Tomoko Iwata

Fibroblast growth factor signaling in development of the cerebral cortex

Despite substantial and exciting recent progress in our understanding of developmental processes in the cerebral cortex, there is still much to be learned about the molecular and cellular mechanisms that account for formation of the cortical structures, and in turn, how the regulation of these mechanisms is linked to cortical functions and behaviors in animals and humans. Fibroblast growth factors (FGFs) are a classic family of growth factors that are important in neural development and whose structures and signaling have been well‐studied molecularly and biochemically. Recent advances have revealed their diverse but specific functions in patterning and neurogenesis during cortical development, as evidenced by multiple experimental approaches using in vivo models. Importantly, changes in FGF signaling during development have been shown to influence structure and function of the cerebral cortex as well as animal behavior, and have been implicated in disorders of nervous system function and intellectual development in humans. For example, disturbance of FGF pathways during development has been implicated in the pathogenesis of autism spectrum disorders. Experimental models with altered cortical structure due to perturbations of FGF signaling present a unique opportunity whereby molecular and cellular mechanisms that underlie cortical function and animal behavior can be directly studied and linked to each other.

Mechanisms of FGFR-mediated carcinogenesis

In this review, the evidence for a role of fibroblast growth factor receptor (FGFR) mediated signalling in carcinogenesis are considered and relevant underlying mechanisms highlighted. FGF signalling mediated by FGFR follows a classic receptor tyrosine kinase signalling pathway and its deregulation at various points of its cascade could result in malignancy. Here we review the accumulating reports that revealed the association of FGF/FGFRs to various types of cancer at a genetic level, along with in vitro and in vivo evidences available so far, which indicates the functional involvement of FGF signalling in tumour formation and progression. An increasing number of drugs against the FGF pathways is currently in clinical testing. We will discuss the strategies for future FGF research in cancer and translational approaches.

Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth

Hepatocyte growth factor (HGF)/Met signaling controls cell migration, growth and differentiation in several embryonic organs and is implicated in human cancer. The physiologic mechanisms that attenuate Met signaling are not well understood. Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons, which suggests a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rac interactive binding site to exert its inhibitory action, which suggests that Mig6 acts, at least in part, distally from Met, possibly by inhibiting Rho-like GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

Fibroblast growth factor receptor 3 kinase domain mutation increases cortical progenitor proliferation via mitogen‐activated protein kinase activation

We have previously shown that mice carrying the K644E kinase domain mutation in fibroblast growth factor receptor 3 (Fgfr3) (EIIa;Fgfr3+/K644E) have enlarged brains with increased proliferation and decreased apoptosis of the cortical progenitors. Despite its unique rostral‐low caudal‐high gradient expression in the cortex, how Fgfr3 temporally and spatially influences progenitor proliferation is unknown. In vivo BrdU labelling now showed that progenitor proliferation was 10–46% higher in the EIIa;Fgfr3+/K644E cortex compared with wild type during embryonic day 11.5 (E11.5)–E13.5. The difference in proliferation between the EIIa;Fgfr3+/K644E and wild‐type cortices was the greatest in the caudal cortex at E12.5 and E13.5. Inhibition of mitogen‐activated or extracellular signal‐regulated protein kinase (MEK) in vitro at E11.5 reduced the proliferation rate of the EIIa;Fgfr3+/K644E cortical progenitors to similar levels observed in the wild type, indicating that the majority of the increase in cell proliferation caused by the Fgfr3 mutation is mitogen‐activated protein kinase (MAPK) pathway‐dependent at this stage. In addition, elevated levels of Sprouty were observed in the EIIa;Fgfr3+/K644E telencephalon at E14.5, indicating the presence of negative feedback that may have suppressed further MAPK activation. We suggest that temporal activation of MAPK is largely responsible for cell proliferation caused by the Fgfr3 mutation during early stages of cortical development.

K-Ras and β-catenin mutations cooperate with Fgfr3 mutations in mice to promote tumorigenesis in the skin and lung, but not in the bladder.

