Fibroblast Growth Factor Receptor 2 (FGFR-2) in gastrointestinal cancerDepartments of Pathology and Integrative Oncological Pathology Nippon Medical School
Characteristics of FGFR-2• Located in chromosome 10q26• Single transmembrane receptor• Alternative splicing isoforms * IIIb and IIIc isoforms (extracellular domain) * C1, C2 and C3 variants (intracellular domain)FGFR-2 has more than 20 alternative splicing isoforms.Expressions and roles of these two types of splicing in extracellular andintracellular domains are closely examined in cancers.
FGFR-2 in Various Cancers Gastric Ca, Breast Ca Lung Ca, Endometrial Ca, Melanoma Gene amplification Missense mutationRisk of Breast and Endometrial Ca Endometrial Ca SNPs in intron 2 Activating mutation FGFR-2 Cervical Ca, Colorectal Ca, Esophageal Ca, Gastric Ca Gastric Ca Pancreatic Ca Overexpression of C3 Increase of IIIb or IIIc Splicing switch from IIIb to IIIc Progression of Prostate Ca. EMT of Bladder Ca. Genetic alterations and overexpression of FGFR-2 have been reported in various. Abnormalities of FGFR-2 are often reported in gastrointestinal cancers.
FGFR-2 Expression in Colorectal Cancer Tissues Percentage of FGFR-2 expression % 70 60 ** 50 40 30 20 10 0 Center lesion Invasive front Matsuda et al., Cancer Lett. 309: 209-219, 2011In colorectal cancer, FGFR2 immunoreactivity was localized at the peripheral lesion of thecancer cell nests, as shown by the arrows.
Inhibitory effects of FGFR-2 in Colorectal Cancer Cell proliferation assay in vitro Subcutaneous tumor in nude mice 0.8 Sc-3 200Absorbance at 450 nm Sc-6 Sh-6 Tumor volume (μm3) 0.6 150 Sh-12 0.4 100 * * 0.2 50 * 0 * 0 0 24 48 (hours) 7 14 21 days Sh-6 and Sh-12: FGFR-2 shRNA stably transfected LoVo cells Matsuda et al., Cancer Lett. 309: 209-219, 2011Mice injected with FGFR2 shRNA-transected LoVo cells had smaller subcutaneous tumors thanmice injected with control cells.These findings suggest that FGFR2 is a therapeutic target for colorectal cancer.
FGFR-2 IIIc in Human Pancreatic Cancer Cases Immuohistochemistry In situ hybridization anti-sense sense Ca Ca Ca Ca Ca Ca Ca: pancreatic cancer Arrows: fibroblastsIn human pancreatic cancer tissues, FGFR2 IIIc and its mRNA were expressed in pancreaticcancer cells and in some fibroblasts close to the cancer cells, as shown by the arrows.
Correlation of Clinicopathological Features and FGFR-2 IIIc Expression in Pancreatic Cancers Overall survival curves Duration to development of liver metastasis after surgery (%) (%) 100 100 P=0.1571 P=0.0071 80 80 60 60 FGFR-2 IIIc (-) 40 FGFR-2 IIIc (-) 40 20 20 FGFR-2 IIIc (+) 0 0 0 500 1000 1500 2000 days 0 500 1000 1500 2000 days FGFR-2 IIIc (+) Ishiwata et al., Am J Pathol 180: 1928-1941, 2012Overall survival curves showed a tendency for poorer prognoses in the FGFR2 IIIc-positivegroups.The duration of the development of liver metastasis after surgery was significantly shorter inIIIc-positive groups than in those of the negative groups.
Preparation of FGFR-2 IIIc Stably Transfected KLM-1 Cells FGFR-2 IIIc mRNA Flow cytometry Mock-3 FGFR2IIIc-6 120 100 80 60 5.35% 12.4% 20 0 Ishiwata et al., Am J Pathol 2012As shown here, IIIc mRNA levels were markedly higher in two IIIc-transfected clones, but notin mock cells. Flow cytometry revealed that higher IIIc-expressing cells on the cell surfacewere more common in IIIc-transfected cells than in mock cells.
Orthotopic Implantation Model of FGFR-2 IIIc-transfected cellsMock-3 * Pancreas tumor (g) 3.0 2.0FGFR-2 IIIc-6 1.0 0In the orthotopic implantation model, IIIc-transfected cells also formed larger primary tumorscompared to the mock cells.Moreover, pancreatic tumor weights were significantly elevated in IIIc-transfected cells comparedto those with mock cells.
Liver Metastasis in Orthotopic Implantation Model Mock-3 2.0 * Liver weight (g) 1.5 1.0 FGFR-2 IIIc-6 0.5 0 Ishiwata et al., Am J Pathol 2012Both the amount and weight of liver metastases were increased in the IIIc-transfected cells.
