expressed in invasive lesions of bladder cancer tissues. Overexpression of STS was associated with the invasion of bladder cancer evidenced by the findings that incidences of STS positive cancers were 21.3% and 41.2% in non-muscle invasive and muscle invasive bladder cancers, respectively (p ¼ 0.0262). STS positive cancers showed shorter RFS and CSS (p ¼ 0.0083, 0.0014, respectively). In multivariate analysis, STS expression level was identified as an independent prognostic factor for CSS (p ¼ 0.043). Furthermore, in vitro knockdown of STS significantly reduced cell migration and invasion capacities of bladder cancer cells accompanied by up-regulation of E-cadherin and down-regulation of vimentin. Interestingly, the expression of androgen receptor (AR) was not correlated with that of STS, pathological stage or survival of patients with bladder cancer, suggesting that AR is not likely to play an important role in the progression of bladder cancer. CONCLUSIONS: The present study demonstrates that STS is associated with the invasion of bladder cancer and is a useful marker for predicting the progression of bladder cancers. STS might play a role in bladder cancer independent from AR signaling pathway. Source of Funding: Grants-in-Aid for Scientific Research (24592406) from Japan Society for the Promotion of Science
PD38-10 BLADDER CANCER EXOSOMES FROM HIGH-GRADE MUSCLE INVASIVE BLADDER CANCER CONTAIN LONG NON-CODING RNA AND MESSENGER RNA Claudia Berrondo*, Jonathan Flax, Victor Kucherov, Aisha Siebert, Thomas Osinski, Alex Rosenberg, Christopher Fucile, Carla Beckham, Rochester, NY INTRODUCTION AND OBJECTIVES: Many long non-coding RNAs (lncRNA) play critical roles in tumor biology. The lncRNA HOX Antisense Intergenic RNA (HOTAIR) is overexpressed in many solid tumors and regulates genes involved in epithelial-to-mesenchyme transition (EMT). There is a need to incorporate lncRNA into biomarker and therapeutic target discovery algorithms. Exosomes may be a good source for biomarkers for UBC because they can be isolated from urine and their contents are protected from degradation. To determine the role of HOTAIR in UBC progression and to identify novel mRNA and lncRNA in urinary exosomes of patients with highgrade muscle invasive (HGMI) UBC. METHODS: HOTAIR was knocked down using lentiviral shRNA. Migration was measured with a wound-scratch assay. Invasion
Vol. 195, No. 4S, Supplement, Monday, May 9, 2016
was measured with trans-well assay and 3D culture system. Tumor, distal normal tissue and urine from patients with HGMI UBC and healthy volunteers (HVs) was obtained with IRB approval. mRNA and lncRNA levels were measured by qRT-PCR. Exosomes were isolated by ultracentrifugation and confirmed with electron microscopy, nanoparticle tracing and Western blot. RNA sequencing was completed using Illumina HiSeq2500 platform with 20,000,000 pair-ended reads and 125 base pair length. RESULTS: UBC cell lines express elevated levels of tumorassociated mRNA and lncRNA (including HOTAIR). The knockdown of HOTAIR in UBC cell lines reduces migration and invasion and correlates with the loss of expression of EMT factors. Tumor from patients with HGMI UBC express elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). Exosomes from UBC cell lines and urinary exosomes from patients with HGMI UBC have elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). RNA sequencing revealed elevated levels of 22 novel mRNA and 4 novel lncRNA in the urinary exosomes of patients with HGMI UBC compared to HVs. qRT-PCR confirmed the differential enrichment of mRNA PDX1, TEDDM1, ZBTB4 and lncRNA HYMA1, LOC100506688 and OXT2-AS1. CONCLUSIONS: UBC cell lines, tumors from patients with HGMI UBC and the exosomes they produce have elevated expression of tumor-associated mRNA and lncRNA (including HOTAIR). HOTAIR is overexpressed in UBC cell lines and UBC patient tumors and is important in UBC progression in vitro through its effects on EMT factor expression, cell migration and invasion. Urinary exosomes from patients with HGMI UBC contain elevated levels of novel mRNA and lncRNA. Taken together, these data suggest that urinary exosomes may contain mRNA and lncRNA that can serve as biomarkers for UBC. Source of Funding: Clinical and Translational Science Institute of University of Rochester and University of Rochester Department of Urology.
