Journal of Surgical Oncology
INHBA Overexpression Indicates Poor Prognosis in Urothelial Carcinoma of Urinary Bladder and Upper Tract HSIANG-YING LEE, MD, MS,1 CHING-CHIA LI, MD,1,2,3,4 CHUN-NUNG HUANG, MD, PhD,1,4 WEI-MING LI, MD, MS,1,3,4,5 HSIN-CHIH YEH, MD, MS,1,2,3,4 HUNG-LUNG KE, MD, MS,1,3,4 KAI-FU YANG, MD,1 PEIR-IN LIANG, MD,6 CHIEN-FENG LI, MD, PhD,7,8,9,10* * AND WEN-JENG WU, MD, PhD1,3,4,11* 1
Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan 3 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Department of Urology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Pingtung Hospital, Ministry of Health and Welfare, Executive Yuan, Pingtung, Taiwan 6 Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 7 Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan 8 National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan 9 Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan 10 Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 11 Department of Urology, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
Background: Urothelial carcinoma (UC) originating from the bladder (UBUC) and upper urinary tract (UTUC) is the most common type of urinary tract tumor. While its pathogenesis remains obscured. Computerizing a published transcriptomic database of UBUC (GSE31684), we identified Inhibin, Beta A (INHBA) as the most significant upregulated gene associated with tumor progression among those associated with growth factor activity (GO:0008083). We therefore analyzed the clinicopathological significance of INHBA expression in UC. Design: QuantiGene assay was used to detect INHBA transcript level in 36 UTUCs and 30 UBUCs. Immunohistochemistry evaluated by H-score was used to determine INHBA protein expression in 340 UTUCs and 296 UBUCs. INHBA expression was correlated with clinicopathological features and disease-specific survival (DSS) and metastasis-free survival (MeFS). Results: Increments of INHBA transcript level was associated with higher pT status in both UTUC and UBUC. INHBA protein overexpression was significantly associated with advanced clinicopathological features in both groups of UC. INHBA overexpression significantly implied inferior DSS (UTUC, P ¼ 0.002; UBUC, P ¼ 0.005) and MeFS (UTUC and UBUC, both P < 0.001) in multivariate analysis. Conclusion: INHBA overexpression implies adverse clinical outcomes for UC, justifying it is a potential prognostic biomarker and a novel therapeutic target in UC.
J. Surg. Oncol. ß 2014 Wiley Periodicals, Inc.
KEY WORDS: urothelial carcinoma; transcriptome; INHBA; prognosis
INTRODUCTION Urothelical carcinoma (UC) originating from the proximal twothirds of the urethra, bladder and upper urinary tract is one of the most common malignancies in the world [1]. UC of the bladder is its most common form and the ninth most common cancer among men in Taiwan [2]. Because the incidence rate is increasing annually, and a substantial portion of patients with advanced or metastatic condition continue to have poor prognoses despite improvements in therapeutic choices. Accordingly, further study is mandatory to identifying novel biomarkers to provide information for clinical prognosis and diagnosis [3–5]. Of the various biomarkers, those involving growth factor activity are most crucial and indispensable for carcinogenesis and has have not been systemically evaluate in UC. For this, we analysed a transcriptomic dataset focusing on those involving the regulation of growth factor activity and identified INHBA as the most significant gene associated with cancer progression in UC. INHBA encodes Inhibin bA which is the subunit of activin and inhibin, which are members of the transforming growth factor-b (TGF-b) superfamily. The combination of homodimer of INHBA by disulfide-link constitutes activin A. Moreover, when INHBA combines with the b and a isoforms, it becomes activin AB and inhibin A, respectively [6]. Activin and inhibin produce opposite effects
ß 2014 Wiley Periodicals, Inc.
