Bladder cancer is the sixth most common cancer in humans. This heterogeneous set of lesions including urothelial carcinoma (Uca) and squamous cell carcinoma (SCC) arise from the urothelium, a stratified epithelium composed of K5-expressing basal cells, intermediate cells and umbrella cells. Superficial Uca lesions are morphologically distinct and exhibit different clinical behaviours: carcinoma in situ (CIS) is a flat aggressive lesion, whereas papillary carcinomas are generally low-grade and non-invasive. Whether these distinct characteristics reflect different cell types of origin is unknown. Here we show using lineage tracing in a murine model of carcinogenesis that intermediate cells give rise primarily to papillary lesions, whereas K5-basal cells are likely progenitors of CIS, muscle-invasive lesions and SCC depending on the genetic background. Our results provide a cellular and genetic basis for the diversity in bladder cancer lesions and provide a possible explanation for their clinical and morphological differences.
This is a preview of subscription content
Siegel, R., Naishadham, D. & Jemal, A. Cancer statistics, 2013. CA Cancer J. Clin. 63, 11–30 (2013).
Prasad, S. M., Decastro, G. J., Steinberg, G. D. & Medscape, Urothelial carcinoma of the bladder: definition, treatment and future efforts. Nat. Rev. Urol. 8, 631–642 (2011).
Dahm, P. & Gschwend, J. E. Malignant non-urothelial neoplasms of the urinary bladder: a review. Eur. Urol. 44, 672–681 (2003).
Goebell, P. J. & Knowles, M. A. Bladder cancer or bladder cancers? Genetically distinct malignant conditions of the urothelium. Urol. Oncol. 28, 409–428 (2010).
Wu, X. R. Urothelial tumorigenesis: a tale of divergent pathways. Nat. Rev. Cancer 5, 713–725 (2005).
Knowles, M. A. Bladder cancer subtypes defined by genomic alterations. Scand. J. Urol. Nephrol. 42, 116–130 (2008).
Castillo-Martin, M., Domingo-Domenech, J., Karni-Schmidt, O., Matos, T. & Cordon-Cardo, C. Molecular pathways of urothelial development and bladder tumorigenesis. Urol. Oncol.-Semin. Ori. 28, 401–408 (2010).
Chan, K. S. et al. Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells. Proc. Natl Acad. Sci. USA 106, 14016–14021 (2009).
Cordon-Cardo, C. Molecular alterations associated with bladder cancer initiation and progression. Scand. J. Urol. Nephrol. Suppl. 218, 154–165 (2008).
Chan, K. S., Volkmer, J. P. & Weissman, I. Cancer stem cells in bladder cancer: a revisited and evolving concept. Curr. Opin. Urol. 20, 393–397 (2010).
Dancik, G. M., Owens, C. R., Iczkowski, K. A. & Theodorescu, D. A cell of origin gene signature indicates human bladder cancer has distinct cellular progenitors. Stem Cells 32, 974–982 (2014).
Volkmer, J. P. et al. Three differentiation states risk-stratify bladder cancer into distinct subtypes. Proc. Natl Acad. Sci. USA 109, 2078–2083 (2012).
Dalbagni, G., Presti, J., Reuter, V., Fair, W. R. & Cordon-Cardo, C. Genetic alterations in bladder cancer. Lancet 342, 469–471 (1993).
Spruck, C. H. III et al. Two molecular pathways to transitional cell carcinoma of the bladder. Cancer Res. 54, 784–788 (1994).
Cheng, L., Cheville, J. C., Neumann, R. M. & Bostwick, D. G. Flat intraepithelial lesions of the urinary bladder. Cancer 88, 625–631 (2000).
Cheng, L. et al. Urothelial papilloma of the bladder. Clinical and biologic implications. Cancer 86, 2098–2101 (1999).
Mitra, A. P. & Cote, R. J. Molecular pathogenesis and diagnostics of bladder cancer. Annu. Rev. Pathol. 4, 251–285 (2009).
Jost, S. P., Gosling, J. A. & Dixon, J. S. The morphology of normal human bladder urothelium. J. Anat. 167, 103–115 (1989).