PD38-11 WHOLE TRANSCRIPTOME ANALYSIS REVEALS MAJOR MOLECULAR PATHWAYS THAT POTENTIALLY DRIVE THE PROGRESSION OF CARCINOMA-IN-SITU TO MUSCLE-INVASIVE BLADDER CANCER Feng He, Yan Liu, Herbert Lepor, Moon-shong Tang, Chuanshu Huang, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: Carcinoma in situ (CIS) of the bladder is a flat, high-grade lesion that poses heightened risk of progression to muscle invasion. However, no biomarker is currently available to reliably predict this progressive step. Identification of the molecular drivers of CIS progression in humans has been hampered in part by the limited amount of fresh materials suitable for whole transcriptome analysis. METHODS: Genetically engineered compound mice with RAS pathway activation and p53 pathway deficiency, each event afflicting about 75% of human muscle-invasive bladder cancer (MIBC), were used as the source of fresh materials for CIS and MIBC. Freshly frozen bladders were subject to frozen sectioning, rapid staining and then laser capture microdissection for CIS and MIBC lesions. Total RNAs were extracted, converted to double stranded cDNA, fluorescein-labelled and hybridized to Affymetrix oligonucleotide mouse genome arrays. Whole transcriptomes were compared between MIBC and CIS using Ingenuity pathway analysis. Selected genes were verified using Real-time PCR, immunofluorescent staining and immunohistochemistry. RESULTS: Compound mice expressing an activated HRAS and deficient for p53 in urothelium developed CIS and MIBC that closely resembled the human counterparts. These lesions were freshly dissected out from lightly stained frozen sections using laser capture microdissection. mRNA and cDNA prepared consecutively were hybridized to mouse genome arrays. Of the 1,847 differentially expressed
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PD38-09 STEROID SULFATASE PREDICTS THE PROGRESSION OF BLADDER CANCER Minoru Kato*, Min Gi, Taro Iguchi, Satoshi Tamada, Hideki Wanibuchi, Tatsuya Nakatani, Osaka, Japan INTRODUCTION AND OBJECTIVES: Androgen signaling is recently suggested to be involved in the growth of bladder cancer. We have previously found that steroid sulfatase (STS) is highly expressed in muscle invasive bladder cancer. STS converts dehydroepiandrosterone sulfate to dehydroepiandrosterone, and is considered one of the key enzymes in androgen signaling pathway. However, the role of STS in bladder cancer has not been elucidated. The purpose of the present study is to determine the clinical and functional significance of STS in bladder cancer. METHODS: STS expression was measured in 170 bladder cancer tissues by immunohistochemistry. Recurrence free survival (RFS) and cancer specific survival (CSS) were evaluated. The functional role of STS on cell proliferation, migration and invasion capacity was evaluated using bladder cancer cell line (TCCSUP and T24) by loss-of-function assay. RESULTS: STS was highly expressed in invasive lesions of bladder cancer tissues. Overexpression of STS was associated with the invasion of bladder cancer evidenced by the findings that incidences of STS positive cancers were 21.3% and 41.2% in non-muscle invasive and muscle invasive bladder cancers, respectively (p ¼ 0.0262). STS positive cancers showed shorter RFS and CSS (p ¼ 0.0083, 0.0014, respectively). In multivariate analysis, STS expression level was identified as an independent prognostic factor for CSS (p ¼ 0.043). Furthermore, in vitro knockdown of STS significantly reduced cell migration and invasion capacities of bladder cancer cells accompanied by up-regulation of E-cadherin and down-regulation of vimentin. Interestingly, the expression of androgen receptor (AR) was not correlated with that of STS, pathological stage or survival of patients with bladder cancer, suggesting that AR is not likely to play an important role in the progression of bladder cancer. CONCLUSIONS: The present study demonstrates that STS is associated with the invasion of bladder cancer and is a useful marker for predicting the progression of bladder cancers. STS might play a role in bladder cancer independent from AR signaling pathway. Source of Funding: Grants-in-Aid for Scientific Research (24592406) from Japan Society for the Promotion of Science
