Mutations in multiple oncogenes including KRAS, CTNNB1, PIK3CA and FGFR2 have been identified in endometrial cancer. The aim of this study was to provide insight into the clinicopathological features associated with patterns of mutation in these genes, a necessary step in planning targeted therapies for endometrial cancer. 466 endometrioid endometrial tumors were tested for mutations in FGFR2, KRAS, CTNNB1, and PIK3CA. The relationships between mutation status, tumor microsatellite instability (MSI) and clinicopathological features including overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier survival analysis and Cox proportional hazard models. Mutations were identified in FGFR2 (48/466); KRAS (87/464); CTNNB1 (88/454) and PIK3CA (104/464). KRAS and FGFR2 mutations were significantly more common, and CTNNB1 mutations less common, in MSI positive tumors. KRAS and FGFR2 occurred in a near mutually exclusive pattern (p = 0.05) and, surprisingly, mutations in KRAS and CTNNB1 also occurred in a near mutually exclusive pattern (p = 0.0002). Multivariate analysis revealed that mutation in KRAS and FGFR2 showed a trend (p = 0.06) towards longer and shorter DFS, respectively. In the 386 patients with early stage disease (stage I and II), FGFR2 mutation was significantly associated with shorter DFS (HR = 3.24; 95% confidence interval, CI, 1.35–7.77; p = 0.008) and OS (HR = 2.00; 95% CI 1.09–3.65; p = 0.025) and KRAS was associated with longer DFS (HR = 0.23; 95% CI 0.05–0.97; p = 0.045). In conclusion, although KRAS and FGFR2 mutations share similar activation of the MAPK pathway, our data suggest very different roles in tumor biology. This has implications for the implementation of anti-FGFR or anti-MEK biologic therapies.
It is known that FGFR2 gene variations confer a risk for breast cancer. FGFR2 and FGF10, the main ligand of FGFR2, are both overexpressed in 5–10% of breast tumors. In our study, we sequenced the most important coding regions of FGFR2 in somatic tumor tissue of 140 sporadic breast cancer patients and performed MLPA analysis to detect copy number variations in FGFR2 and FGF10. We identified one somatic heterozygous missense mutation, p.K660N (c.1980G>C), within the tyrosine kinase domain of FGFR2 in tumor tissue of a sporadic breast cancer patient, which is likely mediated by the FGFR2-IIIb isoform. The presence of wild type and mutated alleles in equal quantities suggests that the mutation has driven clonal amplification of mutant cells. We have analyzed the tyrosine kinase activity of p.K660N and another recently described somatic breast cancer mutation in FGFR2, p.R203C, after expression in HEK293 cells and demonstrated that the intrinsic tyrosine kinase activity of both mutant proteins is strongly increased resulting in elevated phosphorylation and activity of downstream effectors. To our knowledge, this is the first report of functional analysis of somatic breast cancer mutations in FGFR2 providing evidence for the activating nature of FGFR2-mediated signalling in the pathogenesis of breast cancer.
Fibroblast growth factor receptor 4 (FGFR4) is a member of a receptor tyrosine kinase family of enzymes involved in cell cycle control and proliferation. A common single nucleotide polymorphism (SNP) Gly388Arg variant has been associated with increased tumor cell motility and progression of breast cancer, head and neck cancer and soft tissue sarcomas. The present study evaluated the prognostic significance of FGFR4 in oral and oropharynx carcinomas, finding an association of FGFR4 expression and Gly388Arg genotype with tumor onset and prognosis.
Patients and Methods
DNA from peripheral blood of 122 patients with oral and oropharyngeal squamous cell carcinomas was used to determine FGFR4 genotype by PCR-RFLP. Protein expression was assessed by immunohistochemistry (IHC) on paraffin-embedded tissue microarrays.
Presence of allele Arg388 was associated with lymphatic embolization and with disease related premature death. In addition, FGFR4 low expression was related with lymph node positivity and premature relapse of disease, as well as disease related death.