SUMMARY The human fibroblast growth factor receptor 3 (FGFR3) gene is frequently mutated in superficial urothelial cell carcinoma (UCC). To test the functional significance of FGFR3 activating mutations as a ‘driver’ of UCC, we targeted the expression of mutated Fgfr3 to the murine urothelium using Cre-loxP recombination driven by the uroplakin II promoter. The introduction of the Fgfr3 mutations resulted in no obvious effect on tumorigenesis up to 18 months of age. Furthermore, even when the Fgfr3 mutations were introduced together with K-Ras or β-catenin (Ctnnb1) activating mutations, no urothelial dysplasia or UCC was observed. Interestingly, however, owing to a sporadic ectopic Cre recombinase expression in the skin and lung of these mice, Fgfr3 mutation caused papilloma and promoted lung tumorigenesis in cooperation with K-Ras and β-catenin activation, respectively. These results indicate that activation of FGFR3 can cooperate with other mutations to drive tumorigenesis in a context-dependent manner, and support the hypothesis that activation of FGFR3 signaling contributes to human cancer.

Fgfr3 regulates development of the caudal telencephalon

The fibroblast growth factor receptor 3 (Fgfr3) is expressed in a rostrallow to caudalhigh gradient in the developing cerebral cortex. Therefore, we hypothesized that Fgfr3 contributes to the correct morphology and connectivity of the caudal cortex. Overall, the forebrain structures appeared normal in Fgfr3−/− mice. However, cortical and hippocampal volumes were reduced by 26.7% and 16.3%, respectively. Hypoplasia was particularly evident in the caudo‐ventral region of the telencephalon where proliferation was mildly decreased at embryonic day 18.5. Dysplasia of GABAergic neurons in the amygdala and piriform cortex was seen following GAD67 immunohistochemistry. Dye‐tracing studies and diffusion magnetic resonance imaging and tractography detected a subtle thalamocortical tract deficit, and significant decreases in the stria terminalis and lateral arms of the anterior commissure. These results indicate the subtle role of Fgfr3 in formation of caudal regions of the telencephalon affecting some brain projections. Developmental Dynamics 240:1586–1599, 2011.

Fibroblast growth factor receptor 3 activation plays a causative role in urothelial cancer pathogenesis in cooperation with Pten loss in mice

Although somatic mutations and overexpression of the tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) are strongly associated with bladder cancer, evidence for their functional involvement in the pathogenesis remains elusive. Previously we showed that activation of Fgfr3 alone is not sufficient to initiate urothelial tumourigenesis in mice. Here we hypothesize that cooperating mutations are required for Fgfr3‐dependent tumourigenesis in the urothelium and analyse a mouse model in which an inhibitor of Pi3k–Akt signalling, Pten, is deleted in concert with Fgfr3 activation (UroIICreFgfr3+/K644EPtenflox/flox). Two main phenotypical characteristics were observed in the urothelium: increased urothelial thickness and abnormal cellular histopathology, including vacuolization, condensed cellular appearance, enlargement of cells and nuclei, and loss of polarity. These changes were not observed when either mutation was present individually. Expression patterns of known urothelial proteins indicated the abnormal cellular differentiation. Furthermore, quantitative analysis showed that Fgfr3 and Pten mutations cooperatively caused cellular enlargement, while Pten contributed to increased cell proliferation. Finally, FGFR3 overexpression was analysed along the level of phosphorylated mTOR in 66 T1 urothelial tumours in tissue microarray, which supported the occurrence of functional association of these two signalling pathways in urothelial pathogenesis. Taken together, this study provides evidence supporting a functional role of FGFR3 in the process of pathogenesis in urothelial neoplasms. Given the wide availability of inhibitors specific to FGF signalling pathways, our model may open the avenue for FGFR3‐targeted translation in urothelial disease. Copyright

Regulation of cell proliferation and apoptosis in neuroblastoma cells by ccp1, a FGF2 downstream gene