at different stages of cell growth, development and differentiation by acting on the hypothalamic-pituitary-gonadal axis [7]. Activin A was identified as having a role in multiple biological processes, including tumorigenesis and cancer progression [8,9]. Since
Grant sponsor: Ministry of Health and Welfare; Grant numbers: MOHW103-TD-B-111-05, DOH102-TD-M-111-102001; Grant sponsor: Kaohsiung Medical University Hospital; Grant number: KMUH98-8G31; Grant sponsor: Biobank. Hsiang-Ying Lee and Ching-Chia Li contributed equally to this article. Chien-Feng Li and Wen-Jen Wu contributed equally as senior of this work. Competing interests: The authors have no potential conflicts of interest. *Correspondence to: Wen-Jeng Wu, MD, PhD. Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. Fax: þ886-62511235. E-mail: [email protected] **Correspondence to: Chien-Feng Li, MD, PhD. Department of Pathology, Chi-Mei Foundation Medical Center, Tainan, Taiwan. Fax: þ886-6-2511235. E-mail: [email protected] Received 10 August 2014; Accepted 13 October 2014 DOI 10.1002/jso.23836 Published online in Wiley Online Library (wileyonlinelibrary.com).
2
Lee et al.
Fig. 1. Analysis of transcriptome in urothelial carcinoma from public domain dataset (GSE31684). Clustering analysis of genes focusing on growth factor activity (GO:0008083) showed INHBA is the most significantly upregulated gene associated with both increments of pT status. Tissue specimens from tumors with different primary tumor status are indicated on top of the heatmap, and expression levels of upregulated or downregulated genes are designated as a spectrum of brightness of red and green, respectively, with those unaltered in transcript expression coded as black. then, many researchers discovered the overexpression of activin A is association with lung, esophageal, gastric, colon, pancreatic and prostate cancer [9–14]. However, its significant in UC has not been systemically evaluated. Our present study identified INHBA as a candidate gene involving UC progression from public domain datasets, further confirmed by a solid validation using our well-characterized UTUC and UBUC cohorts. To our knowledge, this study is the first to systemically evaluate the expression of INHBA in both bladder and upper urinary tract UCs, and suggests its potential role in tumor progression.
MATERIALS AND METHODS Data Mining to Identify Candidate Transcripts in UC Progression We performed data mining by public domain data from the GEO (National Center Biotechnology information, Bethesda, MD, USA) and identified one dataset, GSE31684 (http://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc ¼ GSE31684), which profiled radical cystectomy specimens of 93 UBUCs using Affymetrix U133 Plus 2.0 Array. To analysis the expression level, we imported the raw CEL files into the Nexus Expression 3 statistical software (BioDiscovery, EI Segundo, CA, USA). All probes were used in the analysis without preselection or filtering. We performed supervised comparative analysis to examine the statistical significance of differentially expressed genes on the basis of primary tumor status (pT). For this purpose, we compared the differential expression between high-stage (pT2-pT4) to low-stage (pTa-pT1) UCs to perform functional profiles focusing on those related to growth factor activity (GO:0008083). Only genes showing significantly differential expression (log2 ration >1 and P < 0.01) were enrolled.
Patients and Tumor Specimens This study was approved by the institutional review board (IRB10302015) of Chi Mei Medical Center. We retrieved urothelial carcinoma cases from the archives of Chi Mei Medical Center archives between 1996 and 2004 for immunohistochemical study and survival analysis. A total of 635 consecutively treated well-characterized urothelial carcinomas were enrolled including 340 tumors originating from the UT and 295 arising from the UB. All patients were treated initially by surgical intervention with curative intent. As a rule, UBUC patients with pT3 or pT4 tumors or with nodal involvement received cisplatin-based adjuvant chemotherapy. However, only 29 of 106 pT3 or pT4 and nodal positive UTUC patients received cisplatin-based adjuvant chemotherapy. The criteria for clinicopathological evaluation Journal of Surgical Oncology
were essentially identical to those in our previous works [15]. Two pathologists (PIL & CFL) re-evaluated hematoxylin-eosin sections of all cases. For the determination of INHBA transcript levels, paraffin blocks from an independent cohort of 36 UTUCs and 30 UBUCs containing high percentage (no less than 70%) of tumor elements were selected and INHBA mRNA was detected with QuantiGene branched DNA assay. Of these, 15 and 21 UTUCs were of pTa-pT1 and pT2 to pT4, respectively; and 15 and 15 UBUCs were of pTa-pT1 and pT2 to pT4, respectively.