Our results propose FGFR4 profile, measured by the Gly388Arg genotype and expression, as a novel marker of prognosis in squamous cell carcinoma of the mouth and oropharynx.
The fibroblast growth factor receptor 3 (FGFR3) gene is known to be frequently mutated in noninvasive urothelial carcinomas of the bladder. In this study, we investigated the expression of FGFR3, Ki-67, and p53 in bladder cancers and the effects of expression on tumor recurrence.
Materials and Methods
Fifty-five cases of primary bladder cancer were examined by immunohistochemistry. The relationship of these markers with various clinicopathological factors, including recurrence, was assessed.
Positivity for cytoplasmic FGFR3 (FGFR3-c) was associated with a lower cancer grade (p=0.022) and stage (p=0.011). Recurrence was more frequent in patients with a higher stage, negative FGFR3-c, and high Ki-67 expression. According to univariate analysis, predictors of recurrence-free survival included the following: age, stage, FGFR-c, Ki-67, and p53. However, none of these was independent from the other parameters in multivariate studies.
The immunohistochemical expression of FGFR3 is not only one of the characteristic features of lower-grade and lower-stage urothelial carcinoma but also a possible marker in predicting disease recurrence.
Carcinoma, transitional cell; Fibroblast growth factor receptor 3; p53 genes; Recurrence
We have previously reported activating mutations of the gene coding for the fibroblast growth factor receptor 3 (FGFR3) in invasive cervical carcinoma. To further analyze the role of FGFR3 in cervical tumor progression, we extended our study to screen a total of 75 invasive tumors and 80 cervical intraepithelial neoplasias (40 low-grade and 40 high-grade lesions).
Using single strand conformation polymorphism (SSCP) followed by DNA sequencing, we found FGFR3 mutation (S249C in all cases) in 5% of invasive cervical carcinomas and no mutation in intraepithelial lesions. These results suggest that, unlike in bladder carcinoma, FGFR3 mutation does not or rarely occur in non invasive lesions. Compared to patients with wildtype FGFR3 tumor, patients with S249C FGFR3 mutated tumors were older (mean age 64 vs. 49.4 years, P = 0.02), and were more likely to be associated with a non-16/18 HPV type in their tumor. Gene expression analysis demonstrated that FGFR3 mutated tumors were associated with higher FGFR3b mRNA expression levels compared to wildtype FGFR3 tumors. Supervised analysis of Affymetrix expression data identified a significant number of genes specifically differentially expressed in tumors with respect to FGFR3 mutation status.
This study suggest that tumors with FGFR3 mutation appear to have distinctive clinical and biological characteristics that may help in defining a population of patients for FGFR3 mutation screening.
Overexpression of FGF receptor 3 (FGFR3) is implicated in the development of t(4;14)-positive multiple myeloma. While FGFR3 is frequently overexpressed and/or activated through mutations in bladder cancer, the functional importance of FGFR3 and its potential as a specific therapeutic target in this disease have not been elucidated in vivo. Here we report that inducible knockdown of FGFR3 in human bladder carcinoma cells arrested cell-cycle progression in culture and markedly attenuated tumor progression in xenografted mice. Further, we developed a unique antibody (R3Mab) that inhibited not only WT FGFR3, but also various mutants of the receptor, including disulfide-linked cysteine mutants. Biochemical analysis and 2.1-Å resolution crystallography revealed that R3Mab bound to a specific FGFR3 epitope that simultaneously blocked ligand binding, prevented receptor dimerization, and induced substantial conformational changes in the receptor. R3Mab exerted potent antitumor activity against bladder carcinoma and t(4;14)-positive multiple myeloma xenografts in mice by antagonizing FGFR3 signaling and eliciting antibody-dependent cell-mediated cytotoxicity (ADCC). These studies provide in vivo evidence demonstrating an oncogenic role of FGFR3 in bladder cancer and support antibody-based targeting of FGFR3 in hematologic and epithelial cancers driven by WT or mutant FGFR3.
Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.
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.
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.
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.
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.
