Darren C. Tomlinson

FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequent in superficial urothelial cell carcinoma (UCC). Ras gene mutations are also found in UCC. As oncogenic activation of both FGFR3 and Ras is predicted to result in stimulation of the mitogen-activated protein kinase (MAPK) pathway, we hypothesized that these might be mutually exclusive events. HRAS mutation has been widely studied in UCC, but all three Ras gene family members have not been screened for mutation in the same sample series. We screened 98 bladder tumours and 31 bladder cell lines for mutations in FGFR3, HRAS, NRAS and KRAS2. FGFR3 mutations were present in 54 tumours (55%) and three cell lines (10%), and Ras gene mutations in 13 tumours (13%) and four cell lines (13%). These included mutations in all three Ras genes; ten in HRAS, four in KRAS2 and four in NRAS and these were not associated with either tumour grade or stage. In no cases were Ras and FGFR3 mutation found together. This mutual exclusion suggests that FGFR3 and Ras gene mutation may represent alternative means to confer the same phenotype on UCC cells. If these events have biological equivalence, Ras mutant invasive UCC may represent a novel subgroup.

FGFR3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer

FGFR3 is frequently activated by mutation in urothelial carcinoma (UC) and represents a potential target for therapy. In multiple myeloma, both over‐expression and mutation of FGFR3 contribute to tumour development. To define the population of UC patients who may benefit from FGFR‐targeted therapy, we assessed both mutation and receptor over‐expression in primary UCs from a population of new patients. Manual or laser capture microdissection was used to isolate pure tumour cell populations. Where present, non‐invasive and invasive components in the same section were microdissected. A screen of the region of the highest tumour stage in each sample yielded a mutation frequency of 42%. Mutations comprised 61 single and five double mutations, all in hotspot codons previously identified in UC. There was a significant association of mutation with low tumour grade and stage. Subsequently, non‐invasive areas from the 43 tumours with both non‐invasive and invasive components were analysed separately; 18 of these had mutation in at least one region, including nine with mutation in all regions examined, eight with mutation in only the non‐invasive component and one with different mutations in different regions. Of the eight with mutation in only the non‐invasive component, six were predicted to represent a single tumour and two showed morphological dissimilarity of fragments within the block, indicating the possible presence of distinct tumour clones. Immunohistochemistry showed over‐expression of FGFR3 protein in many tumours compared to normal bladder and ureteric controls. Increased expression was associated with mutation (85% of mutant tumours showed high‐level expression). Overall, 42% of tumours with no detectable mutation showed over‐expression, including many muscle‐invasive tumours. This may represent a non‐mutant subset of tumours in which FGFR3 signalling contributes to the transformed phenotype and which may benefit from FGFR‐targeted therapies. Copyright

Small molecule FGF receptor inhibitors block FGFR-dependent urothelial carcinoma growth in vitro and in vivo

Background:Activating mutations of FGFR3 are frequently identified in superficial urothelial carcinoma (UC) and increased expression of FGFR1 and FGFR3 are common in both superficial and invasive UC.Methods:The effects of inhibition of receptor activity by three small molecule inhibitors (PD173074, TKI-258 and SU5402) were investigated in a panel of bladder tumour cell lines with known FGFR expression levels and FGFR3 mutation status.Results:All inhibitors prevented activation of FGFR3, and inhibited downstream MAPK pathway signalling. Response was related to FGFR3 and/or FGFR1 expression levels. Cell lines with the highest levels of FGFR expression showed the greatest response and little or no effect was measured in normal human urothelial cells or in UC cell lines with activating RAS gene mutations. In sensitive cell lines, the drugs induced cell cycle arrest and/or apoptosis. IC50 values for PD173074 and TKI-258 were in the nanomolar concentration range compared with micromolar concentrations for SU5402. PD173074 showed the greatest effects in vitro and in vivo significantly delayed the growth of subcutaneous bladder tumour xenografts.Conclusion:These results indicate that inhibition of FGFR1 and wild-type or mutant FGFR3 may represent a useful therapeutic approach in patients with both non-muscle invasive and muscle invasive UC.

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

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.

Fibroblast Growth Factor Receptor 1 Promotes Proliferation and Survival via Activation of the Mitogen-Activated Protein Kinase Pathway in Bladder Cancer

Fibroblast growth factor receptors (FGFR) play key roles in proliferation, differentiation, and tumorigenesis. Many urothelial carcinomas contain activating point mutations or increased expression of FGFR3. However, little is known about the role of other FGFRs. We examined FGFR expression in telomerase-immortalized normal human urothelial cells, urothelial carcinoma cell lines, and tumor samples and showed that FGFR1 expression is increased in a high proportion of cell lines and tumors independent of stage and grade. To determine the role of FGFR1 in low-stage bladder cancer, we overexpressed FGFR1 in telomerase-immortalized normal human urothelial cells and examined changes in proliferation and cell survival in response to FGF2. FGFR1 stimulation increased proliferation and reduced apoptosis. To elucidate the mechanistic basis for these alterations, we examined the signaling cascades activated by FGFR1. FRS2alpha and PLCgamma were activated in response to FGF2, leading to activation of the mitogen-activated protein kinase pathway. The level of mitogen-activated protein kinase activation correlated with the level of cyclin D1, MCL1, and phospho-BAD, which also correlated with FGFR-induced proliferation and survival. Knockdown of FGFR1 in urothelial carcinoma cell lines revealed differential FGFR1 dependence. JMSU1 cells were dependent on FGFR1 expression for survival but three other cell lines were not. Two cell lines (JMSU1 and UMUC3) were dependent on FGFR1 for growth in soft agar. Only one of the cell lines tested (UMUC3) was frankly tumorigenic; here, FGFR1 knockdown inhibited tumor growth. Our results indicate that FGFR1 has significant effects on urothelial cell phenotype and may represent a useful therapeutic target in some cases of urothelial carcinoma.