PD38-10 BLADDER CANCER EXOSOMES FROM HIGH-GRADE MUSCLE INVASIVE BLADDER CANCER CONTAIN LONG NON-CODING RNA AND MESSENGER RNA Claudia Berrondo*, Jonathan Flax, Victor Kucherov, Aisha Siebert, Thomas Osinski, Alex Rosenberg, Christopher Fucile, Carla Beckham, Rochester, NY INTRODUCTION AND OBJECTIVES: Many long non-coding RNAs (lncRNA) play critical roles in tumor biology. The lncRNA HOX Antisense Intergenic RNA (HOTAIR) is overexpressed in many solid tumors and regulates genes involved in epithelial-to-mesenchyme transition (EMT). There is a need to incorporate lncRNA into biomarker and therapeutic target discovery algorithms. Exosomes may be a good source for biomarkers for UBC because they can be isolated from urine and their contents are protected from degradation. To determine the role of HOTAIR in UBC progression and to identify novel mRNA and lncRNA in urinary exosomes of patients with highgrade muscle invasive (HGMI) UBC. METHODS: HOTAIR was knocked down using lentiviral shRNA. Migration was measured with a wound-scratch assay. Invasion
Vol. 195, No. 4S, Supplement, Monday, May 9, 2016
was measured with trans-well assay and 3D culture system. Tumor, distal normal tissue and urine from patients with HGMI UBC and healthy volunteers (HVs) was obtained with IRB approval. mRNA and lncRNA levels were measured by qRT-PCR. Exosomes were isolated by ultracentrifugation and confirmed with electron microscopy, nanoparticle tracing and Western blot. RNA sequencing was completed using Illumina HiSeq2500 platform with 20,000,000 pair-ended reads and 125 base pair length. RESULTS: UBC cell lines express elevated levels of tumorassociated mRNA and lncRNA (including HOTAIR). The knockdown of HOTAIR in UBC cell lines reduces migration and invasion and correlates with the loss of expression of EMT factors. Tumor from patients with HGMI UBC express elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). Exosomes from UBC cell lines and urinary exosomes from patients with HGMI UBC have elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). RNA sequencing revealed elevated levels of 22 novel mRNA and 4 novel lncRNA in the urinary exosomes of patients with HGMI UBC compared to HVs. qRT-PCR confirmed the differential enrichment of mRNA PDX1, TEDDM1, ZBTB4 and lncRNA HYMA1, LOC100506688 and OXT2-AS1. CONCLUSIONS: UBC cell lines, tumors from patients with HGMI UBC and the exosomes they produce have elevated expression of tumor-associated mRNA and lncRNA (including HOTAIR). HOTAIR is overexpressed in UBC cell lines and UBC patient tumors and is important in UBC progression in vitro through its effects on EMT factor expression, cell migration and invasion. Urinary exosomes from patients with HGMI UBC contain elevated levels of novel mRNA and lncRNA. Taken together, these data suggest that urinary exosomes may contain mRNA and lncRNA that can serve as biomarkers for UBC. Source of Funding: Clinical and Translational Science Institute of University of Rochester and University of Rochester Department of Urology.
PD38-11 WHOLE TRANSCRIPTOME ANALYSIS REVEALS MAJOR MOLECULAR PATHWAYS THAT POTENTIALLY DRIVE THE PROGRESSION OF CARCINOMA-IN-SITU TO MUSCLE-INVASIVE BLADDER CANCER Feng He, Yan Liu, Herbert Lepor, Moon-shong Tang, Chuanshu Huang, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: Carcinoma in situ (CIS) of the bladder is a flat, high-grade lesion that poses heightened risk of progression to muscle invasion. However, no biomarker is currently available to reliably predict this progressive step. Identification of the molecular drivers of CIS progression in humans has been hampered in part by the limited amount of fresh materials suitable for whole transcriptome analysis. METHODS: Genetically engineered compound mice with RAS pathway activation and p53 pathway deficiency, each event afflicting about 75% of human muscle-invasive bladder cancer (MIBC), were used as the source of fresh materials for CIS and MIBC. Freshly frozen bladders were subject to frozen sectioning, rapid staining and then laser capture microdissection for CIS and MIBC lesions. Total RNAs were extracted, converted to double stranded cDNA, fluorescein-labelled and hybridized to Affymetrix oligonucleotide mouse genome arrays. Whole transcriptomes were compared between MIBC and CIS using Ingenuity pathway analysis. Selected genes were verified using Real-time PCR, immunofluorescent staining and immunohistochemistry. RESULTS: Compound mice expressing an activated HRAS and deficient for p53 in urothelium developed CIS and MIBC that closely resembled the human counterparts. These lesions were freshly dissected out from lightly stained frozen sections using laser capture microdissection. mRNA and cDNA prepared consecutively were hybridized to mouse genome arrays. Of the 1,847 differentially expressed