FGFR3; FGFR1; tyrosine kinase inhibitor; urothelial carcinoma; PD173074; TKI-258
Fibroblast growth factor receptors (FGFRs) play key roles in proliferation, differentiation and tumorigenesis. Many urothelial carcinomas (UC) contain activating point mutations or increased expression of FGFR3. However, little is known about the role of other FGFRs. We have examined FGFR expression in immortalised normal human urothelial cells (TERT-NHUC), UC cell lines and tumor samples and demonstrated 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 over-expressed FGFR1 in TERT-NHUC, 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. FRS2α and PLCγ were activated in response to FGF2, leading to activation of the MAPK pathway. The level of MAPK activation correlated with the level of cyclinD1, MCL1 and phosphorylated BAD, which also correlated with FGFR-induced proliferation and survival. Knockdown of FGFR1 in UC cell lines revealed differential FGFR1-dependence. JMSU1 cells were dependent on FGFR1 expression for survival but 3 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 and 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 UC.
FGFR1; urothelial cell carcinoma; therapeutic target
Evolution of unresponsiveness to homeostasis-promoting signals from the microenvironment is a hallmark of malignant tumor cells. In Dunning R3327 model rat prostate tumors that are comprised of distinct stromal and epithelial compartments, progression from non-malignant, androgen-responsive tumors to malignancy is characterized by loss of compartmentation coincident with a loss of resident epithelial cell FGFR2IIIb that receives instructive signals from stromal FGF7 and FGF10. Restoration of FGFR2IIIb to malignant tumor cells restores responsiveness to stromal cells, restores distinct stromal and epithelial compartments and retards malignant progression. Cultured stromal cells from two compartment tumors are comprised of smooth muscle α−actin-positive cells that express predominantly FGFR3 and fibroblast-like cells devoid of α−actin and FGFR3. Here we show that it is primarily the smooth muscle cell-like α−actin-expressing stromal cells that survive, morphologically differentiate and delay tumor incidence and size in the presence of malignant cells in which FGFR2IIIb has been restored. Expression of FGFR3 by transfection in the fibroblast-like stromal cells conferred ability to respond similar to the smooth muscle cell-like stromal cells in which FGFR3 is normally resident. These results highlight the importance of the two-way communication back and forth between stroma and epithelium that is mediated by signaling within the FGFR family during progression to malignancy.
cell-cell communication; Dunning tumors; tumor microenvironment; prostate cancer; receptor tyrosine kinases; stromal-epithelial interactions; tissue homeostasis
FGF signaling is associated with breast cancer and is required for mammary placode formation in the mouse. In this study, we employed a genetic mosaic analysis based on Cre-mediated recombination to investigate FGF receptor 2 (Fgfr2) function in the postnatal mammary gland. Mosaic inactivation of Fgfr2 by the MMTVCre transgene enabled us to compare the behavior of Fgfr2 null and Fgfr2 heterozygous cells in the same gland. Fgfr2 null cells were at a competitive disadvantage to their Fgfr2 heterozygous neighbors in the highly proliferative terminal end buds (TEBs) at the invasion front, owing to a negative effect of loss of Fgfr2 function on cell proliferation. However, Fgfr2 null cells were tolerated in mature ducts. In these genetic mosaic mammary glands, the epithelial network is apparently built by TEBs that over time are composed of a progressively larger proportion of Fgfr2-positive cells. However, subsequently, most cells lose Fgfr2 function, presumably due to additional rounds of Cre-mediated recombination. Using an independent strategy to create mosaic mammary glands, which employed an adenovirus-Cre that acts only once, we confirmed that Fgfr2 null cells were out-competed by neighboring Fgfr2 heterozygous cells. Together, our data demonstrate that Fgfr2 functions in the proliferating and invading TEBs, but it is not required in the mature ducts of the pubertal mammary gland.
Cre recombinase; Epithelial-mesenchymal crosstalk; FGF signaling; Mosaic analysis; Terminal end buds
Loss of FGFRs results in skin abnormalities due to activation of keratinocytes and epidermal T cells.