Gandhi, D. et al. Retinoid signalling in progenitors controls specification and regeneration of the urothelium. Dev. Cell 26, 469–482 (2013).
Yamany, T., van Batavia, J. & Mendelsohn, C. Formation and regeneration of the urothelium. Curr. Opin. Organ Transplant. (2014).
Khandelwal, P., Abraham, S. N. & Apodaca, G. Cell biology and physiology of the uroepithelium. Am. J. Physiol. Renal. Physiol. 297, F1477–F1501 (2009).
Sun, T. T., Liang, F. X. & Wu, X. R. Uroplakins as markers of urothelial differentiation. Adv. Exp. Med. Biol. 462, 7–18 (1999) discussion 103–114
Kong, X-T. et al. Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases. J. Cell. Biol. 167, 1195–1204 (2004).
Jost, S. P. Cell cycle of normal bladder urothelium in developing and adult mice. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 57, 27–36 (1989).
Jost, S. P. Renewal of normal urothelium in adult mice. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 51, 65–70 (1986).
Kreft, M. E., Hudoklin, S., Jezernik, K. & Romih, R. Formation and maintenance of blood-urine barrier in urothelium. Protoplasma 246, 3–14 (2010).
Mysorekar, I. U., Mulvey, M. A., Hultgren, S. J. & Gordon, J. I. Molecular regulation of urothelial renewal and host defenses during infection with uropathogenic Escherichia coli. J. Biol. Chem. 277, 7412–7419 (2002).
Farsund, T. & Dahl, E. Cell kinetics of mouse urinary bladder epithelium. III. A histologic and ultrastructural study of bladder epithelium during regeneration after a single dose of cyclophosphamide, with special reference to the mechanism by which polyploid cells are formed. Virchows Arch. B Cell Pathol. 26, 215–223 (1978).
Jost, S. P. & Potten, C. S. Urothelial proliferation in growing mice. Cell Tissue Kinet. 19, 155–160 (1986).
Shin, K. et al. Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma. Nat. Cell Biol. 16, 469–478 (2014).
Shin, K. et al. Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder. Nature 472, 110–114 (2011).
Indra, A. K. et al. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen- inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 27, 4324–4327 (1999).
Shen, T. H. et al. A BAC-based transgenic mouse specifically expresses an inducible Cre in the urothelium. PLoS ONE 7, e35243 (2012).
Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71 (1999).
Cancer Genome Atlas Research Network, Comprehensive molecular characterization of urothelial bladder carcinoma. Nature 507, 315–322 (2014).
Shariat, S. F. et al. Prognostic value of P53 nuclear accumulation and histopathologic features in T1 transitional cell carcinoma of the urinary bladder. Urology 56, 735–740 (2000).
Gao, J. et al. p53 deficiency provokes urothelial proliferation and synergizes with activated Ha-ras in promoting urothelial tumorigenesis. Oncogene 23, 687–696 (2004).
Choi, W. et al. Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy. Cancer Cell 25, 152–165 (2014).
Ogawa, K. et al. Comparison of uroplakin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats and mice. Toxicol. Pathol. 27, 645–651 (1999).
Vasconcelos-Nobrega, C., Colaco, A., Lopes, C. & Oliveira, P. A. Review: BBN as an urothelial carcinogen. In vivo 26, 727–739 (2012).
Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).
Zupancic, D., Ovcak, Z., Vidmar, G. & Romih, R. Altered expression of UPIa, UPIb, UPII, and UPIIIa during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats. Virchows Arch. (2011).
Ozaki, K. et al. High susceptibility of p53(+/ −) knockout mice in N-butyl-N-(4-hydroxybutyl)nitrosamine urinary bladder carcinogenesis and lack of frequent mutation in residual allele. Cancer Res. 58, 3806–3811 (1998).
He, Z., Kosinska, W., Zhao, Z. L., Wu, X. R. & Guttenplan, J. B. Tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine as the basis for urothelial carcinogenesis. Mutat. Res. 742, 92–95 (2012).
Bertram, J. S. & Craig, A. W. Specific induction of bladder cancer in mice by butyl-(4-hydroxybutyl)-nitrosamine and the effects of hormonal modifications on the sex difference in response. Eur. J. Cancer 8, 587–594 (1972).