QuantiGene (branched DNA) Assay to Determine INHBA Transcript Level For quantification of INHBA mRNA expression, probes were design for INHBA for use in QuantiGene Multiplex 2.0 assay systems (Panomics, Inc.) under manufacturer’s instructions. In brief, probe set oligonucleotides were mixed with the lysed paraffin sections, and the mixture was then added to an assay well in a 96-well plate coated with capture probe oligonucleotide. Target RNA was captured during an overnight incubation at 55 °C. Unbound material was removed by threerun washes with 300 ml of wash buffer followed by subsequent hybridization of DNA amplifier molecules, followed by three washes after each incubation. After the final wash, the dioxetane alkaline phosphatase substrate Lumiphos Plus (Lumingen Inc., Southfield, MI) was added the reaction wells. After a short incubation, luminescent signal was detected by using Luminex 100 (Luminex, TX) microplate luminometer. The detection level of INHBA transcript was further adjusted with POLR2A expression.
Immunohistochemical Staining INHBA Tissue sections of 4 mm thickness were cut onto precoated slides from paraffin-embedded tissue blocks, followed by deparaffinization, rehydration, and antigen retrieval as previously described [15]. Endogenous peroxidase was blocked and subsequently incubated with a primary antibody targeting INHBA (H-120, Santa Cruz, CA) at a dilution of 1:50 for one hour. Primary antibodies were detected by using the DAKO ChemMate EnVision Kit (K5001, Carpinteria, CA, USA). The presence of brown chromogen in cytoplasm of target cells indicated positive immunoreactivity. We used testicular tissue which known to express INHBA as a positive control. Incubation without the primary antibody was used as a negative control.
Interpretation and Scoring of INHBA Two pathologists (PIL & CFL), blinded for the clinical information and follow-up data, evaluated the expression status of INHBA. Scoring of
INHBA Overexpression Indicates Poor Prognosis
3
TABLE I. Summary of Differentially Expressed Genes Associated With Growth Factor Activity in the Transcriptome of Urothelial Carcinoma of Urinary Bladder (GSE31684) Comparing T2-4 to Ta-T1 Probe
Log ratio
P-value
Gene symbol
Biological process
Molecular function
210511_s_at
2.0373
INHBA Overexpression Indicates Poor Prognosis in Urothelial Carcinoma of Urinary Bladder and Upper Tract HSIANG-YING LEE, MD, MS,1 CHING-CHIA LI, MD,1,2,3,4 CHUN-NUNG HUANG, MD, PhD,1,4 WEI-MING LI, MD, MS,1,3,4,5 HSIN-CHIH YEH, MD, MS,1,2,3,4 HUNG-LUNG KE, MD, MS,1,3,4 KAI-FU YANG, MD,1 PEIR-IN LIANG, MD,6 CHIEN-FENG LI, MD, PhD,7,8,9,10* * AND WEN-JENG WU, MD, PhD1,3,4,11* 1
Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan 3 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Department of Urology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Pingtung Hospital, Ministry of Health and Welfare, Executive Yuan, Pingtung, Taiwan 6 Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 7 Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan 8 National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan 9 Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan 10 Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 11 Department of Urology, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
Background: Urothelial carcinoma (UC) originating from the bladder (UBUC) and upper urinary tract (UTUC) is the most common type of urinary tract tumor. While its pathogenesis remains obscured. Computerizing a published transcriptomic database of UBUC (GSE31684), we identified Inhibin, Beta A (INHBA) as the most significant upregulated gene associated with tumor progression among those associated with growth factor activity (GO:0008083). We therefore analyzed the clinicopathological significance of INHBA expression in UC. Design: QuantiGene assay was used to detect INHBA transcript level in 36 UTUCs and 30 UBUCs. Immunohistochemistry evaluated by H-score was used to determine INHBA protein expression in 340 UTUCs and 296 UBUCs. INHBA expression was correlated with clinicopathological features and disease-specific survival (DSS) and metastasis-free survival (MeFS). Results: Increments of INHBA transcript level was associated with higher pT status in both UTUC and UBUC. INHBA protein overexpression was significantly associated with advanced clinicopathological features in both groups of UC. INHBA overexpression significantly implied inferior DSS (UTUC, P ¼ 0.002; UBUC, P ¼ 0.005) and MeFS (UTUC and UBUC, both P < 0.001) in multivariate analysis. Conclusion: INHBA overexpression implies adverse clinical outcomes for UC, justifying it is a potential prognostic biomarker and a novel therapeutic target in UC.
