Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Knockdown by shRNA identifies S249C mutant FGFR3 as a potential therapeutic target in bladder cancer

Oncogene volume 26, pages 5889–5899 (2007)Cite this article

Abstract

More than 60% of low-grade non-invasive papillary urothelial cell carcinomas contain activating point mutations of fibroblast growth factor receptor 3 (FGFR3). The phenotypic consequences of constitutive activation of FGFR3 in bladder cancer have not been elucidated and further studies are required to confirm the consequences of inhibiting receptor activity in urothelial cells. We measured FGFR3 transcript levels and demonstrated that transcript levels were significantly more abundant in low-stage and grade tumours. We identified a tumour cell line, 97-7, expressing the most common FGFR3 mutation (S249C) at similar FGFR3 transcript levels to low-stage and grade tumours. In these cells, S249C FGFR3 protein formed stable homodimers and was constitutively phosphorylated. We used retrovirus-mediated delivery of shRNA to knockdown S249C FGFR3. This induced cell flattening, decreased cell proliferation and reduced clonogenicity on plastic and in soft agar. However, no effects of knockdown of wild-type FGFR3 were observed in telomerase immortalized normal human urothelial cells, indicating possible dependence of the tumour cell line on mutant FGFR3. Re-expression of S249C FGFR3 in shRNA-expressing 97-7 cells resulted in a reversal of phenotypic changes, confirming the specificity of the shRNA. These results indicate that targeted inhibition of S249C FGFR3 may represent a useful therapeutic approach in superficial bladder cancer.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  • Adar R, Monsonego-Ornan E, David P, Yayon A . (2002). Differential activation of cysteine-substitution mutants of fibroblast growth factor receptor 3 is determined by cysteine localization. J Bone Miner Res 17: 860–868.

    Article  CAS  PubMed  Google Scholar 

  • Bakkar AA, Wallerand H, Radvanyi F, Lahaye JB, Pissard S, Lecerf L et al. (2003). FGFR3 and TP53 gene mutations define two distinct pathways in urothelial cell carcinoma of the bladder. Cancer Res 63: 8108–8112.

    CAS  PubMed  Google Scholar 

  • Bernard-Pierrot I, Brams A, Dunois-Larde C, Caillault A, Diez de Medina SG, Cappellen D et al. (2006). Oncogenic properties of the mutated forms of fibroblast growth factor receptor 3b. Carcinogenesis 27: 740–747.

    Article  CAS  PubMed  Google Scholar 

  • Billerey C, Chopin D, Aubriot-Lorton MH, Ricol D, Gil Diez de Medina S, Van Rhijn B et al. (2001). Frequent FGFR3 mutations in papillary non-invasive bladder (pTa) tumors. Am J Pathol 158: 1955–1959.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y et al. (2006). 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 3: 199–204.

    Article  CAS  PubMed  Google Scholar 

  • Cappellen D, De Oliveira C, Ricol D, de Medina S, Bourdin J, Sastre-Garau X et al. (1999). Frequent activating mutations of FGFR3 in human bladder and cervix carcinomas. Nat Genet 23: 18–20.

    Article  CAS  PubMed  Google Scholar 

  • Chapman EJ, Hurst CD, Pitt E, Chambers P, Aveyard JS, Knowles MA . (2006). Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway. Oncogene 25: 5037–5045.

    Article  CAS  PubMed  Google Scholar 

  • Chen J, Lee BH, Williams IR, Kutok JL, Mitsiades CS, Duclos N et al. (2005). FGFR3 as a therapeutic target of the small molecule inhibitor PKC412 in hematopoietic malignancies. Oncogene 24: 8259–8267.

    Article  CAS  PubMed  Google Scholar 

  • Chesi M, Bergsagel PL, Kuehl WM . (2002). The enigma of ectopic expression of FGFR3 in multiple myeloma: a critical initiating event or just a target for mutational activation during tumor progression. Current Opinion Hematol 9: 288–293.

    Article  Google Scholar 

  • Chesi M, Brents LA, Ely SA, Bais C, Robbiani DF, Mesri EA et al. (2001). Activated fibroblast growth factor receptor 3 is an oncogene that contributes to tumor progression in multiple myeloma. Blood 97: 729–736.

