J Histochem Cytochem. 2016 Jan;64(1):7-17.
Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation
Nobuhiro Ueno1, Akio Shimizu1, Michiyuki Kanai2, Yugo Iwaya3, Shugo Ueda4, Jun Nakayama5, Misuzu Kurokawa Seo1
1Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan
2Digestive Disease Center, Hanwasumiyoshi General Hospital, Osaka, Japan
3Department of Gastroenterology, Shinshu University School of Medicine, Matsumoto 390-8621, Japan
4Department of Gastroenterological Surgery and Oncology, Kitano Hospital, Osaka 530-8480, Japan
5Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto 390-8621, Japan
Deregulated expression of fibroblast growth factor receptors (FGFRs) and their ligands plays critical roles in tumorigenesis. The gene expression of an alternatively spliced isoform of FGFR3, FGFR3IIIc, was analyzed by RT-PCR. in esophageal carcinoma (EC) patients, including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC), The incidence of FGFR3IIIc was higher in EC (12/16, 75%) than in non-cancerous mucosa (NCM) (6/16, 38%; p = 0.073). Indeed, immunohistochemical analysis of early stage ESCC showed that carcinoma cells expressing FGFR3IIIc were stained with SCC-112, a tumor marker, and Ki67, a cell proliferation marker, suggesting that expression of FGFR3IIIc promotes cell proliferation. We used EC-GI-10 cells endogenously expressing FGFR3IIIc as a model of ESCC to provide a mechanistic insight into the role of FGFR3IIIc in ESCC. The knockdown of endogenous FGFR3 by siRNA treatment significantly abrogated cell proliferation, while FGFR3IIIc overexpression enhanced it. EC-GI-10 cells in ESCC and EC patients endogenously express FGF2, a specific ligand for FGFR3IIIc, suggesting that the upregulated expression of FGFR3IIIc creates autocrine FGF signaling in ESCC. Taking together, FGFR3IIIc may be an early stage tumor marker and a molecular target for ESCC therapy.
KEYWORDS: biological tumor marker; cell proliferation; esophageal cancer; fibroblast growth factor receptor 3; molecular target therapy
Fibroblast growth factor (FGF) receptor (FGFR) is a tyrosine kinase receptor that promotes cell proliferation, differentiation, migration, and survival by binding to FGFs 1. In cancer, overexpression or amplification of FGFR, switching between alternatively spliced isoforms, or mutation of FGFR isoforms triggers carcinogenesis or malignant progression 2. Therefore, FGFR-specific inhibitors have been developed for such cancers 3, 4. For example, AZD4547 is being evaluated in a phase II clinical trial for FGFR1- or FGFR2-amplified cancers, including squamous non-small cell lung cancer, gastric cancer, esophageal cancer, or breast cancer 3, 5.
Two alternatively spliced isoforms of FGFR3 possess an alternative sequence for the C-terminal half of the third Ig domain (IgIII), encoded by a separate 5′-exon IIIa and 3′-exon, either IIIb or IIIc (Figure 1A and 1B), which determines FGF specificity 6-8.
Figure 1 A schematic drawing of FGFR3 isoforms and alternative splicing of the transcripts. (A) The FGFR3 gene has three exons for the IgIIIa, IIIb, and IIIc domains. The FGFR3IIIb isoform is generated by alternative splicing between exons 8 and 10, which encode the C-terminal half of IgIIIb and the transmembrane region, respectively. On the other hand, the FGFR3IIIc isoform is generated by alternative splicing between exons 7 and 9, which encode the C-terminal half of IgIIIa and the other C-terminal half of IgIIIc, respectively. (B) Amino acid sequences of IgIIIb and IgIIIc. Amino acids that are identical in IgIIIb and IgIIIc are highlighted in yellow.
In Japan, the 5-year relative survival rates for esophageal cancer patients are is approximately 30%, suggesting that the esophageal cancer is has a poorer prognosis compared than that of most other cancers9. However, the 5-year relative survival rate for stage I esophageal cancer patients is approximately 80%; thus, it is important to find a beneficial molecular target for esophageal cancer therapy at an early stage9.