J. Surg. Oncol. ß 2014 Wiley Periodicals, Inc.
KEY WORDS: urothelial carcinoma; transcriptome; INHBA; prognosis
INTRODUCTION Urothelical carcinoma (UC) originating from the proximal twothirds of the urethra, bladder and upper urinary tract is one of the most common malignancies in the world [1]. UC of the bladder is its most common form and the ninth most common cancer among men in Taiwan [2]. Because the incidence rate is increasing annually, and a substantial portion of patients with advanced or metastatic condition continue to have poor prognoses despite improvements in therapeutic choices. Accordingly, further study is mandatory to identifying novel biomarkers to provide information for clinical prognosis and diagnosis [3–5]. Of the various biomarkers, those involving growth factor activity are most crucial and indispensable for carcinogenesis and has have not been systemically evaluate in UC. For this, we analysed a transcriptomic dataset focusing on those involving the regulation of growth factor activity and identified INHBA as the most significant gene associated with cancer progression in UC. INHBA encodes Inhibin bA which is the subunit of activin and inhibin, which are members of the transforming growth factor-b (TGF-b) superfamily. The combination of homodimer of INHBA by disulfide-link constitutes activin A. Moreover, when INHBA combines with the b and a isoforms, it becomes activin AB and inhibin A, respectively [6]. Activin and inhibin produce opposite effects
ß 2014 Wiley Periodicals, Inc.
at different stages of cell growth, development and differentiation by acting on the hypothalamic-pituitary-gonadal axis [7]. Activin A was identified as having a role in multiple biological processes, including tumorigenesis and cancer progression [8,9]. Since
Grant sponsor: Ministry of Health and Welfare; Grant numbers: MOHW103-TD-B-111-05, DOH102-TD-M-111-102001; Grant sponsor: Kaohsiung Medical University Hospital; Grant number: KMUH98-8G31; Grant sponsor: Biobank. Hsiang-Ying Lee and Ching-Chia Li contributed equally to this article. Chien-Feng Li and Wen-Jen Wu contributed equally as senior of this work. Competing interests: The authors have no potential conflicts of interest. *Correspondence to: Wen-Jeng Wu, MD, PhD. Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. Fax: þ886-62511235. E-mail: [email protected] **Correspondence to: Chien-Feng Li, MD, PhD. Department of Pathology, Chi-Mei Foundation Medical Center, Tainan, Taiwan. Fax: þ886-6-2511235. E-mail: [email protected] Received 10 August 2014; Accepted 13 October 2014 DOI 10.1002/jso.23836 Published online in Wiley Online Library (wileyonlinelibrary.com).
2
Lee et al.
Fig. 1. Analysis of transcriptome in urothelial carcinoma from public domain dataset (GSE31684). Clustering analysis of genes focusing on growth factor activity (GO:0008083) showed INHBA is the most significantly upregulated gene associated with both increments of pT status. Tissue specimens from tumors with different primary tumor status are indicated on top of the heatmap, and expression levels of upregulated or downregulated genes are designated as a spectrum of brightness of red and green, respectively, with those unaltered in transcript expression coded as black. then, many researchers discovered the overexpression of activin A is association with lung, esophageal, gastric, colon, pancreatic and prostate cancer [9–14]. However, its significant in UC has not been systemically evaluated. Our present study identified INHBA as a candidate gene involving UC progression from public domain datasets, further confirmed by a solid validation using our well-characterized UTUC and UBUC cohorts. To our knowledge, this study is the first to systemically evaluate the expression of INHBA in both bladder and upper urinary tract UCs, and suggests its potential role in tumor progression.
