Homodimerization controls the fibroblast growth factor 9 subfamily's receptor binding and heparan sulfate-dependent diffusion in the extracellular matrix

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.

Inhibition of activated fibroblast growth factor receptor 2 in endometrial cancer cells induces cell death despite PTEN abrogation

KRAS activation and PTEN inactivation are frequent events in endometrial tumorigenesis, occurring in 10% to 30% and 26% to 80% of endometrial cancers, respectively. Because we have recently shown activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 16% of endometrioid endometrial cancers, we sought to determine the genetic context in which FGFR2 mutations occur. Analysis of 116 primary endometrioid endometrial cancers revealed that FGFR2 and KRAS mutations were mutually exclusive, whereas FGFR2 mutations were seen concomitantly with PTEN mutations. Here, we show that shRNA knockdown of FGFR2 or treatment with a pan-FGFR inhibitor, PD173074, resulted in cell cycle arrest and induction of cell death in endometrial cancer cells with activating mutations in FGFR2. This cell death in response to FGFR2 inhibition occurred within the context of loss-of-function mutations in PTEN and constitutive AKT phosphorylation, and was associated with a marked reduction in extracellular signal-regulated kinase 1/2 activation. Together, these data suggest that inhibition of FGFR2 may be a viable therapeutic option in endometrial tumors possessing activating mutations in FGFR2, despite the frequent abrogation of PTEN in this cancer type.

Brivanib alaninate, a dual inhibitor of vascular endothelial growth factor receptor and fibroblast growth factor receptor tyrosine kinases, induces growth inhibition in mouse models of human hepatocellular carcinoma

PURPOSE: Hreceptor (VEGFR) and FGF receptor (FGFR) signaling pathways. EXPERIMENTAL DESIGN: Six different s.c. patient-derived HCC xenografts were implanted into mice. Tumor growth was evaluated in mice treated with brivanib compared with control. The effects of brivanib on apoptosis and cell proliferation were evaluated by immunohistochemistry. The SK-HEP1 and HepG2 cells were used to investigate the effects of brivanib on the VEGFR-2 and FGFR-1 signaling pathways in vitro. Western blotting was used to determine changes in proteins in these xenografts and cell lines. RESULTS: Brivanib significantly suppressed tumor growth in five of six xenograft lines. Furthermore, brivanib-induced growth inhibition was associated with a decrease in phosphorylated VEGFR-2 at Tyr(1054/1059), increased apoptosis, reduced microvessel density, inhibition of cell proliferation, and down-regulation of cell cycle regulators. The levels of FGFR-1 and FGFR-2 expression in these xenograft lines were positively correlated with its sensitivity to brivanib-induced growth inhibition. In VEGF-stimulated and basic FGF stimulated SK-HEP1 cells, brivanib significantly inhibited VEGFR-2, FGFR-1, extracellular signal-regulated kinase 1/2, and Akt phosphorylation. CONCLUSION: This study provides a strong rationale for clinical investigation of brivanib in patients with HCC.

Loss-of-function fibroblast growth factor receptor-2 mutations in melanoma

We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer.

FGFR2 mutations are rare across histologic subtypes of ovarian cancer

OBJECTIVE: Ovarian cancer is the leading cause of death from gynecologic malignancies in the Western world. Fibroblast growth factor receptor (FGFR) signaling has been implicated to play a role in ovarian tumorigenesis. Mutational activation of one member of this receptor family, FGFR2, is a frequent event in endometrioid endometrial cancer. Given the similarities in the histologic and molecular genetics of ovarian and endometrial cancers, we hypothesized that activating FGFR2 mutations may occur in a subset of endometrioid ovarian tumors, and possibly other histotypes. METHODS: Six FGFR2 exons were sequenced in 120 primary ovarian tumors representing the major histologic subtypes. RESULTS: FGFR2 mutation was detected at low frequency in endometrioid (1/46, 2.2%) and serous (1/41, 2.4%) ovarian cancer. No mutations were detected in clear cell, mucinous, or mixed histology tumors or in the ovarian cancer cell lines tested. Functional characterization of the FGFR2 mutations confirmed that the mutations detected in ovarian cancer result in receptor activation. CONCLUSIONS: Despite the low incidence of FGFR2 mutations in ovarian cancer, the two FGFR2 mutations identified in ovarian tumors (S252W, Y376C) overlap with the oncogenic mutations previously identified in endometrial tumors, suggesting activated FGFR2 may contribute to ovarian cancer pathogenesis in a small subset of ovarian tumors.