Fibroblast growth factors (FGFs) are master regulators of organogenesis and tissue homeostasis. In this study, we used different combinations of FGF receptor (FGFR)-deficient mice to unravel their functions in the skin. Loss of the IIIb splice variants of FGFR1 and FGFR2 in keratinocytes caused progressive loss of skin appendages, cutaneous inflammation, keratinocyte hyperproliferation, and acanthosis. We identified loss of FGF-induced expression of tight junction components with subsequent deficits in epidermal barrier function as the mechanism underlying the progressive inflammatory skin disease. The defective barrier causes activation of keratinocytes and epidermal γδ T cells, which produce interleukin-1 family member 8 and S100A8/A9 proteins. These cytokines initiate an inflammatory response and induce a double paracrine loop through production of keratinocyte mitogens by dermal cells. Our results identify essential roles for FGFs in the regulation of the epidermal barrier and in the prevention of cutaneous inflammation, and highlight the importance of stromal–epithelial interactions in skin homeostasis and disease.
FGFR3 mutations are common in low grade urothelial carcinoma and represent a potential therapeutic target in this disease. Their incidence and functional role in high grade urothelial carcinoma (HGUC), which displays an increased propensity for recurrence and muscularis propria invasion, is less well defined. We developed a mass spectrometry based genotyping assay to define the incidence of FGFR3 mutations in a large clinically annotated set of urothelial carcinomas. FGFR3 mutations were found in 17% of HGUC versus 84% of low-grade lesions. Retrospective pathologic review of the class of FGFR3 mutant HGUC revealed unique histologic features characterized by a bulky, exophytic component with branching papillary architecture as well as irregular nuclei with a koilocytoid appearance. The predictive value of this histologic appearance was confirmed using a prospective set of 49 additional HGUC. Prospective histologic review was able to correctly predict for the presence of an FGFR3 mutation in 13 of 24 HGUC specimens that exhibited the distinct morphology (54%). All 25 specimens lacking the defined histologic features were FGFR3 wild-type for a negative predictive value of 100%. Macrodissection of individual tumors confirmed the presence of the FGFR3 mutant allele in non-invasive and invasive, low and high-grade regions of individual tumors and in the lymph node metastases of patients whose tumors possessed the characteristic morphologic signature, suggesting that FGFR3 mutations are not restricted to the more clinically indolent regions of HGUCs. These data suggest that histologic screening of HGUC followed by confirmatory genotyping can be used to enrich for the population of HGUC most likely to harbor activating mutations in the FGFR3 receptor tyrosine kinase. Histologic review could thus aid in the development of targeted inhibitors of FGFR3 by facilitating the identification of the subset of patients most likely to harbor activating mutations in the FGFR3 gene.
fibroblast growth factor receptor-3; FGFR3; urothelial carcinoma; morphology
Apert syndrome is a congenital disorder characterized by severe skull malformations and caused by one of two missense mutations, S252W and P253R, on fibroblast growth factor receptor 2 (FGFR2). The molecular bases underlying differential Apert syndrome phenotypes are still poorly understood and it is unclear why cleft palate is more frequent in patients carrying the S252W mutation. Taking advantage of Apert syndrome mouse models, we performed a novel combination of morphometric, histological and immunohistochemical analyses to precisely quantify distinct palatal phenotypes in Fgfr2+/S252W and Fgfr2+/P253R mice. We localized regions of differentially altered FGF signaling and assessed local cell patterns to establish a baseline for understanding the differential effects of these two Fgfr2 mutations. Palatal suture scoring and comparative 3D shape analysis from high resolution μCT images of 120 newborn mouse skulls showed that Fgfr2+/S252W mice display relatively more severe palate dysmorphologies, with contracted and more separated palatal shelves, a greater tendency to fuse the maxillary-palatine sutures and aberrant development of the inter-premaxillary suture. These palatal defects are associated with suture-specific patterns of abnormal cellular proliferation, differentiation and apoptosis. The posterior region of the developing palate emerges as a potential target for therapeutic strategies in clinical management of cleft palate in Apert syndrome patients.