Inactivation of the Rb pathway and overexpression of both isoforms of E2F3 are obligate events in bladder tumours with 6p22 amplification

E2F3 and CDKAL1 are candidate genes from the 6p22 region frequently amplified in bladder cancer. Expression of E2F3 isoforms (E2F3a and b) and CDKAL1 were examined and modulated in 6p22-amplified bladder cell lines. Eight lines with amplification showed overexpression of both E2F3 isoforms and CDKAL1. shRNA-mediated knockdown of CDKAL1 had no effect on proliferation. Knockdown of E2F3a or E2F3b alone induced antiproliferative effects, with the most significant effect on proliferation being observed when both isoforms were knocked down together. As E2Fs interact with the Rb tumour suppressor protein, Rb expression was analysed. There was a striking relationship between 6p22.3 amplification, E2F3 overexpression and lack of Rb expression. This was also examined in primary bladder tumours. Array-CGH detected 6p22.3 amplification in 8/91 invasive tumours. Five were studied in more detail. Four showed 13q14.2 loss (including RB1) and expressed no Rb protein. In the fifth, 13q was unaltered but the CDKN2A locus was deleted. This tumour was negative for p16 and positive for Rb protein. As p16 is a negative regulator of the Rb pathway, its loss represents an alternative mechanism for inactivation. Indeed, a phospho-specific Rb antibody showed much Rb protein in a hyperphosphorylated (inactive) form. We conclude that inactivation of the Rb pathway is required in addition to E2F3 overexpression in this subset of bladder tumours.

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin–proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

A Decade of FGF Receptor Research in Bladder Cancer: Past, Present, and Future Challenges

Fibroblast growth factors (FGFs) orchestrate a variety of cellular functions by binding to their transmembrane tyrosine-kinase receptors (FGFRs) and activating downstream signalling pathways, including RAS/MAPK, PLCγ1, PI3K, and STATs. In the last ten years, it has become clear that FGF signalling is altered in a high proportion of bladder tumours. Activating mutations and/or overexpression of FGFR3 are common in urothelial tumours with low malignant potential and low-stage and -grade urothelial carcinomas (UCs) and are associated with a lower risk of progression and better survival in some subgroups. FGFR1 is not mutated in UC, but overexpression is frequent in all grades and stages and recent data indicate a role in urothelial epithelial-mesenchymal transition. In vitro and in vivo studies have shown that FGFR inhibition has cytotoxic and/or cytostatic effects in FGFR-dependent bladder cancer cells and FGFR-targeted agents are currently being investigated in clinical studies for the treatment of UC. Urine-based tests detecting common FGFR3 mutations are also under development for surveillance of low-grade and -stage tumours and for general population screening. Overall, FGFRs hold promise as therapeutic targets, diagnostic and prognostic markers, and screening tools for early detection and clinical management of UC.

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

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that play key roles in proliferation, differentiation, and tumorigenesis. FGFR3 was identified as the major family member expressed in both normal human urothelium and cultured normal human urothelial (NHU) cells and was expressed as the IIIb isoform. We also identified a splice variant, FGFR3 Delta8-10, lacking exons encoding the COOH-terminal half of immunoglobulin-like domain III and the transmembrane domain. Previous reports have assumed that this is a cancer-specific splice variant. We showed that FGFR3 Delta8-10 is a normal transcript in NHU cells and is translated, N-glycosylated, and secreted. Primary urothelium expressed high levels of FGFR3 transcripts. In culture, levels were reduced in actively proliferating cells but increased at confluence and as cells approached senescence. Cells overexpressing FGFR3 IIIb showed FGF1-induced proliferation, which was inhibited by the addition of FGFR3 Delta8-10. In bladder tumor cell lines derived from aggressive carcinomas, there were significant alterations in the relative expression of isoforms including an overall decrease in the proportion of FGFR3 Delta8-10 and predominant expression of FGFR3 IIIc in some cases. In summary, alternative splicing of FGFR3 IIIb in NHU cells represents a normal mechanism to generate a transcript that regulates proliferation and in bladder cancer, the ratio of FGFR3 isoforms is significantly altered.

Mutant fibroblast growth factor receptor 3 induces intracellular signaling and cellular transformation in a cell type- and mutation-specific manner

Although activating mutations of fibroblast growth factor receptor 3 (FGFR3) are frequent in bladder tumors, little information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum. We investigated the phenotypic and signaling consequences of three FGFR3 mutations (S249C, Y375C, and K652E) in immortalized normal human urothelial cells (TERT-NHUC) and mouse fibroblasts (NIH-3T3). In TERT-NHUC, all mutant forms of FGFR3 induced phosphorylation of FRS2α and ERK1/2, but not AKT or SRC. PLCγ1 phosphorylation was only observed in TERT-NHUC expressing the common S249C and Y375C mutations, and not the rare K652E mutation. Cells expressing S249C and Y375C FGFR3 displayed an increased saturation density, related to increased proliferation and viability. This effect was significantly dependent on PLCγ1 signaling and undetectable in cells expressing K652E FGFR3, which failed to phosphorylate PLCγ1. In contrast to TERT-NHUC, expression of mutant FGFR3 in NIH-3T3 resulted in phosphorylation of Src and Akt. In addition, all forms of mutant FGFR3 were able to phosphorylate Plcγ1 and induce morphological transformation, cell proliferation, and anchorage-independent growth. Our results indicate that the effects of mutant FGFR3 are both cell type specific and mutation specific. Mutant FGFR3 may confer a selective advantage in the urothelium by overcoming normal contact inhibition of proliferation.