MATERIALS AND METHODS Data Mining to Identify Candidate Transcripts in UC Progression We performed data mining by public domain data from the GEO (National Center Biotechnology information, Bethesda, MD, USA) and identified one dataset, GSE31684 (http://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc ¼ GSE31684), which profiled radical cystectomy specimens of 93 UBUCs using Affymetrix U133 Plus 2.0 Array. To analysis the expression level, we imported the raw CEL files into the Nexus Expression 3 statistical software (BioDiscovery, EI Segundo, CA, USA). All probes were used in the analysis without preselection or filtering. We performed supervised comparative analysis to examine the statistical significance of differentially expressed genes on the basis of primary tumor status (pT). For this purpose, we compared the differential expression between high-stage (pT2-pT4) to low-stage (pTa-pT1) UCs to perform functional profiles focusing on those related to growth factor activity (GO:0008083). Only genes showing significantly differential expression (log2 ration >1 and P < 0.01) were enrolled.
Patients and Tumor Specimens This study was approved by the institutional review board (IRB10302015) of Chi Mei Medical Center. We retrieved urothelial carcinoma cases from the archives of Chi Mei Medical Center archives between 1996 and 2004 for immunohistochemical study and survival analysis. A total of 635 consecutively treated well-characterized urothelial carcinomas were enrolled including 340 tumors originating from the UT and 295 arising from the UB. All patients were treated initially by surgical intervention with curative intent. As a rule, UBUC patients with pT3 or pT4 tumors or with nodal involvement received cisplatin-based adjuvant chemotherapy. However, only 29 of 106 pT3 or pT4 and nodal positive UTUC patients received cisplatin-based adjuvant chemotherapy. The criteria for clinicopathological evaluation Journal of Surgical Oncology
were essentially identical to those in our previous works [15]. Two pathologists (PIL & CFL) re-evaluated hematoxylin-eosin sections of all cases. For the determination of INHBA transcript levels, paraffin blocks from an independent cohort of 36 UTUCs and 30 UBUCs containing high percentage (no less than 70%) of tumor elements were selected and INHBA mRNA was detected with QuantiGene branched DNA assay. Of these, 15 and 21 UTUCs were of pTa-pT1 and pT2 to pT4, respectively; and 15 and 15 UBUCs were of pTa-pT1 and pT2 to pT4, respectively.
QuantiGene (branched DNA) Assay to Determine INHBA Transcript Level For quantification of INHBA mRNA expression, probes were design for INHBA for use in QuantiGene Multiplex 2.0 assay systems (Panomics, Inc.) under manufacturer’s instructions. In brief, probe set oligonucleotides were mixed with the lysed paraffin sections, and the mixture was then added to an assay well in a 96-well plate coated with capture probe oligonucleotide. Target RNA was captured during an overnight incubation at 55 °C. Unbound material was removed by threerun washes with 300 ml of wash buffer followed by subsequent hybridization of DNA amplifier molecules, followed by three washes after each incubation. After the final wash, the dioxetane alkaline phosphatase substrate Lumiphos Plus (Lumingen Inc., Southfield, MI) was added the reaction wells. After a short incubation, luminescent signal was detected by using Luminex 100 (Luminex, TX) microplate luminometer. The detection level of INHBA transcript was further adjusted with POLR2A expression.
Immunohistochemical Staining INHBA Tissue sections of 4 mm thickness were cut onto precoated slides from paraffin-embedded tissue blocks, followed by deparaffinization, rehydration, and antigen retrieval as previously described [15]. Endogenous peroxidase was blocked and subsequently incubated with a primary antibody targeting INHBA (H-120, Santa Cruz, CA) at a dilution of 1:50 for one hour. Primary antibodies were detected by using the DAKO ChemMate EnVision Kit (K5001, Carpinteria, CA, USA). The presence of brown chromogen in cytoplasm of target cells indicated positive immunoreactivity. We used testicular tissue which known to express INHBA as a positive control. Incubation without the primary antibody was used as a negative control.
Interpretation and Scoring of INHBA Two pathologists (PIL & CFL), blinded for the clinical information and follow-up data, evaluated the expression status of INHBA. Scoring of
INHBA Overexpression Indicates Poor Prognosis
3
TABLE I. Summary of Differentially Expressed Genes Associated With Growth Factor Activity in the Transcriptome of Urothelial Carcinoma of Urinary Bladder (GSE31684) Comparing T2-4 to Ta-T1 Probe
Log ratio
P-value
Gene symbol
Biological process
Molecular function
210511_s_at
2.0373