Fibroblast growth factor receptor 3 (FGFR3), highly conserved in both humans and murine, is one of key tyrosine kinase receptors for FGF. FGFR3 is expressed in different tissues, including cartilage, brain, kidney, and intestine at different development stages. Conventional knockout of Fgfr3 alleles leads to short life span, and overgrowth of bone. In clinic, human FGFR3 mutations are responsible for three different types of chondrodysplasia syndromes including achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). For better understanding of the roles of FGFR3 in different tissues at different stages of development and in pathological conditions, we generated Fgfr3 conditional knockout mice in which loxp sites flank exons 9-10 in the Fgfr3 allele. We also demonstrated that Cre-mediated recombination using Col2a1-Cre, a Cre line expressed in chondrocyte during bone development, results in specific deletion of the gene in tissues containing cartilage. This animal model will be useful to study distinct roles of FGFR3 in different tissues at different ages.
FGFR3; conditional knock out; Cre-Loxp; gene targeting
Fibroblast growth factor receptor-2 (FGFR-2) plays an important role in tumorigenesis. In thyroid cancer it has been observed a FGFR-2 down-modulation, but the role of this receptor has not been yet clarified. Therefore, we decided to examine the expression of both FGFR-2 isoform, FGFR-2-IIIb and FGFR-2-IIIc, in different histological thyroid variants such as hyperplasia, follicular adenoma and papillary carcinoma. Immunohistochemistry and quantitative Real-Time PCR analyses were performed on samples of hyperplasia, follicular adenoma and papillary carcinoma, compared with normal thyroid tissue. Thyroid hyperplasia did not show statistically significant reduction in FGFR-2 protein and mRNA levels. Interestingly, in both follicular adenoma and papillary carcinoma samples we observed a strongly reduced expression of both FGFR-2 isoforms. We speculate that FGFR-2 down-modulation might be an early event in thyroid carcinogenesis. Furthermore, we suggest the potential use of FGFR-2 as an early marker for thyroid cancer diagnosis.
Due to frequent mutations in certain cancers, FGFR3 gene is considered as an oncogene. However, in some normal tissues, FGFR3 can limit cell growth and promote cell differentiation. Thus, FGFR3 action appears paradoxical.
FGFR3 expression was forced in pancreatic cell lines. The receptor exerted dual effects: it suppressed tumor growth in pancreatic epithelial-like cells and had oncogenic properties in pancreatic mesenchymal-like cells. Distinct exclusive pathways were activated, STATs in epithelial-like cells and MAP Kinases in mesenchymal-like cells. Both FGFR3 splice variants had similar effects and used the same intracellular signaling. In human pancreatic carcinoma tissues, levels of FGFR3 dropped in tumors.
In tumors from epithelial origin, FGFR3 signal can limit tumor growth, explaining why the 4p16.3 locus bearing FGFR3 is frequently lost and why activating mutations of FGFR3 in benign or low grade tumors of epithelial origin are associated with good prognosis. The new hypothesis that FGFR3 can harbor both tumor suppressive and oncogenic properties is crucial in the context of targeted therapies involving specific tyrosine kinase inhibitors (TKIs). TKIs against FGFR3 might result in adverse effects if used in the wrong cell context.
FGFR3; Pancreatic cancer; Tumor suppressor; Oncogene; MAP kinases; STAT
Rhabdomyosarcoma (RMS) is a malignancy with features of skeletal muscle, and the most common soft-tissue sarcoma of childhood. Survival for high risk groups is ~30% at 5 years and there are no durable therapies tailored to its genetic aberrations. During genetic modeling of the common RMS variants, embryonal (eRMS) and alveolar (aRMS), we noted that the RTK FGFR4 was upregulated as an early event in aRMS. Herein, we evaluated the expression of FGFR4 in eRMS compared to aRMS, and whether FGFR4 had similar or distinct roles in their tumorigenesis.