BackgroundCoiled-coil domain containing 115 (Ccdc115) or coiled coil protein-1 (ccp1) was previously identified as a downstream gene of Fibroblast Growth Factor 2 (FGF2) highly expressed in embryonic and adult brain. However, its function has not been characterised to date. Here we hypothesized that ccp1 may be a downstream effecter of FGF2, promoting cell proliferation and protecting from apoptosis.MethodsForced ccp1 expression in mouse embryonic fibroblast (MEF) and neuroblastoma SK-N-SH cell line, as well as down-regulation of ccp1 expression by siRNA in NIH3T3, was used to characterize the role of ccp1.ResultsCcp1 over-expression increased cell proliferation, whereas down-regulation of ccp1 expression reduced it. Ccp1 was able to increase cell proliferation in the absence of serum. Furthermore, ccp1 reduced apoptosis upon withdrawal of serum in SK-N-SH. The mitogen-activated protein kinase (MAPK) or ERK Kinase (MEK) inhibitor, U0126, only partially inhibited the ccp1-dependent BrdU incorporation, indicating that other signaling pathway may be involved in ccp1-induced cell proliferation. Induction of Sprouty (SPRY) upon FGF2 treatment was accelerated in ccp1 over-expressing cells.ConclusionsAll together, the results showed that ccp1 regulates cell number by promoting proliferation and suppressing cell death. FGF2 was shown to enhance the effects of ccp1, however, it is likely that other mitogenic factors present in the serum can also enhance the effects. Whether these effects are mediated by FGF2 influencing the ccp1 function or by increasing the ccp1 expression level is still unclear. At least some of the proliferative regulation by ccp1 is mediated by MAPK, however other signaling pathways are likely to be involved.

FGFR3 mutation increases bladder tumourigenesis by suppressing acute inflammation: Suppression of urothelial inflammation by FGFR3

Recent studies of muscle‐invasive bladder cancer show that FGFR3 mutations are generally found in a luminal papillary tumour subtype that is characterised by better survival than other molecular subtypes. To better understand the role of FGFR3 in invasive bladder cancer, we examined the process of tumour development induced by the tobacco carcinogen OH‐BBN in genetically engineered models that express mutationally activated FGFR3 S249C or FGFR3 K644E in the urothelium. Both occurrence and progression of OH‐BBN‐driven tumours were increased in the presence of an S249C mutation compared to wild‐type control mice. Interestingly, at an early tumour initiation stage, the acute inflammatory response in OH‐BBN‐treated bladders was suppressed in the presence of an S249C mutation. However, at later stages of tumour progression, increased inflammation was observed in S249C tumours, long after the carcinogen administration had ceased. Early‐phase neutrophil depletion using an anti‐Ly6G monoclonal antibody resulted in an increased neutrophil‐to‐lymphocyte ratio at later stages of pathogenesis, indicative of enhanced tumour pathogenesis, which supports the hypothesis that suppression of acute inflammation could play a causative role. Statistical analyses of correlation showed that while initial bladder phenotypes in morphology and inflammation were FGFR3‐dependent, increased levels of inflammation were associated with tumour progression at the later stage. This study provides a novel insight into the tumour‐promoting effect of FGFR3 mutations via regulation of inflammation at the pre‐tumour stage in the bladder. Copyright

12-P017 Fgfr3 regulates development of the caudal cerebral cortex

Structural changes caused by altered Fibroblast Growth Factor (FGF) signaling during embryonic development have been shown to influence cortical function and animal behavior. FGFs are known to control patterning and neurogenesis, the initial two key events that define forebrain structures. We hypothesized that one of the four high-affinity FGF receptors, Fgfr3, may regulate rostrocaudal forebrain patterning and neurogenesis, and that this function may be shared with other FGFR subtypes through co-regulation of downstream pathways. Here we investigated the phenotype of the cerebral cortex and hippocampus in Fgfr3 knockout mice in embryonic and early postnatal stages. We found that Fgfr3 homozygous knockout mice (Fgfr3 / ) rarely survived beyond birth on a C57/Bl6 background. However, the production efficiency improved upon backcrossing onto a CD1 background. A reduction in the brain weight and size was detected in Fgfr3 / in comparison to +/ and +/+ littermate controls as early as E16.5, and more prominently at E18.5/P0.5. The overall structure appeared largely normal in the Fgfr3 / cortex and hippocampus at postnatal day 7.5. However, the volume of Fgfr3 / was reduced compared to controls by 26.7% and 22.5% in the cortex and hippocampus, respectively (n = 3). This reduction is largely comparable to that observed in mouse models with Fgfr1 deletion. Finally, diffusion magnetic resonance imaging and tractography revealed a decrease in caudal cortical tracts crossing the midline via the anterior commissure. All together, these results suggest that Fgfr3 is important for formation of caudal regions of the cerebral cortex.