A three-gene methylation marker panel for the nodal metastatic risk assessment of muscle-invasive bladder cancer
- 45 Downloads
In this study, we aimed to identify a DNA methylation pattern suitable for prognosis assessment of muscle-invasive bladder cancer and to investigate metastasis-associated processes regulated by DNA methylation.
Genome-wide methylation analysis was performed on 23 muscle-invasive bladder tumors by microarray analysis. Validation was performed by the qAMP technique in two different patient cohorts (n = 32 and n = 100). mRNA expression was analyzed in 12 samples. Protein expression was determined using tissue microarrays of 291 patients. Bladder cancer cell lines T24 and 253JB-V were used for functional analyses.
Microarray analyses revealed KISS1R, SEPT9 and CSAD as putative biomarkers with hypermethylation in node-positive tumors. The combination of the three genes predicted the metastatic risk with sensitivity of 73% and specificity of 71% in cohort 1, and sensitivity of 82% and specificity of 54% in cohort 2. mRNA expression differences were detected for KISS1R (p = 0.04). Protein expression of KISS1R was significantly reduced (p < 0.001). Knockdown of SEPT9v3 resulted in increased cell migration by 28% (p = 0.04) and increased invasion by 22% (p = 0.004). KISS1R overexpression resulted in decreased cell migration (25%, p = 0.1).
We identified a methylation marker panel suitable to differentiate between patients with positive and negative lymph nodes at time of cystectomy. This enables a risk assessment for patients who potentially benefit from extended lymph node resection as well as from neoadjuvant chemotherapy and could improve the survival rates. Furthermore, we examined the impact of putative markers on tumor behavior. Hence, KISS1R and SEPT9 could represent a starting point for the development of novel therapy approaches.
KeywordsMuscle-invasive bladder cancer Methylation Biomarker Metastasis
Compliance with ethical standards
Conflict of interest
Authors declare that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Human and animal rights statement
This article does not contain any studies with animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
- Connolly D, Yang Z, Castaldi M, Simmons N, Oktay MH, Coniglio S, Fazzari MJ, Verdier-Pinard P, Montagna C (2011) Septin 9 isoform expression, localization and epigenetic changes during human and mouse breast cancer progression. Breast Cancer Res 13(4):R76. https://doi.org/10.1186/bcr2924 CrossRefPubMedCentralGoogle Scholar
- Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P, Steplewski K, Shabon U, Miller JE, Middleton SE, Darker JG, Larminie CGC, Wilson S, Bergsma DJ, Emson P, Faull R, Philpott KL, Harrison DC (2001) AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 276(31): 28969–28975. https://doi.org/10.1074/jbc.M102743200 CrossRefGoogle Scholar
- Stathatos N, Bourdeau I, Espinosa AV, Saji M, Vasko VV, Burman KD, Stratakis CA, Ringel MD (2005) KiSS-1/G protein-coupled receptor 54 metastasis suppressor pathway increases myocyte-enriched calcineurin interacting protein 1 expression and chronically inhibits calcineurin activity. J Clin Endocrinol Metab 90(9):5432–5440. https://doi.org/10.1210/jc.2005-0963 CrossRefGoogle Scholar
- van Hoesel AQ, Sato Y, Elashoff DA, Turner RR, Giuliano AE, Shamonki JM, Kuppen PJ, van de Velde CJ, Hoon DS (2013) Assessment of DNA methylation status in early stages of breast cancer development. Br J Cancer 108(10):2033–2038. https://doi.org/10.1038/bjc.2013.136 CrossRefPubMedCentralGoogle Scholar
- van Kessel KE, van de Werken HJ, Lurkin I, Ziel-van der Made AC, Zwarthoff EC, Boormans JL (2017) A reported 20-gene expression signature to predict lymph node-positive disease at radical cystectomy for muscle-invasive bladder cancer is clinically not applicable. PLoS One 12(3):e0174039. https://doi.org/10.1371/journal.pone.0174039 CrossRefPubMedCentralGoogle Scholar
- Witjes JA, Comperat E, Cowan NC, De Santis M, Gakis G, Lebret T, Ribal MJ, Van der Heijden AG, Sherif A, European Association of U (2014) EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines. Eur Urol 65(4): 778–792. https://doi.org/10.1016/j.eururo.2013.11.046 CrossRefGoogle Scholar