    Article  CAS  PubMed  Google Scholar 

  • Chesi M, Nardini E, Brents LA, Schrock E, Ried T, Kuehl WM et al. (1997). Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet 16: 260–264.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chesi M, Nardini E, Lim RS, Smith KD, Kuehl WM, Bergsagel PL . (1998). The t(4;14) translocation in myeloma dysregulates both FGFR3 and a novel gene, MMSET, resulting in IgH/MMSET hybrid transcripts. Blood 92: 3025–3034.

    CAS  PubMed  Google Scholar 

  • d’Avis PY, Robertson SC, Meyer AN, Bardwell WM, Webster MK, Donoghue DJ . (1998). Constitutive activation of fibroblast growth factor receptor 3 by mutations responsible for the lethal skeletal dysplasia thanatophoric dysplasia type I. Cell Growth Differ 9: 71–78.

    PubMed  Google Scholar 

  • Eswarakumar VP, Lax I, Schlessinger J . (2005). Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 16: 139–149.

    Article  CAS  PubMed  Google Scholar 

  • Gomez-Roman JJ, Saenz P, Molina M, Cuevas Gonzalez J, Escuredo K, Santa Cruz S et al. (2005). Fibroblast growth factor receptor 3 is overexpressed in urinary tract carcinomas and modulates the neoplastic cell growth. Clin Cancer Res 11: 459.

    CAS  PubMed  Google Scholar 

  • Grand EK, Chase AJ, Heath C, Rahemtulla A, Cross NC . (2004). Targeting FGFR3 in multiple myeloma: inhibition of t(4;14)-positive cells by SU5402 and PD173074. Leukemia 18: 962–966.

    Article  CAS  PubMed  Google Scholar 

  • Hart KC, Robertson SC, Donoghue DJ . (2001). Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation. Mol Biol Cell 12: 931–942.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hart KC, Robertson SC, Kanemitsu MY, Meyer AN, Tynan JA, Donoghue DJ . (2000). Transformation and Stat activation by derivatives of FGFR1, FGFR3, and FGFR4. Oncogene 19: 3309–3320.

    Article  CAS  PubMed  Google Scholar 

  • Holmang S, Hedelin H, Anderstrom C, Johansson SL . (1995). The relationship among multiple recurrences, progression and prognosis of patients with stages Ta and T1 transitional cell cancer of the bladder followed for at least 20 years. J Urol 153: 1823–1826; discussion 1826–1827.

    Article  CAS  PubMed  Google Scholar 

  • Intini D, Baldini L, Fabris S, Lombardi L, Ciceri G, Maiolo AT et al. (2001). Analysis of FGFR3 gene mutations in multiple myeloma patients with t(4;14). Br J Haematol 114: 362–364.

    Article  CAS  PubMed  Google Scholar 

  • Jebar AH, Hurst CD, Tomlinson DC, Johnston C, Taylor CF, Knowles MA . (2005). FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma. Oncogene 24: 5218–5225.

    Article  CAS  PubMed  Google Scholar 

  • Keats JJ, Maxwell CA, Taylor BJ, Hendzel MJ, Chesi M, Bergsagel PL et al. (2005). Overexpression of transcripts originating from the MMSET locus characterizes all t(4;14)(p16;q32)-positive multiple myeloma patients. Blood 105: 4060–4069.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kimura T, Suzuki H, Ohashi T, Asano K, Kiyota H, Eto Y . (2001). The incidence of thanatophoric dysplasia mutations in FGFR3 gene is higher in low-grade or superficial bladder carcinomas. Cancer 92: 2555–2561.

    Article  CAS  PubMed  Google Scholar 

  • Knowles MA . (2006). Molecular subtypes of bladder cancer: Jekyll and Hyde or chalk and cheese? Carcinogenesis 27: 361–373.

    Article  CAS  PubMed  Google Scholar 

  • Kurth KH, Denis L, Bouffioux C, Sylvester R, Debruyne FM, Pavone-Macaluso M et al. (1995). Factors affecting recurrence and progression in superficial bladder tumours. Eur J Cancer 31A: 1840–1846.