FGFR2 as a molecular target in endometrial cancer

Although molecularly targeted therapies have been effective in some cancer types, no targeted therapy is approved for use in endometrial cancer. The recent identification of activating mutations in fibroblast growth factor receptor 2 (FGFR2) in endometrial tumors has generated a new avenue for the development of targeted therapeutic agents. The majority of the mutations identified are identical to germline mutations in FGFR2 and FGFR3 that cause craniosynostosis and hypochondroplasia syndromes and result in both ligand-independent and ligand-dependent receptor activation. Mutations that predominantly occur in the endometrioid subtype of endometrial cancer, are mutually exclusive with KRAS mutation, but occur in the presence of PTEN abrogation. In vitro studies have shown that endometrial cancer cell lines with activating FGFR2 mutations are selectively sensitive to a pan-FGFR inhibitor, PD173074. Several agents with activity against FGFRs are currently in clinical trials. Investigation of these agents in endometrial cancer patients with activating FGFR2 mutations is warranted.

Vibrational spectroscopic analysis of the mineral crandallite CaAl3(PO4)2(OH)5•(H2O) from the Jenolan Caves, Australia

The mineral crandallite CaAl3(PO4)2(OH)5•(H2O) has been identified in deposits found in the Jenolan Caves, New South Wales, Australia by using a combination of X-ray diffraction and Raman spectroscopic techniques. A comparison is made between the vibrational spectra of crandallite found in the Jenolan Caves and a standard crandallite. Raman and infrared bands are assigned to PO43- and HPO42- stretching and bending modes. The predominant features are the internal vibrations of the PO43 and HPO42- groups. A mechanism for the formation of crandallite is presented and the conditions for the formation are elucidated.

The structure of the mineral leogangite Cu10(OH)6(SO4)(AsO4)4•8H2O–implications for arsenic accumulation and removal

The objective of this research is to determine the molecular structure of the mineral leogangite. The formation of the types of arsenosulphate minerals offers a mechanism for arsenate removal from soils and mine dumps. Raman and infrared spectroscopy have been used to characterise the mineral. Observed bands are assigned to the stretching and bending vibrations of (SO4)2- and (AsO4)3- units, stretching and bending vibrations of hydrogen bonded (OH)- ions and Cu2+-(O,OH) units. The approximate range of O-H...O hydrogen bond lengths is inferred from the Raman spectra. Raman spectra of leogangite from different origins differ in that some spectra are more complex, where bands are sharp and the degenerate bands of (SO4)2- and (AsO4)3- are split and more intense. Lower wavenumbers of  H2O bending vibration in the spectrum may indicate the presence of weaker hydrogen bonds compared with those in a different leogangite samples. The formation of leogangite offers a mechanism for the removal of arsenic from the environment.

A Raman spectroscopic study of the mono-hydrogen phosphate mineral dorfmanite Na2(PO3OH)•2H2O – and in comparison with brushite

Aspects of the molecular structure of the mineral dorfmanite Na2(PO3OH)•2H2O were determined by Raman spectroscopy. The mineral originated from the Kedykverpakhk Mt., Lovozero, Kola Peninsula, Russia. Raman bands are assigned to the hydrogen phosphate units. The intense Raman band at 949 cm-1 and the less intense band at 866 cm-1 are assigned to the PO3 and POH stretching vibrations. Bands at 991, 1066 and 1141 cm-1 are assigned to the ν3 antisymmetric stretching modes. Raman bands at 393, 413 and 448 cm-1 and 514, 541 and 570 cm-1 are attributed to the ν2 and ν4 bending modes of the HPO4 units, respectively. Raman bands at 3373, 3443 and 3492 cm-1 are assigned to water stretching vibrations. POH stretching vibrations are identified by bands at 2904, 3080 and 3134 cm-1. Raman spectroscopy has proven very useful for the study of the structure of the mineral dorfmanite.