In the current study, the expressions of FGFR3IIIb, FGFR3IIIc, and other FGFRs were assessed in esophageal carcinoma (EC) and neighboring non-cancerous mucosa (NCM) using RT-PCR analysis. It was found that the incidence of FGFR3IIIc expression in EC was 2-fold compared to that in NCM, but no difference was observed in the incidence of expressions of FGFR3IIIb and other FGFRs between EC and NCM.
Immunohistochemical analysis with anti-FGFR3IIIc antibody was performed to determine whether FGFR3IIIc was expressed in EC cells. We found that the cells expressing SCC-112, a tumor marker, were positively stained by FGFR3IIIc at an early stage (stage 0) of ESCC, and these cells were also shown to colocalize with Ki-67, a cell proliferation marker. These results indicate that the enhanced expression of FGFR3IIIc in ESCC may promote cell proliferation.
To examine whether the enhanced expression of FGFR3IIIc promotes esophageal cancer cell proliferation, EC-GI-10 cells, an EC cell line, were used for in vitro proliferation analysis. The knockdown of endogenous FGFR3 in EC-GI-10 cells by siRNA (siFGFR3) significantly reduced cell proliferation. Interestingly, overexpression of FGFR3IIIc by lentiviral infection enhanced cell proliferation, whereas that of FGFR3IIIb did not. As an additional finding, the enhanced cell proliferation induced by FGFR3IIIc expression was significantly suppressed by AZD4547, an FGFR-specific kinase inhibitor (Figure 2A), suggesting that upregulation of FGFR3IIIc promoted cell proliferation (Figure 2B).
RT-PCR analysis showed that EC-GI-10 cells, ESCC, and NCM obtained from ESCC patients expressed FGF2, indicating that FGFR3IIIc expression in ESCC expressing FGFR3IIIc-specific FGFs, such as FGF2, may create an autocrine signaling pathway to stimulate their own proliferation.
Figure 2 FGFR3 activation and FGFR-targeting AZD4547. (A) FGF binding to FGFR3 causes their assembly into dimers, which enables the two receptor cytoplasmic domains to cross-phosphorylate each other on multiple tyrosine residues (Y577, Y647/648, Y724, Y760, and Y770). These autophosphorylated tyrosines on the intracellular domain of FGFR3 generate signals through, for example, the Ras-MAPK, PI3K-Akt, STATS, and PLC-γ-PKC pathways to induce cell proliferation, survival, differentiation, and migration. AZD4547, an FGFR kinase inhibitor, competes for the adenosine triphosphate (ATP)-binding site and reduces the kinase activity of FGFR, suppressing cancer cell proliferation. (B) The cell proliferation of FGFR3IIIc-overexpressing EC-GI-10 cells (siFGFR3 + FGFR3IIIc) was significantly stronger than that of siFGFR3-EC-GI-10 cells (siFGFR3, by 2-fold), whereas the overexpression of FGFR3IIIb (siFGFR3 + FGFR3IIIb) was not. AZD4547 at 30 and 100 nmol/L suppressed the enhanced cell proliferation associated with FGFR3IIIc expression by approximately 60% and 70%, respectively. The results are shown as the mean ± S.E. of quintuplicate experiments [#, p < 0.05 versus the siFGFR3 cells, t-test, N.S., not significant, and *, p < 0.05 versus the FGFR3IIIc cells without AZD4547 (−), Dunnett’s multiple comparison test].
Key Findings: This study reports for the first time that:
- FGFR3IIIc was expressed in ESCC patients at stage 0, but not in normal esophageal epithelium cells.
- FGFR3IIIc expression in ESCC promoted cell proliferation by endogenous FGFR3IIIc-specific FGFs, such as FGF2, via an autocrine pathway.
- The enhanced cell proliferative activity with FGFR3IIIc expression in ESCC was suppressed by AZD4547, an FGFR-specific tyrosine kinase inhibitor.
These findings provide clear evidence of the importance of FGFR3IIIc as an early stage tumor marker as well as a molecular target for ESCC therapy.
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