Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation.

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.

Novel type II collagen reporter mice: New tool for assessing collagen 2α1 expression in vivo and in vitro.

We report the generation of a new mouse strain harboring a Col2-pd2EGFP reporter transgene; pd2EGFP has a much shorter half-life than EGFP, making it a near real-time reporter for Col2α1 expression in vivo and in vitro. In the post-natal growth plate, pd2EGFP fluorescence was expressed in almost all proliferative chondrocytes and in some hypertrophic chondrocytes based on localization with type X collagen. In articular cartilage, pd2EGFP fluorescence diminished over time, nicely illustrating the decrease of type II collagen synthesis in articular chondrocytes during growth. Monolayers of FACS-sorted chondrocytes from P1-2 mice showed faster loss of pd2EGFP compared to EGFP, reflecting rapid chondrocyte de-differentiation. High-density culture of FACS-pd2EGFP- growth plate chondrocytes revealed the typical temporal expression pattern in which type II collagen preceded type X collagen matrix deposition. The Col2-pd2EGFP reporter mouse will be a valuable tool for studies of growth plate chondrocyte biology.

FGFs in endochondral skeletal development.

The mammalian skeleton developments and grows through two complementary pathways: membranous ossification, which gives rise to the calvarial bones and distal clavicle, and endochondral ossification, which is responsible for the bones of the limbs, girdles, vertebrae, face and base of the skull and the medial clavicle. Fibroblast growth factors (FGFs) and their cognate FGF receptors (FGFRs) play important roles in regulating both pathways. However, the details of how FGF signals are initiated, propagated and modulated within the developing skeleton are only slowly emerging. This prospect will focus on the current understanding of these events during endochondral skeletal development with special attention given to concepts that have emerged in the past few years.

Sprouty 2 disturbs FGFR3 degradation in thanatophoric dysplasia type II: a severe form of human achondroplasia.

Thanatophoric dysplasia is a member of the achondroplasia family of human skeletal dysplasias, which result from FGFR3 mutations that exaggerate this receptor's inhibitory influence on chondrocyte proliferation and differentiation in the skeletal growth plate. We have previously reported that defective lysosomal degradation of activated receptor contributes to the gain-of-function of the mutant FGFR3. We now provide evidence that this disturbance is mediated by the receptor's kinase activity and involves constitutive induction and activation of Spry2. Our findings suggest that activated Spry2 may interfere with c-Cbl-mediated ubiquitination of FGFR3 by sequestering c-Cbl. They provide novel insight into the pathogenesis of this group of human skeletal dysplasias and identify a mechanism that potentially could be targeted therapeutically.

Preferential use of imaging modalities in staging newly diagnosed rectal cancer: a survey of US radiation oncologists.

BACKGROUND: Accurate staging is crucial for management of patients with newly diagnosed rectal cancer. Endorectal ultrasound (EUS) has been the standard modality in the United States for decades, with magnetic resonance imaging (MRI) now preferred by national guidelines. Positron emission tomography (PET), conversely, is not recommended. The current utilization of imaging modalities by American radiation oncologists in staging newly diagnosed rectal cancer is unknown. METHODS: American radiation oncologists completed an anonymous institutional review board-approved online survey probing their imaging preferences for initial staging of rectal cancer patients. RESULTS: We received 220 responses from American radiation oncologists, with 39% in academic centers and with 45% seeing more than 10 rectal cancer patients per year. Most respondents utilize all three imaging modalities for rectal cancer staging-EUS, MRI and positron emission tomography/computed tomography (PET/CT). Fifty-two percent and 38% of respondents are high utilizers of EUS and MRI, respectively, defined as ordering these tests at least 75% of the time. Forty seven percent were high PET utilizers. The latter was associated with practice in a private setting (P=0.015) and being within 10 years from residency training completion (P<0.01). CONCLUSIONS: Our analysis reveals a dramatic discordance among national guidelines and the practice patterns among American radiation oncologists. More rely on PET for initial staging of rectal cancer patients than on pelvic MRI. Further research needs to determine the most effective imaging work-up of patients with an initial diagnosis of rectal cancer.

Does a fine line exist between regional and metastatic pelvic lymph nodes in rectal cancer-striking discordance between national guidelines and treatment recommendations by US radiation oncologists.