Freedman, N. D., Silverman, D. T., Hollenbeck, A. R., Schatzkin, A. & Abnet, C. C. Association between smoking and risk of bladder cancer among men and women. J. Am. Med. Assoc. 306, 737–745 (2011).
Madeb, R. & Messing, E. M. Gender, racial and age differences in bladder cancer incidence and mortality. Urol. Oncol. 22, 86–92 (2004).
Hsu, J. W. et al. Decreased tumorigenesis and mortality from bladder cancer in mice lacking urothelial androgen receptor. Am. J. Pathol. 182, 1811–1820 (2013).
Edgecombe, A., Nguyen, B. N., Djordjevic, B., Belanger, E. C. & Mai, K. T. Utility of cytokeratin 5/6, cytokeratin 20, and p16 in the diagnosis of reactive urothelial atypia and noninvasive component of urothelial neoplasia. Appl. Immunohistochem. Mol Morphol. 20, 264–271 (2012).
Yin, H., He, Q., Li, T. & Leong, A. S. Cytokeratin 20 and Ki-67 to distinguish carcinoma in situ from flat non-neoplastic urothelium. Appl. Immunohistochem. Mol. Morphol. 14, 260–265 (2006).
Montironi, R. & Lopez-Beltran, A. The 2004 WHO classification of bladder tumors: A summary and commentary. Int. J. Surg. Pathol. 13, 143–153 (2005).
Montironi, R., Mazzucchelli, R., Scarpelli, M., Lopez-Beltran, A. & Cheng, L. Morphological diagnosis of urothelial neoplasms. J. Clin. Pathol. 61, 3–10 (2008).
Cheville, J. C. et al. Inverted urothelial papilloma: is ploidy, MIB-1 proliferative activity, or p53 protein accumulation predictive of urothelial carcinoma? Cancer 88, 632–636 (2000).
McConkey, D. J. et al. Molecular genetics of bladder cancer: Emerging mechanisms of tumor initiation and progression. Urol. Oncol. 28, 429–440 (2010).
Morrison, C. D. et al. Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer. Proc. Natl Acad. Sci. USA 111, E672–E681 (2014).
Herr, H. W. Tumor progression and survival of patients with high grade, noninvasive papillary (TaG3) bladder tumors: 15-year outcome. J. Urol. 163, 60–61 (2000).
Gui, Y. et al. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder. Nat. Genet. 43, 875–878 (2011).
Kaufman, D. S., Shipley, W. U. & Feldman, A. S. Bladder cancer. Lancet 374, 239–249 (2009).
Bamgbola, O. F. Urinary schistosomiasis. Pediatr. Nephrol. (2014)10.1007/s00467-013-2723-1
Michaud, D. S. Chronic inflammation and bladder cancer. Urol. Oncol. 25, 260–268 (2007).
Locke, J. R., Hill, D. E. & Walzer, Y. Incidence of squamous cell carcinoma in patients with long-term catheter drainage. J. Urol. 133, 1034–1035 (1985).
Hammes, J. S., Bestoso, J. T. & Sharma, A. Squamous cell carcinoma in situ arising at the exit site of a tunneled catheter. Am. J. Kidney Dis. 44, e43–e46 (2004).
Delnay, K. M., Stonehill, W. H., Goldman, H., Jukkola, A. F. & Dmochowski, R. R. Bladder histological changes associated with chronic indwelling urinary catheter. J. Urol. 161, 1106–1108 (1999).
Ray, D. et al. Transcriptional profiling of the bladder in urogenital schistosomiasis reveals pathways of inflammatory fibrosis and urothelial compromise. PLoS Neglect. Trop. Dis. 6, e1912 (2012).
Donehower, L. A. et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215–221 (1992).
We thank W. Finstad for help with data collection, D. Metzger and P. Chambon for the Krt5CreERT2 line, F. H. Li for technical assistance, the Herbert Irving Comprehensive Cancer Center Histology Core facility for paraffin embedding and the Herbert Irving Comprehensive Cancer Center Microscopy Core for help with confocal microscopy. We thank C. Abate-Shen for critical reading of the manuscript and M. Benson, T. Owczarek and J. Mckiernan for helpful discussions. This work was funded by The TJ Martell Foundation (C.M.) and a Dean’s Research Fellowship from Columbia University (T.Y.).