    Article  CAS  PubMed  Google Scholar 

  • Logie A, Dunois-Larde C, Rosty C, Levrel O, Blanche M, Ribeiro A et al. (2005). Activating mutations of the tyrosine kinase receptor FGFR3 are associated with benign skin tumors in mice and humans. Human Mol Genetics 14: 1153.

    Article  CAS  Google Scholar 

  • Martinez-Torrecuadrada J, Cifuentes G, Lopez-Serra P, Saenz P, Martinez A, Casal JI . (2005). Targeting the extracellular domain of fibroblast growth factor receptor 3 with human single-chain Fv antibodies inhibits bladder carcinoma cell line proliferation. Clin Cancer Res 11: 6280–6290.

    Article  CAS  PubMed  Google Scholar 

  • Matsukura S, Jones PA, Takai D . (2003). Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acids Research 31: e77.

    Article  PubMed  PubMed Central  Google Scholar 

  • Matsumoto M, Ohtsuki Y, Ochii K, Seike Y, Iseda N, Sasaki T et al. (2004). Fibroblast growth factor receptor 3 protein expression in urothelial carcinoma of the urinary bladder, exhibiting no association with low-grade and/or non-invasive lesions. Oncol Rep 12: 967.

    CAS  PubMed  Google Scholar 

  • Mhawech-Fauceglia P, Cheney RT, Fischer G, Beck A, Herrmann FR . (2006). FGFR3 and p53 protein expressions in patients with pTa and pT1 urothelial bladder cancer. Eur J Surg Oncol 32: 231–237.

    Article  CAS  PubMed  Google Scholar 

  • Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F et al. (1996). Receptor specificity of the fibroblast growth factor family. J Biol Chem 271: 15292–15297.

    Article  CAS  PubMed  Google Scholar 

  • Parkin DM, Bray F, Ferlay J, Pisani P . (2005). Global cancer statistics, 2002. CA Cancer J Clin 55: 74–108.

    Article  PubMed  Google Scholar 

  • Paterson JL, Li Z, Wen XY, Masih-Khan E, Chang H, Pollett JB et al. (2004). Preclinical studies of fibroblast growth factor receptor 3 as a therapeutic target in multiple myeloma. Br J Haematol 124: 595–603.

    Article  CAS  PubMed  Google Scholar 

  • Richelda R, Ronchetti D, Baldini L, Cro L, Viggiano L, Marzella R et al. (1997). A novel chromosomal translocation t(4; 14)(p16.3; q32) in multiple myeloma involves the fibroblast growth-factor receptor 3 gene. Blood 90: 4062–4070.

    CAS  PubMed  Google Scholar 

  • Ronchetti D, Greco A, Compasso S, Colombo G, Dell’Era P, Otsuki T et al. (2001). Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations. Oncogene 20: 3553–3562.

    Article  CAS  PubMed  Google Scholar 

  • Sarkar S, Julicher KP, Burger MS, Della Valle V, Larsen CJ, Yeager TR et al. (2000). Different combinations of genetic/epigenetic alterations inactivate the p53 and pRb pathways in invasive human bladder cancers. Cancer Res 60: 3862–3871.

    CAS  PubMed  Google Scholar 

  • Scotet E, Houssaint E . (1995). The choice between alternative IIIb and IIIc exons of the FGFR-3 gene is not strictly tissue-specific. Biochim Biophys Acta 1264: 238–242.

    Article  PubMed  Google Scholar 

  • Sibley K, Bell S, Knowles MA . (2000). Redefining a critical region of LOH on 4p16.3 in bladder cancer. Genes Chromosomes Cancer 29: 378–379.

    Article  CAS  PubMed  Google Scholar 

  • Southgate J, Hutton KA, Thomas DF, Trejdosiewicz LK . (1994). Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab Invest 71: 583–594.

    CAS  PubMed  Google Scholar 

  • Strausberg RL, Simpson AJ, Old LJ, Riggins GJ . (2004). Oncogenomics and the development of new cancer therapies. Nature 429: 469–474.