Human RMS cell lines and tumor tissue were analyzed for FGFR4 expression by immunoblot and IHC. Genetic and pharmacologic loss-of-function of FGFR4 using virally-transduced shRNAs and the FGFR small molecule inhibitor PD173074, respectively, were used to study the role of FGFR4 in RMS cell lines in vitro and xenografts in vivo. Expression of the anti-apoptotic protein BCL2L1 was also examined.
FGFR4 is expressed in both RMS subtypes, but protein expression is higher in aRMS. The signature aRMS gene fusion product, PAX3-FOXO1, induced FGFR4 expression in primary human myoblasts. In eRMS, FGFR4 loss-of-function reduced cell proliferation in vitro and xenograft formation in vivo. In aRMS, it diminished cell survival in vitro. In myoblasts and aRMS, FGFR4 was necessary and sufficient for expression of BCL2L1, while in eRMS, this induction was not observed, suggesting differential FGFR4 signaling.
These studies define dichotomous roles for FGFR4 in RMS subtypes, and support further study of FGFR4 as a therapeutic target.
FGFR4; PAX3-FOXO1; Rhabdomyosarcoma; Pediatric cancers
The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4–FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4–mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.
Background & Aims
Fibroblast growth factor receptor (FGFR) 4 controls bile acid metabolism and protects the liver from fibrosis, but the roles of FGFR1 and FGFR2 in the adult liver are largely unknown. We investigated the functions and mechanisms of action of these receptors in liver homeostasis, regeneration, and fibrosis.
We generated mice with hepatocytes that lack FGFR1 and FGFR2 and subjected them to acute and chronic carbon tetrachloride-induced liver injury and partial hepatectomy; mice were also injected with FGF7. We performed histology, histomorphometry, real-time reverse transcription PCR, and immunoblot analyses.
In hepatocytes, loss of FGFR1 and FGFR2 eliminated responsiveness to FGF7 and related FGF family members, but did not affect toxin-induced liver injury and fibrosis. However, mortality after partial hepatectomy increased because of severe hepatocyte necrosis. These effects appeared to be mediated by a failure of hepatocyes to induce the expression of the transcriptional regulators Dbp and Tef upon liver surgery; this affected expression of their target genes, which encode detoxifying cytochrome P450 enzymes. We found that Dbp and Tef expression was directly controlled by FGFR signalling in hepatocytes. As a consequence of the reduced expression of genes that control detoxification, the liver tissue that remained after partial hepatectomy failed to efficiently metabolize endogenous compounds and the drugs applied for anaesthesia/analgesia.
We identified a new, cytoprotective effect of FGFR1 and FGFR2 in the regenerating liver and suggest the use of recombinant FGF7 to increase survival of patients after surgical resection of large amounts of liver tissue.
liver disease; cirrhosis; drug toxicity; cytoprotection
Fibroblast growth factor (FGF) family signaling mediates cell-to-cell communication in development and organ homeostasis in adults. Of the FGF receptor (FGFR) isotypes, FGFR4 is the sole resident isotype present in mature parenchymal hepatocytes. FGFR1 that is normally associated with activated nonparenchymal cells appears ectopically in hepatoma cells. Ectopic expression and chronic activity of FGFR1 in hepatocytes accelerates diethylnitrosamine (DEN)-initiated hepatocarcinogenesis by driving unrestrained cell proliferation and tumor angiogenesis. Hepatocyte FGFR4 mediates liver’s role in systemic cholesterol/bile acid and lipid metabolism and affects proper hepatolobular restoration after damage without effect on cell proliferation. Here we ask whether FGFR4 plays a role in progression of hepatocellular carcinoma (HCC). We report that although spontaneous HCC was not detected in livers of FGFR4-deficient mice, the ablation of FGFR4 accelerated DEN-induced hepatocarcinogenesis. In contrast to FGFR1 that induced a strong mitogenic response and depressed rate of cell death in hepatoma cells, FGFR4 failed to induce a mitogenic response and increased the rate of cell death. FGFR1 but not FGFR4 induced cyclin D1 and repressed p27 expression. Analysis of activation of Erk, JNK and PI3K-related AKT signaling pathways indicated that in contrast to FGFR1, FGFR4 failed to sustain Erk activation and did not activate AKT. These differences may underlie the opposing effects of FGFR1 and FGFR4. These results suggest that in contrast to ectopic FGFR1 that is a strong promoter of hepatoma, resident FGFR4 that mediates differentiated hepatocyte metabolic functions also serves to suppress hepatoma progression.