The authors declare no competing financial interests.
Integrated supplementary information
A bar graph showing the percentage of labelled cells after Tamoxifen (Tm)-induction in Upk2LZ, Krt5Tom and Krt5LZ mice. Tamoxifen dependent recombination in Upk2LZ mice was on average 29% (SD = 8.5,n = 3 mice); 39% on average in K5Tom mice (SD = 15,n = 8), and 28% in K5LZ mice (SD = 10,n = 3). Analysis of Tamoxifen-treated adult Upk2CreERT2;LacZ;p53+/− (Upk2LZ mice) revealed Cre-dependent recombination in 29% of the Intermediate and Superficial cell populations (n = 3 animals, SD = 8.5), and labelled cells were rare in the basal layer (less than or equal to 0.05%; 9/17,454, n = 15). Analysis of Krt5CreERT2LacZ;p53+/− (K5LZ) and Krt5CreERT2;mTom(K5Tom) lines indicates that in both, recombination was Tamoxifen-dependent and specific (Fig. 1g, h, k, l). We observed expression of the lineage tag on average in 39% of the K5-basal cell population in K5Tom mice (n = 8, SD = 15) and 28% of K5-basal cells in K5LZ mice (n = 3, SD = 10). Analysis of the distribution of labelled K5-basal cells and their descendants in Tamoxifen-induced K5Tom and K5LZ mice revealed that Cre-dependent recombination was selectively localized; rare labelled cells were present in the intermediate layer and no detectable labelled cells in the superficial layer (2/13352, labelled Intermediate cells, 0.01%; 0/13352 Superficial cells, n = 16).
Supplementary Figure 2 Characteristic response to BBN treatment separated by weeks of BBN exposure, sex and p53 status.
(A-F) H&E stained section through p53+/− male mice demonstrating CIS at 16 weeks (A, B), a high grade papillary lesion at 20 weeks (C, D) and invasion at 24 weeks (E, F). (G-L) H&E stained section through p53+/− female mice demonstrating dysplasia at 16 weeks (G, H), CIS at 20 weeks (I, J) and invasion at 24 weeks (K, L). (M-R) H&E stained section through p53+/+ male mice demonstrating squamous metaplasia at 16 weeks (M, N), SCC at 20 weeks (O, P) and invasive SCC at 24 weeks (Q, R). (S-X) H&E stained section through p53+/+ female mice demonstrating squamous metaplasia and SCC at 16 weeks (S, T), papillary SCC at 20 weeks (U, V) and SCC invasion into the muscle and fat at 24 (W, X) weeks. Abbreviations: CIS = Carcinoma in situ; SCC = Squamous cell carcinoma. Magnifications: A, C, E, G, I, K, M, O, Q, S, U, W X04; B, H, J X20 zoomed; D, F, N, P, R, T, V, X X20; L X10. Scale bars: A, C, E, G, I, K, M, O, Q, S, U, W 500 μm; B, H, J: 50 μm; D, F, N, P, R, T, V, X: 100 μm; L: 200 μm.
About this article
Cite this article
Van Batavia, J., Yamany, T., Molotkov, A. et al. Bladder cancers arise from distinct urothelial sub-populations. Nat Cell Biol 16, 982–991 (2014). https://doi.org/10.1038/ncb3038
Combined exome and transcriptome sequencing of non-muscle-invasive bladder cancer: associations between genomic changes, expression subtypes, and clinical outcomes
Genome Medicine (2022)
Targeting WD repeat domain 5 enhances chemosensitivity and inhibits proliferation and programmed death-ligand 1 expression in bladder cancer
Journal of Experimental & Clinical Cancer Research (2021)
Dominant role of CDKN2B/p15INK4B of 9p21.3 tumor suppressor hub in inhibition of cell-cycle and glycolysis
Nature Communications (2021)
Development and validation of a molecular prognostic index of bladder cancer based on immunogenomic landscape analysis
Cancer Cell International (2020)
Hypermethylation of FOXA1 and allelic loss of PTEN drive squamous differentiation and promote heterogeneity in bladder cancer