    Article  CAS  PubMed  Google Scholar 

  • Tomlinson DC, L’Hote CG, Kennedy W, Pitt E, Knowles MA . (2005). Alternative splicing of fibroblast growth factor receptor 3 produces a secreted isoform that inhibits fibroblast growth factor-induced proliferation and is repressed in urothelial carcinoma cell lines. Cancer Res 65: 10441–10449.

    Article  CAS  PubMed  Google Scholar 

  • Trudel S, Ely S, Farooqi Y, Affer M, Robbiani DF, Chesi M et al. (2004). Inhibition of fibroblast growth factor receptor 3 induces differentiation and apoptosis in t(4;14) myeloma. Blood 103: 3521–3528.

    Article  CAS  PubMed  Google Scholar 

  • Trudel S, Li ZH, Wei E, Wiesmann M, Chang H, Chen C et al. (2005). CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the potential treatment of t(4;14) multiple myeloma. Blood 105: 2941.

    Article  CAS  PubMed  Google Scholar 

  • Trudel S, Stewart AK, Rom E, Wei E, Li ZH, Kotzer S et al. (2006). The inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t(4;14) multiple myeloma cells. Blood 107: 4039–4046.

    Article  CAS  PubMed  Google Scholar 

  • van Rhijn BW, Lurkin I, Chopin DK, Kirkels WJ, Thiery JP, van der Kwast TH et al. (2003). Combined microsatellite and FGFR3 mutation analysis enables a highly sensitive detection of urothelial cell carcinoma in voided urine. Clin Cancer Res 9: 257–263.

    CAS  PubMed  Google Scholar 

  • van Rhijn BW, Lurkin I, Radvanyi F, Kirkels WJ, van der Kwast TH, Zwarthoff EC . (2001). The fibroblast growth factor receptor 3 (FGFR3) mutation is a strong indicator of superficial bladder cancer with low recurrence rate. Cancer Res 61: 1265–1268.

    CAS  PubMed  Google Scholar 

  • Voorhoeve PM, Agami R . (2003). The tumor-suppressive functions of the human INK4A locus. Cancer Cell 4: 311–319.

    Article  CAS  PubMed  Google Scholar 

  • Webster MK, Donoghue DJ . (1997). FGFR activation in skeletal disorders: too much of a good thing. Trends Genet 13: 178–182.

    Article  CAS  PubMed  Google Scholar 

  • Weinstein IB . (2002). Cancer. Addiction to oncogenes – the Achilles heal of cancer. Science 297: 63–64.

    Article  CAS  PubMed  Google Scholar 

  • Wu XR . (2005). Urothelial tumorigenesis: a tale of divergent pathways. Nat Rev Cancer 5: 713–725.

    Article  CAS  PubMed  Google Scholar 

  • Zhu L, Somlo G, Zhou B, Shao J, Bedell V, Slovak ML et al. (2005). Fibroblast growth factor receptor 3 inhibition by short hairpin RNAs leads to apoptosis in multiple myeloma. Mol Cancer Ther 4: 787.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr Catherine Reznikoff for generously providing the cell lines 97-7, 97-18, 97-29, 96-1, 97-24 and 97-1, Dr D Podolsky for providing FGFR3b cDNA, Dr R Agami for pRS-hyg and Dr Eva Pitt for excellent tissue culture support. This work was funded by Cancer Research UK.

Author information

Authors and Affiliations

  1. Cancer Research UK Clinical Centre, St James's University Hospital, Leeds, UK

    D C Tomlinson, C D Hurst & M A Knowles

Authors
  1. D C Tomlinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C D Hurst
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M A Knowles
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M A Knowles.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tomlinson, D., Hurst, C. & Knowles, M. Knockdown by shRNA identifies S249C mutant FGFR3 as a potential therapeutic target in bladder cancer. Oncogene 26, 5889–5899 (2007). https://doi.org/10.1038/sj.onc.1210399

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.onc.1210399

Keywords

This article is cited by

Search

Advanced search

Quick links