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

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.

Figure 1

Expression levels of FGFR3 in bladder tumours and bladder cancer cell lines. FGFR3 transcript levels were measured by real-time RT-PCR and normalised to SDHA. Values represent fold difference compared to FGFR3 transcript levels in NHUC. A, FGFR3 transcript levels were measured in 34 pTa, 19 pT1 and 12 pT2 bladder tumours. B, the tumours were separated according to grade; 31 grade 2 and 33 grade 3. Only one grade 1 tumour was harvested and therefore not included in the graph. C, FGFR3 transcript levels were measured in 26 bladder cancer cell lines and the fold change was represented using log₁₀. The bars for bladder cancer cell lines containing FGFR3 mutations are shown in grey.

Figure 2

Retrovirus-mediated delivery of shRNA in bladder cancer cells. A, FGFR3 transcript levels in 97-7 cells transduced with vector, non-specific shRNA or FGFR3 shRNA retroviruses were measured by real-time RT-PCR. B, lysates from 97-7 cells harvested three and seven weeks post-transduction and FGFR3 protein levels and examined by western blot.

Figure 3

S249C FGFR3b is dimerized and phosphorylated and its knockdown induces cell flattening. A, protein lysates from TERT-NHUC over-expressing FGFR3b and 97-7 were western blotted with (+) and without (−) denaturation. (−) samples contained no ß-mercaptoethanol and were not heat denatured. S249C mutation caused stabilization of dimers compared to wildtype protein (arrows). B, protein lysates were immunoprecipitated with FGFR3 E antibody and blotted with anti-phosphotyrosine 4G10 (pTYR) antibody. Blots were stripped and re-probed with the FGFR3 B9 antibody as a loading control. C, phase contrast micrograph of 97-7 cells ten days post-transduction. (bar=100μm). D, immunofluorescence images of alpha-tubulin demonstrating changes in the cytoskeleton of S249C FGFR3b knockdown cells compared to non-specific shRNA (bar=30μm).

Figure 4

Differential effects of knockdown of S249C FGFR3b in tumour cells and of wild-type FGFR3b in normal cells. 97-7 cells (A) or TERT-NHUC (B) transduced with vector, non-specific shRNA or FGFR3 shRNA retroviruses were plated at 3×10⁴ cells per well in triplicate in full medium and counted at three time points up to day six. Cells expressing vector are represented by square points and a solid line, cells expressing non-specific shRNA are represented by triangle points and a dashed line, cells expressing FGFR3 shRNA are represented by round points and a solid line.

Figure 5

FGFR3 knockdown reduces clonogenicity of 97-7 cells. A, 12 day colonies from cells transduced with vector, non-specific or shRNA retroviruses (3000 cells plated). B, viable colonies in agarose were stained with p-iodonitrotretrazolium violet. The grey and black bars represent the number of viable colonies < 1mm² or > 1mm², respectively.

Figure 6

Re-expression of S249C FGFR3b reverses phenotypic changes in FGFR3 knockdown 97-7 cells. A, phase contrast micrographs of 97-7 cells expressing shRNA and shRNA expressing cells transduced with retrovirus expressing S249C FGFR3b (bar=100μm). Pictures at ten days post transduction. B, western blot showing FGFR3b expression in 97-7 cells and in shRNA cells re-expressing S249C FGFR3b. C, fold change in cell number over four days. D, Number of colonies at low density and in soft agar. For low density, 500 cells were plated in 10 cm dishes and colonies counted on day 12. For soft agar, viable colonies were stained with p-iodonitrotretrazolium violet at 21 days. Graph represents the number of viable colonies per 10cm².