BACKGROUND: Management of rectal cancer with involved lateral pelvic lymph nodes (LPLNs) at the time of diagnosis-the stage we refer institutionally to as Stage 3.5-is controversial. The American Joint Committee on Cancer's 7th edition classifies internal iliac lymph nodes (LNs) as regional (Stage III), but both external and common iliac LNs as metastatic (Stage IV). However, in many Asian countries all LPLNs are considered regional and patients are treated with curative intent, with literature supporting improved outcomes with LPLN dissection. Management patterns of these patients by US radiation oncologists (ROs) are unknown. METHODS: American ROs completed an anonymous institutional review board-approved online questionnaire regarding rectal cancer management. RESULTS: Among the 220 completed responses, 45% treat more than 10 patients annually and 39% work in academia. We found 10.5% and 34.2% recommend biopsy of clinically involved internal and common iliac LNs, respectively. The vast majority of responders-98.6% and 94.5%-treat involved internal and common iliac LNs with curative intent, respectively. Respondents recommend treatment intensification to involved internal iliac LNs by dissection of the nodal basin (88.2%) and radiation therapy (RT) boost (59.1%), and treatment intensification to involved common iliac LNs by LN dissection (76.4%) and RT boost (63.6%). CONCLUSIONS: Our analysis reveals that the vast majority of US ROs approach patients with involved LPLNs, both regional (internal iliac) and metastatic (common iliac), with curative intent. They recommend treatment intensification with surgical resection and/or RT boost to involved nodes. Prospective clinical trials need to determine the appropriate management of patients with Stage 3.5 rectal cancer.

Safety and Efficacy of Accelerated Hypofractionation and Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma Patients With Varying Degrees of Hepatic Impairment.

PURPOSE: To report the toxicities and outcomes for stereotactic body radiation therapy (SBRT) and accelerated hypofractionated radiation therapy (AHRT) in patients with Child-Pugh (CP) class A, B, or C and albumin-bilirubin (ALBI) score 1, 2, or 3 hepatocellular carcinoma. METHODS AND MATERIALS: We retrospectively reviewed the data from 146 patients with hepatocellular carcinoma who had undergone SBRT (50 Gy in 5 fractions) or AHRT (45 Gy in 18 fractions). The primary endpoint was liver toxicity, defined as an increase in the CP score of ≥2 within 6 months of radiation therapy. The secondary endpoints of ALBI change, overall survival, and local control were also calculated. RESULTS: The median follow-up was 23 months (range 1-59). Most received SBRT (72%), and 28% received AHRT. Of all 146 patients, 45 (31%) had a CP score elevation of ≥2 within 6 months of radiation therapy (RT) (27 patients [28%] with baseline CP-A/B7 and 18 [35%] with baseline CP-B8/B9/C cirrhosis; P = .45). On multivariate analysis, neither baseline CP nor ALBI score was predictive of toxicity. No patient with a decline in liver functionality of CP ≥2 within 6 months of RT returned to baseline at later time points. Eleven grade 4 toxicities were observed. The mean change in the raw ALBI score at ∼6 months was similar for all baseline ALBI groups. Twenty-two patients underwent orthotopic liver transplantation after RT, 13 of whom had baseline CP-B8/B9/C liver functionality. For all patients, the 1- and 2-year treated-lesion local control was greater for SBRT than for AHRT (2-year 94% vs 65%, P < .0001). CONCLUSIONS: The tolerability of SBRT or AHRT as measured by a CP score decline of ≥2 within 6 months of RT was similar across baseline liver functionality groups. Compared with AHRT, SBRT was associated with superior local control. Because the true tolerability of limited-volume RT for patients with CP-B or CP-C cirrhosis is unknown, prospective trials validating its safety and efficacy are warranted.

Ligand activation leads to regulated intramembrane proteolysis of fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (FGFR3) is a major negative regulator of bone growth that inhibits the proliferation and differentiation of growth plate chondrocytes. Activating mutations of its c isoform cause dwarfism in humans; somatic mutations can drive oncogenic transformation in multiple myeloma and bladder cancer. How these distinct activities arise is not clear. FGFR3 was previously shown to undergo proteolytic cleavage in the bovine rib growth plate, but this was not explored further. Here, we show that FGF1 induces regulated intramembrane proteolysis (RIP) of FGFR3. The ectodomain is proteolytically cleaved (S1) in response to ligand-induced receptor activation, but unlike most RIP target proteins, it requires endocytosis and does not involve a metalloproteinase. S1 cleavage generates a C-terminal domain fragment that initially remains anchored in the membrane, is phosphorylated, and is spatially distinct from the intact receptor. Ectodomain cleavage is followed by intramembrane cleavage (S2) to generate a soluble intracellular domain that is released into the cytosol and can translocate to the nucleus. We identify the S1 cleavage site and show that γ-secretase mediates the S2 cleavage event. In this way we demonstrate a mechanism for the nuclear localization of FGFR3 in response to ligand activation, which may occur in both development and disease.

FGFs in endochondral skeletal development.

The mammalian skeleton forms and grows through two developmental pathways: membranous ossification, which gives rise to calvarial bones and the distal clavicle, and endochondral ossification, which is responsible for the bones of the limbs, girdles, vertebrae, face, base of the skull and the medial clavicle. The regulation of both pathways is extremely complex, and the rules that govern it are still emerging. However, it has become clear that fibroblast growth factors (FGFs) and their cognate receptors (FGFRs) play essential roles. This review focuses on the roles of FGFs and FGFRs in endochondral skeletal development, with special attention given to concepts that have emerged in the past few years.