Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

Activating mutations in FGFR3 tyrosine kinase cause several forms of human skeletal dysplasia. Although the mechanisms of FGFR3 action in cartilage are not completely understood, it is believed that the STAT1 transcription factor plays a central role in pathogenic FGFR3 signaling. Here, we analyzed STAT1 activation by the N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants associated with skeletal dysplasias. In a cell-free kinase assay, only K650M and K650E-FGFR3 caused activatory STAT1(Y701) phosphorylation. Similarly, in RCS chondrocytes, HeLa, and 293T cellular environments, only K650M and K650E-FGFR3 caused strong STAT1 activation. Other FGFR3 mutants caused weak (HeLa) or no activation (293T and RCS). This contrasted with ERK MAP kinase activation, which was strongly induced by all six mutants and correlated with the inhibition of proliferation in RCS chondrocytes. Thus the ability to activate STAT1 appears restricted to the K650M and K650E-FGFR3 mutants, which however account for only a small minority of the FGFR3-related skeletal dysplasia cases. Other pathways such as ERK should therefore be considered as central to pathological FGFR3 signaling in cartilage

Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

Activating mutations in FGFR3 tyrosine kinase cause several forms of human skeletal dysplasia. Although the mechanisms of FGFR3 action in cartilage are not completely understood, it is believed that the STAT1 transcription factor plays a central role in pathogenic FGFR3 signaling. Here, we analyzed STAT1 activation by the N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants associated with skeletal dysplasias. In a cell-free kinase assay, only K650M and K650E-FGFR3 caused activatory STAT1(Y701) phosphorylation. Similarly, in RCS chondrocytes, HeLa, and 293T cellular environments, only K650M and K650E-FGFR3 caused strong STAT1 activation. Other FGFR3 mutants caused weak (HeLa) or no activation (293T and RCS). This contrasted with ERK MAP kinase activation, which was strongly induced by all six mutants and correlated with the inhibition of proliferation in RCS chondrocytes. Thus the ability to activate STAT1 appears restricted to the K650M and K650E-FGFR3 mutants, which however account for only a small minority of the FGFR3-related skeletal dysplasia cases. Other pathways such as ERK should therefore be considered as central to pathological FGFR3 signaling in cartilage

Apoptosis inhibitor 5 (API-5; AAC-11; FIF) is upregulated in human carcinomas in vivo

Apoptosis inhibitor 5 (API-5) is a 55 kDa nuclear protein with potent anti-apoptotic signaling in tumor cells in vitro. In this study, we analyzed the expression of the API-5 protein in vivo in a broad spectrum of human carcinomas, including those of the colon, lung, liver, kidney, pancreas, stomach and esophagus using tumor tissues obtained during tumor resection. The results showed significant upregulation of API-5 expression in biopsies of lung (23%, n=13) and colorectal tumors (33%, n=27) in comparison with biopsies from the adjacent normal tissue. Colon cancer biopsies were used to study the cell populations with an upregulated level of expression of API-5 more closely. Using a magnetic bead-based selection for the epithelial cell marker EpCAM, we purified epithelial cells from the tumor and control tissues and analyzed these cells for API-5 expression by western immunoblotting. We observed that EpCAM-positive tumor cells expressed API-5 in all three colorectal cancer cases tested, in contrast to the control EpCAM-positive and EpCAM-negative cells isolated from the control or tumor tissues. These data suggest that the expression of the API-5 protein is upregulated in tumor epithelial cells and may serve as a prognostic marker in colorectal cancer

NF449 Is a Novel Inhibitor of Fibroblast Growth Factor Receptor 3 (FGFR3) Signaling Active in Chondrocytes and Multiple Myeloma Cells*

The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders, including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity toward FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAPK activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared non-competitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site

The effect of FGFR3 mutants on RCS chondrocyte proliferation in context of STAT1 and ERK1/2 activation.

<p>(A) RCS chondrocytes transfected with vectors expressing wild-type FGFR3, activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650E and K650M), and kinase-inactive mutant K508M were grown for 48 hours and analyzed for the indicated molecules by western blotting. Note differential STAT1 and ERK activation by the activating FGFR3 mutants. Cells transfected with empty plasmid (pcDNA3) serve as negative control for transfection. (B) RCS chondrocytes were transfected as described in (A), grown for 72 hours and counted. Note the inhibition of RCS growth by wild-type FGFR3 as well as the activating mutants, as compared to cells transfected either by kinase-inactive K508M-FGFR3 or an empty plasmid. The data represent an average from four individually transfected wells with indicated standard deviation. The cell count difference compared between cells transfected with wild-type FGFR3 and empty plasmid, as well as the cell count difference between cells transfected with wild-type FGFR3 and N540K, G380R, R248C, Y373C, K650M and K650E mutants, were statistically significant (Student's <i>t</i>-test, <i>p</i><0.01). (C) The experiment shown on (B) was repeated five times to eliminate the variance associated with differential transfection efficiency. The differences in percentages of growth compared between cells transfected with wild-type FGFR3 and empty plasmid, and between cells transfected with wild-type FGFR3 and N540K, G380R, R248C, Y373C, K650M and K650E mutants, were statistically significant (Student's <i>t</i>-test, <i>p</i><0.01).</p

STAT1 activation by FGFR3 mutants in a cell-free kinase assay.

<p>Full-length wild-type FGFR3 or its activating mutants N540K, G380R, R248C, Y373C, K650M and K650E were expressed in CHO cells, activated by brief FGF2 treatment and purified by immunoprecipitation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003961#s3" target="_blank">Materials and Methods</a>. Immunocomplexes were subjected to kinase assay with recombinant STAT1 as a substrate. Cells transfected with GFP vector serve as negative control for immunoprecipitation. Samples utilizing recombinant FGFR3 tyrosine kinase domain (TK) or those with omitted ATP serve as positive or negative control for kinase assay, respectively. Note that only K650M and K650E-FGFR3 mutants cause STAT1 phosphorylation, as evidenced by western blotting with antibody recognizing STAT1 only when phosphorylated at Y701 (P-Y701-STAT1). FGFR3 and STAT1 western blottings serve as controls for kinase or substrate quantity. Note the appearance of both immature and mature (glycosylated) FGFR3 forms expressed by CHO cells.</p

STAT1 and ERK1/2 activation by FGFR3 mutants.

<p>(A) The N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants used in this study all cause FGFR3-skeletal dysplasias and signal through ERK MAP kinase in contrast to STAT1, that is activated mostly by the K650M and K650E-FGFR3 mutants. (B) According to the study compiling the clinical data of 591 patients suffering from FGFR3-related skeletal dysplasia <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003961#pone.0003961-PassosBueno1" target="_blank">[1]</a>, the STAT1-activating K650M and K650E account for as little as 4.9% of cases. It is therefore unlikely that activation of STAT1 plays a central role in FGFR3-related skeletal dysplasias as currently believed, but rather represents a signaling feature unique to small subset of patients carrying the K650M and K650E mutations.</p

STAT5 activation by FGFR3 mutants.

<p>RCS chondrocytes transfected with vectors expressing wild-type FGFR3, activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M and K650E), and kinase-inactive mutant K508M were grown for 24 hours and analyzed for the indicated molecules by western blotting. Note the significant STAT5(Y694) phosphorylation induced by K650M and K650E-FGFR3. The membrane used for P-STAT5-Y694 detection was reprobed with antibody recognizing STAT5 regardless of its phosphorylation. Arrow indicates P-STAT5(Y694) or STAT5 signal. FGFR3 and ACTIN western blottings serve as loading controls. Cells transfected by GFP vector serve as negative control for transfection.</p