The molecular structure of the mineral sarmientite Fe2(AsO4,SO4)2(OH)6•5H2O : implications for arsenic accumulation and removal

Sarmientite is an environmental mineral; its formation in soils enables the entrapment and immobilisation of arsenic. The mineral sarmientite is often amorphous making the application of X-ray diffraction difficult. Vibrational spectroscopy has been applied to the study of sarmientite. Bands are attributed to the vibrational units of arsenate, sulphate, hydroxyl and water. Raman bands at 794, 814 and 831 cm−1 are assigned to the ν3 (AsO4)3− antisymmetric stretching modes and the ν1 symmetric stretching mode is observed at 891 cm−1. Raman bands at 1003 and 1106 cm−1 are attributed to vibrations. The Raman band at 484 cm−1 is assigned to the triply degenerate (AsO4)3− bending vibration. The high intensity Raman band observed at 355 cm−1 (both lower and upper) is considered to be due to the (AsO4)3−ν2 bending vibration. Bands attributed to water and OH stretching vibrations are observed.

Vibrational spectroscopy of the multi anion mineral zykaite Fe4(AsO4)(SO4)(OH)•15H2O – implications for arsenate removal

Some minerals are colloidal and are poorly diffracting . Vibrational spectroscopy offers one of the few methods for the assessment of the structure of these types of minerals. Among this group of minerals is zykaite with formula Fe4(AsO4)(SO4)(OH)•15H2O. The objective of this research is to determine the molecular structure of the mineral zykaite using vibrational spectroscopy. Raman and infrared bands are attributed to the AsO43-, SO42- and water stretching vibrations. The sharp band at 3515 cm-1 is assigned to the stretching vibration of the OH units. This mineral offers a mechanism for the formation of more crystalline minerals such as scorodite and bukovskyite. Arsenate ions can be removed from aqueous systems through the addition of ferric compounds such as ferric chloride. This results in the formation of minerals such as zykaite and pitticite (Fe3+,AsO4,SO4,H2O).

Raman spectroscopy of the multi anion mineral arsentsumebite Pb2Cu(AsO4)(SO4)(OH) and in comparison with tsumebite Pb2Cu(PO4)(SO4)(OH)

The mineral arsentsumebite Pb2Cu(AsO4)(SO4)(OH), a copper arsenate-sulfate hydroxide of the brackebuschite group has been characterised by Raman spectroscopy. The brackebuschite mineral group are a series of monoclinic arsenates, phosphates and vanadates of the general formula A2B(XO4)(OH,H2O), where A may be Ba, Ca, Pb, Sr, while B may be Al, Cu2+,Fe2+, Fe3+, Mn2+, Mn3+, Zn and XO4 may be AsO4, PO4, SO4,VO4. Bands are assigned to the stretching and bending modes of SO42- AsO43- and HOAsO3 units. Raman spectroscopy readily distinguishes between the two minerals arsentsumebite and tsumebite. Raman bands attributed to arsenate are not observed in the Raman spectrum of tsumebite. Phosphate bands found in the Raman spectrum of tsumebite are not found in the Raman spectrum of arsentsumebite. Raman spectroscopy readily distinguishes the two minerals tsumebite and arsentsumebite.

Raman spectroscopy of the multi-anion mineral mallestigite Pb3Sb5+(SO4)(AsO4)(OH)6•3H2O – a mineral of archaeological significance

Some minerals are formed which show poorly defined X-ray diffraction patterns. Vibrational spectroscopy offers one of the few methods for the assessment of the structure of the oxyanions in such minerals. Among this group of minerals is mallestigite with formula Pb3Sb5+(SO4)(AsO4)(OH)6•3H2O. The objective of this research is to determine the molecular structure of the mineral mallestigite using vibrational spectroscopy. Raman and infrared bands are attributed to the AsO43- , SO42- and water stretching vibrations. Mallestigite is a mineral formed in ancient waste dumps such as occurs at Mallestiger, Carinthia, Austria and as such is a mineral of archaeological significance.

Vibrational spectroscopic analysis of taranakite (K,NH4)Al3(PO4)3(OH) •9(H2O) from the Jenolan Caves, Australia

Many phosphate containing minerals are found in the Jenolan Caves. Such minerals are formed by the reaction of bat guano and clays from the caves. Among these cave minerals is the mineral taranakite (K,NH4)Al3(PO4)3(OH)•9(H2O) which has been identified by X-ray diffraction. Jenolan Caves taranakite has been characterised by Raman spectroscopy. Raman and infrared bands are assigned to H2PO4-, OH and NH stretching vibrations. By using a combination of XRD and Raman spectroscopy, the existence of taranakite in the caves has been proven.