FGFR3 Mutational Activation Can Induce Luminal-like Papillary Bladder Tumor Formation and Favors a Male Sex Bias.

BACKGROUND: Bladder cancer (BCa) is more common in men and presents differences in molecular subtypes based on sex. Fibroblast growth factor receptor 3 (FGFR3) mutations are enriched in the luminal papillary muscle-invasive BCa (MIBC) and non-MIBC subtypes. OBJECTIVE: To determine whether FGFR3 mutations initiate BCa and impact BCa male sex bias. DESIGN, SETTING, AND PARTICIPANTS: We developed a transgenic mouse model expressing the most frequent FGFR3 mutation, FGFR3-S249C, in urothelial cells. Bladder tumorigenesis was monitored in transgenic mice, with and without carcinogen exposure. Mouse and human BCa transcriptomic data were compared. INTERVENTION: Mutant FGFR3 overexpression in mouse urothelium and siRNA knockdown in cell lines, and N-butyl-N(4-hydroxybutyl)-nitrosamine (BBN) exposure. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Impact of transgene dosage on tumor frequency, synergy with BBN treatment, and FGFR3 pathway activation were analyzed. The sex-specific incidence of FGFR3-mutated tumors was evaluated in mice and humans. FGFR3 expression in FGFR3-S249C mouse urothelium and in various human epithelia was measured. Mutant FGFR3 regulation of androgen (AR) and estrogen (ESR1) receptor activity was evaluated, through target gene expression (regulon) and reporter assays. RESULTS AND LIMITATIONS: FGFR3-S249C expression in mice induced low-grade papillary BCa resembling human luminal counterpart at histological, genomic, and transcriptomic levels, and promoted BBN-induced basal BCa formation. Mutant FGFR3 expression levels impacted tumor incidence in mice, and mutant FGFR3-driven human tumors were restricted to epithelia presenting high normal FGFR3 expression levels. BCa male sex bias, also found in our model, was even higher in human FGFR3-mutated tumors compared with wild-type tumors and was associated with higher AR and lower ESR1 regulon activity. Mutant FGFR3 expression inhibited both ESR1 and AR activity in mouse tumors and human cell lines, demonstrating causation only between FGFR3 activation and low ESR1 activity in tumors. CONCLUSIONS: Mutant FGFR3 initiates luminal papillary BCa formation and favors BCa male sex bias, potentially through FGFR3-dependent ESR1 downregulation. Patients with premalignant lesions or early-stage BCa could thus potentially benefit from FGFR3 targeting. FGFR3 expression level in epithelia could account for FGFR3-driven carcinoma tissue specificity. PATIENT SUMMARY: By developing a transgenic mouse model, we showed that gain-of-function mutations of FGFR3 receptor, among the most frequent genetic alterations in bladder cancer (BCa), initiate BCa formation. Our results could support noninvasive detection of FGFR3 mutations and FGFR3 targeting in early-stage bladder lesions.

Exposure of human megakaryocytes to high shear rates accelerates platelet production.

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation.

Oncogenic properties of the mutated forms of fibroblast growth factor receptor 3b.

Germinal activating mutations of FGFR3 are responsible for several forms of dwarfism due to the inhibitory effect of FGFR3 on bone growth. Surprisingly, identical somatic activating mutations have been found at the somatic level in tumours: at high frequency in benign epithelial tumours (seborrheic keratosis, urothelial papilloma) and in low-grade, low-stage urothelial carcinomas, and at a lower frequency in other types of urothelial carcinoma, in cervix carcinoma, and in haematological cancer, multiple myeloma. FGFR3 exists as two isoforms, FGFR3b and FGFR3c, differs in ligand specificity and tissue expression. FGFR3b is the main form in epithelial cells and derived tumours, whereas FGFR3c is the main form in mesenchyme-derived cells and multiple myeloma. Several lines of evidence suggest that mutated FGFR3c has transforming properties. Although mutated FGFR3b is mostly found in benign epithelial tumours or carcinomas of low malignant potential, we present evidence here that mutated FGFR3b is oncogenic. All bladder tumours presenting FGFR3 mutations expressed this receptor more strongly than normal urothelium or non-mutated tumours. NIH-3T3 cells transfected with a mutated form of FGFR3b--FGFR3b-S249C, the most common mutation in bladder tumours--presented a spindle-cell morphology, grew in soft agar and gave rise to tumours when xenografted into nude mice. We identified one line of 17 bladder cell lines tested (MGH-U3) that expressed a mutated form of FGFR3b, FGFR3b-Y375C. We showed using siRNA and SU5402, an FGFR inhibitor, that the tumour properties of MGH-U3 depended on mutated receptor activity. Thus, in two different models, mutated FGFR3b presents oncogenic properties.

Absence of FGFR3 mutations in urinary bladder tumours of rats and mice treated with N-butyl-N-(-4-hydroxybutyl)nitrosamine.

Frequent activating mutations of FGFR3 (fibroblast growth factor receptor 3) are found in human urothelial cell carcinomas, particularly in superficial papillary tumours (in 74%-84% of pTaG1-G2), but not in carcinomas in situ (CIS) and at a low rate in invasive tumours (in 16%-21% of pT1-4). In mice and rats, BBN (N-butyl-N-(4-hydroxybutyl)nitrosamine) specifically induces bladder tumours. In rats, superficial papillary tumours are mostly observed. In mice, tumour progression follows the CIS pathway: CIS are first observed, followed by tumours that invade surrounding muscle. Therefore, we looked for FGFR3 mutations in these two animal models of bladder cancer. Only the FGFR3b isoform is expressed in human urothelium and derived tumours. We identified the FGFR3b isoform in rats for the first time and showed that this is the main isoform expressed in the bladder urothelium and derived carcinomas in mice and rats, as in humans. SSCP and sequence analysis of FGFR3b showed sequence changes (polymorphisms or silent mutations) in four BBN-induced rat and mouse bladder tumours. The absence of activating mutations of FGFR3 in the mouse model was in agreement with the fact that mouse BBN-induced bladder tumour progression mimics the CIS pathway. The absence of FGFR3 mutations in the rat bladder tumours suggests that, at least at the genetic level, rat superficial papillary tumours differ from their human counterparts.

Activating mutations of the tyrosine kinase receptor FGFR3 are associated with benign skin tumors in mice and humans.

Specific germline activating point mutations in the gene encoding the tyrosine kinase receptor FGFR3 (fibroblast growth factor receptor 3) result in autosomal dominant human skeletal dysplasias. The identification in multiple myeloma and in two epithelial cancers-bladder and cervical carcinomas-of somatic FGFR3 mutations identical to the germinal activating mutations found in skeletal dysplasias, together with functional studies, have suggested an oncogenic role for this receptor. Although acanthosis nigricans, a benign skin tumor, has been found in some syndromes associated with germinal activating mutations of FGFR3, the role of activated FGFR3 in the epidermis has never been investigated. Here, we targeted an activated receptor mutant (S249C FGFR3) to the basal cells of the epidermis of transgenic mice. Mice expressing the transgene developed benign epidermal tumors with no sign of malignancy. These skin lesions had features in common with acanthosis nigricans and other benign human skin tumors, including seborrheic keratosis, one of the most common benign epidermal tumors in humans. We therefore screened a series of 62 cases of seborrheic keratosis for FGFR3 mutations. A large proportion of these tumors (39%) harbored somatic activating FGFR3 mutations, identical to those associated with skeletal dysplasia syndromes and bladder and cervical neoplasms. Our findings directly implicate FGFR3 activation as a major cause of benign epidermal tumors in humans.