Satu Vainikka

Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction.

Fibroblast growth factor (FGF) receptor (FGFR) gene family consists of at least four receptor tyrosine kinases that transduce signals important in a variety of developmental and physiological processes related to cell growth and differentiation. Here we have characterized the binding of different FGFs to FGFR‐4. Our results establish an FGF binding profile for FGFR‐4 with aFGF having the highest affinity, followed by K‐FGF/hst‐1 and bFGF. In addition, FGF‐6 was found to bind to FGFR‐4 in ligand competition experiments. Interestingly, the FGFR‐4 gene was found to encode only the prototype receptor in a region where both FGFR‐1 and FGFR‐2 show alternative splicing leading to differences in their ligand binding specificities and to secreted forms of these receptors. Ligands binding to FGFR‐4 induced receptor autophosphorylation and phosphorylation of a set of cellular polypeptides, which differed from those phosphorylated in FGFR‐1‐expressing cells. Specifically, the FGFR‐1‐expressing cells showed a considerably more extensive tyrosine phosphorylation of PLC‐gamma than the FGFR‐4‐expressing cells. Structural and functional specificity within the FGFR family exemplified by FGFR‐4 may help to explain how FGFs perform their diverse functions.

Diverse receptors for fibroblast growth factors

The development and maintenance of multicellular organisms requires a complex interplay between cells in different tissues. Many of the factors mediating cell-cell communication are polypeptides, which were originally identified because of their ability to stimulate cell growth. In addition to growth signalling several of these factors have been observed to modulate cell survival, chemotaxis and differentiation both in vitro and in vivo. Fibroblast growth factors are a good example of polypeptide mitogens eliciting a wide variety of responses depending on the target cell type. Our knowledge of the cell surface receptors mediating the effects of FGFs has recently expanded remarkably. Perhaps not surprisingly, the complexity of the FGF family and FGF induced responses is reflected as diversity and redundancy of the FGF receptors.

Association of a 85-kDa Serine Kinase with Activated Fibroblast Growth Factor Receptor-4

Fibroblast growth factors (FGFs) transduce a variety of biological signals via four distinct tyrosine kinase receptors. We have characterized the phosphorylation of FGF receptor 4 (FGFR-4) and its association with a putative substrate, p85, using transfected L6 myoblast and NIH3T3 fibroblast cell lines. FGFR-4 was phosphorylated in vivo and in vitro mainly on serine and threonine residues in several peptides and to a lower degree on tyrosine residues. When analyzed further by in-gel kinase assay, immunoprecipitates of ligand-activated FGFR-4 contained a serine autophosphorylated polypeptide doublet of 85 kDa. Analysis of the major autophosphorylation site Y754F mutant of FGFR-4 showed that binding of p85 and its serine phosphorylation were independent of receptor autophosphorylation at this site. Okadaic acid treatment increased the basal autophosphorylation activity of p85 but decreased FGFR-4 tyrosine phosphorylation. In contrast, orthovanadate treatment increased the tyrosine phosphorylation of FGFR-4. These data show that a serine kinase is associated with activated FGFR-4 and suggest a role for serine phosphorylation in FGFR-4 function.

Production and characterization of the extracellular domain of recombinant human fibroblast growth factor receptor 4.

Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.

Abstract 2080: Inhibiting androgen receptor-associated Src signaling by VAL201 inhibits prostate cancer metastasis in an orthotopic mouse model.