Effects of siRNA targeting FGFR-2 IIIc on PANC-1 cell growth FGFR2 IIIc/18S rRNA 1.2 qRT-PCR Cell proliferation rates 1.0 0.8 1.6 Absorbance at 450nm 0.6 0.4 ** ** 1.4 1.2 * ** 0.2 1.0 0 0.8 0.6 0.4 Flow cytometry 0.2 0 Negative s275290 s275191 7.37% 1.85% 1.06%The siRNA decreased IIIc mRNA and IIIc protein expression, and the proliferation rates of thesecells were lower than those in control siRNA transfected cells.
Effects of Polyclonal anti-FGFR-2 IIIc on Pancreatic Cancer Cell Growth and Migration Inhibition of cell proliferation Inhibition of migration Migrated distance (μm) PANC-1 KLM-1% inhibition of untreated control 1 10 50 100 100 (mg/ml) 600 *** 0 400 5 10 200 15 * 0 20 (%) * PANC-1: High FGFR-2 IIIc expression cell PANC-1 KLM-1 KLM-1: Low FGFR-2 IIIc expression cell Ishiwata et al., Am J Pathol 2012 A significant inhibition of proliferation was observed after the addition of the anti-FGFR2 IIIc antibody in PANC-1 cells. The anti-FGFR2 IIIc antibody also inhibited the migration of PANC-1 cells.
Immunoreactivity of FGFR-2 IIIc Survival Curves of FGFR-2IIIc high and low colorectal cancer (%) (%) Immunoreactivity of FGFR-2 IIIc P=0.0265 100 *# 100 FGFR-2 IIIc (-) * 50 50 FGFR-2 IIIc (+) 0 0 1000 2000 3000 (days) Matsuda et al., Mol Cancer Ther 11: 2010-2020, 2012The percentages of FGFR2 IIIc-positive cells in these lesions increased in the order shown here.The group in which FGFR2 IIIc was highly expressed in colorectal cancer cells, showed shortersurvival rates.
FGFR-2 IIIc-transfected DLD-1 cells in vivo Subcutaneous tumors Orthotopic implanted tumors Mock-1 Wild * Mock-1 2000 **Tumor volume (mm3) Fgfr-9 Tumor volume (mm3) 600 400 * 1000 200 Fgfr-9 0 0 7 14 21 day Matsuda et al., Mol Cancer Ther, 2012 In subcutaneous transplantation studies, FGFR2 IIIc transfected colorectal cancer cells formed larger tumors than mock cells. Furthermore, FGFR2 IIIc transfected cells formed larger tumors in the orthotopic implantation model, as shown by the arrows. One out of three animals with IIIc transfected cells showed metastasis in their small intestines, as shown by the white arrow.
Inhibition of CRC cell growth using human monoclonal anti-FGFR-2 IIIc antibody Western blot Cell proliferation LoVo HCT-15 untreated rhIIIb rhIIIc GFP FGFR-2 IIIcFGFR-2 IIIc Absorbance at 450nm 0.8human IgG * 0.6 * 1.0 0.4 * 0.5 * 0.2 0 0 24 48 (hrs) 24 48 (hrs) Matsuda et al., Mol Cancer Ther, 2012Human monoclonal anti-FGFR2 IIIc antibody was prepared by phage display method.For possible clinical applications in the future, we prepared a human monoclonal anti-FGFR2 IIIcantibody using the phage-display method. The antibody reacted with recombinant human FGFR2IIIc, but it did not react with recombinant FGFR2 IIIb. Cell proliferation was significantly inhibitedby the addition of the human monoclonal anti-FGFR2 IIIc antibodies.
Inhibition of CRC cell migration using human monoclonal anti-FGFR-2 IIIc antibody Boyden chamber Time lapse x105 LoVo HCT-15 LoVo HCT-15 * * 6 400 * Mean distance (μm) 5 *Cells/well 300 4 3 200 2 100 1 0 0In both Boyden chamber and time lapse analyses, cell migration was also significantly inhibitedby the addition of the human monoclonal anti-FGFR2 IIIc antibodies.These findings suggest that FGFR2 IIIc plays important roles in the carcinogenesis of colorectalcancer and tumor progression. Matsuda et al., Mol Cancer Ther, 2012
Summary of FGFR-2 IIIcClinicopathological studies IHC analysis of FGFR-2 IIIc Pancreatic cancer Liver metastasis Colorectal cancer Overall survival↓ Experimental studies FGFR-2 IIIc- transfected cell Growth Migration Invasion Metastasis Attachment Pancreatic Cancer ↑ ↑ ↑ ↑ → Colorectal Cancer ↑ ↑ ↑ ↑ ↓Clinicopathologically, FGFR2-IIIc correlated with liver metastasis in pancreatic cancer and overall survival incolorectal cancers. Experimental studies using transplantation models showed that FGFR-2 IIIc contributes tothe aggressiveness of these cancers.