FGF; cholesterol metabolism; hepatocellular carcinoma; liver adenoma; metabolism; tyrosine kinase signaling
Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer.
Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We have identified a total of 11 different FGFR2 mutations in 3/10 (30%) of endometrial cell lines and 19/187 (10%) of primary uterine tumors. Mutations were seen primarily in tumors of the endometrioid histologic subtype (18/115 cases investigated, 16%). The majority of the somatic mutations identified were identical to germline activating mutations in FGFR2 and FGFR3 that cause Apert Syndrome, Beare–Stevenson Syndrome, hypochondroplasia, achondroplasia and SADDAN syndrome. The two most common somatic mutations identified were S252W (in eight tumors) and N550K (in five samples). Four novel mutations were identified, three of which are also likely to result in receptor gain-of-function. Extensive functional analyses have already been performed on many of these mutations, demonstrating they result in receptor activation through a variety of mechanisms. The discovery of activating FGFR2 mutations in endometrial carcinoma raises the possibility of employing anti-FGFR molecularly targeted therapies in patients with advanced or recurrent endometrial carcinoma.
endometrial cancer; mutation; FGFR2; apert syndrome
In partnership with exclusively the epithelial FGFR2IIIb isotype and a structurally-specific heparan sulfate motif, stromal-derived FGF7 delivers both growth-promoting and growth-limiting differentiation signals to epithelial cells that promote cellular homeostasis between stromal and epithelial compartments. Intercompartmental homeostasis supported by FGF7/FGFR2IIIb is subverted in many solid epithelial tumors. The normally mesenchymal-derived homologue FGFR1 drives proliferation and a progressive tumor-associated phenotype when it appears ectopically in epithelial cells. In order to understand the mechanism underlying the unique biological effects of FGFR2IIIb, we developed an inducible FGFR2IIIb expression system that is specifically dependent on FGF7 for activation in an initially unresponsive cell line to avoid selection for only the growth-promoting aspects of FGFR2IIIb signaling. We then determined FGF7/FGFR2IIIb signaling-specific tyrosine phosphorylated proteins within 5 min after FGF7 stimulation by phosphopeptide immunoaffinity purification and nano-LC-MS/MS. The FGF7/FGFR2 pair caused tyrosine phosphorylation of multiple proteins that have been implicated in the growth stimulating activities of FGFR1 that included multi-substrate organizers FRS2α and IRS4, ERK2 and phosphatases SHP2 and SHIP2. It uniquely phosphorylated CDK2 and phosphatase PTPN18 on sites involved in the attenuation of cell proliferation, and several factors that maintain nuclear-cytosolic relationships (emerin and LAP2), protein structure and other cellular fine structures as well as some proteins of unknown functions. Several of the FGF7/FGFR2IIIb-specific targets have been associated with maintenance of function and tumor suppression and disruption in tumors. In contrast, a number of pTyr substrates associated with FGF2/FGFR1 that are generally associated with intracellular Ca2+-phospholipid signaling, membrane and cytoskeletal plasticity, cell adhesion, migration and the tumorigenic phenotype were not observed with FGF7/FGFR2IIIb. Our findings provide specific downstream targets for dissection of causal relationships underlying the distinct role of FGF7/FGFR2IIIb signaling in epithelial cell homeostasis.
receptor tyrosine kinases; signaling networks; stromal-epithelial homeostasis; tumor suppression; tyrosine phosphatases
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.