VAL201 is a specific inhibitor of androgen receptor (AR) and estrogen receptor (ER) associated src signaling that has shown promising effects to reduce prostate cancer growth, and the compound is considered as a potential treatment for castration-resistant prostate cancer. Inhibition of src by VAL201 takes place after androgen binding, allowing inhibition of growth without blocking desirable receptor-dependent transcriptional activity, and thereby eliminating the majority of side effects associated with androgen deprivation therapies. The ER positive human prostate cancer cell line PC-3 is usually cited as AR negative, but there is evidence of low levels of AR expression as a form without transcriptional activity that could associate with src. We have studied the effects of Val201 on PC-3 cell proliferation in vitro and growth and metastasis in vivo in an orthotopic xenograft model. The proliferation effects were studied for 100 pM, 1 nM, 10 nM, 100 nM and 1 μM concentrations of VAL201 by measuring WST-1 values at days 3, 5, 7 and 9. Groups with vehicle and 1 μM gemcitabine as reference compound were included in the study. The xenograft study was performed with 6-7 week-old immunodeficient BALB/c nude mice that were allocated to 6 groups (with n=15/group) according to the body weight, one group receiving vehicle and the others VAL201 at doses 0.04, 0.4, 4, 10 and 20 mg/kg. PC-3 cells in Matrigel were inoculated orthotopically into the prostate. Subcutaneous dosing was started at day 1 and continued daily for 28 days. The mice were weighed twice a week. Orthotopic tumors were measured by caliper and the prostate and the regional lymph nodes were harvested at sacrifice. Metastases in lymph nodes were determined from HE stained paraffin sections. VAL201 showed dose-dependent inhibition of PC-3 cell proliferation that was statistically significant with all doses above 100 pM. In the xenograft study VAL201 had no effect on body weight. Statistically significant effects on orthotopic tumor growth were not observed despite of a 35% decrease observed in tumor volume with the 0.4 mg/kg dose. However, 0.04 and 0.4 mg/kg doses of VAL201 showed a significant 50% inhibition of the development of lymph node metastases. As a conclusion, VAL201 inhibited proliferation of PC-3 cells in vitro and development of lymph node metastases in a xenograft model, demonstrating its potential in inhibiting prostate cancer growth and metastasis without adverse effects associated with androgen deprivation. Citation Format: George S. Morris, Mari I. Suominen, Katja M. Fagerlund, Jukka-Pekka Rissanen, Jussi M. Halleen, Satu Vainikka. Inhibiting androgen receptor-associated Src signaling by VAL201 inhibits prostate cancer metastasis in an orthotopic mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2080. doi:10.1158/1538-7445.AM2013-2080

Abstract 922: Inhibiting androgen receptor associated Src signaling with VAL201 inhibits breast cancer growth in an orthotopic xenograft model.

VAL201 is a specific inhibitor of androgen receptor (AR) and estrogen receptor (ER) associated src signaling that has shown promising effects to reduce prostate cancer growth in cancer cell cultures and animal models. Inhibition of src by VAL201 takes place after androgen binding, allowing inhibition of growth without blocking desirable receptor-dependent transcriptional activity, and thereby eliminating the majority of side effects associated with androgen deprivation therapies. Majority of breast cancers are ER positive, and the hormonal treatment includes depletion of estrogen by antiestrogens or aromatase inhibitors (AIs). However, 88% of ER positive and 20-30% of ER negative breast cancers are AR positive, and recently some have been shown to be dependent on AR signaling. Furthermore, AI therapy can increase the androgen levels, as androgens are no longer converted to estrogens. We have studied the effects of Val201 on breast cancer growth in a xenograft model using estrogen-dependent, AR expressing MCF-7 breast cancer cells. Five-week old athymic nude mice were allocated to groups according to body weight (n=10/group). Slow releasing pellets containing 0.72 mg of 17β-estradiol for 60 day release were implanted one day before the cell inoculation. MCF-7 human mammary adenocarcinoma cells were cultured in standard cell culture conditions until semiconfluent and inoculated into the inguinal mammary fat pad at day 0. Vehicle and VAL201 at doses 0.004, 0.04, 0.4 and 4.0 mg/kg were administered sc daily for 28 days starting at day 1. Tumor growth was monitored by caliper measurements three times a week, and the tumors were weighed at sacrifice. VAL201 had no effect on body weight. Tumor volume growth curves demonstrated a dose-dependent inhibition of tumor growth by VAL201. Similar responses enabled combining the treatment groups to a low-dose group (including the groups receiving 0.004 and 0.04 mg/kg VAL201) and a high-dose group (including the groups receiving 0.4 and 4.0 mg/kg VAL201) to increase statistical power in further analyses. The combined groups demonstrated that VAL201 decreased tumor volume and weight. As a conclusion, VAL201 showed inhibition of breast cancer growth in this orthotopic xenograft model, demonstrating its potential as a novel therapeutic agent for breast cancer. The growth inhibition was established in the presence of estrogen, underlining the importance of androgen receptor-associated src signaling even without estrogen depletion. Citation Format: Mari I. Suominen, George S. Morris, Katja M. Fagerlund, Jukka-Pekka Rissanen, Satu Vainikka, Jussi M. Halleen. Inhibiting androgen receptor associated Src signaling with VAL201 inhibits breast cancer growth in an orthotopic xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 922. doi:10.1158/1538-7445.AM2013-922