Differential effects of polymorphic alleles of FGF receptor 4 on colon cancer growth and metastasis

Abstract

A gly(388)arg polymorphism (rs351855) in the transmembrane domain of the fibroblast growth factor receptor (FGFR4) is associated with increased risk, staging, and metastasis in several different types of cancer. To specifically assess the impact of the polymorphic FGFR4 in colorectal cancer (CRC), we engineered CRC cell lines with distinct endogenous expression patterns to overexpress either the FGFR4(gly) or FGFR4(arg) alleles. The biologic analyses revealed an oncogenic importance for both polymorphic alleles, but FGFR4(gly) was the stronger inducer of tumor growth, whereas FGFR4(arg) was the stronger inducer of migration. An evaluation of clinical specimens revealed that FGFR4 was upregulated in 20/71 patients independent of gly(388)arg status. There was no correlation between the presence of an FGFR4(arg) allele and CRC or polyp risk in 3,471 participants of the CORSA study. However, among 182 patients with CRC, FGFR4(arg)-carriers had a fivefold higher risk of tumors that were stage II or greater. Together, our results established that both allelic forms of FGFR4 exert an oncogenic impact and may serve equally well as therapeutic targets in CRC. One important implication of our findings is that FGFR4(arg)-carriers are at a higher risk for more aggressive tumors and therefore may profit from early detection measures.

Figure 1

Impact of FGFR4 overexpression on tumor cell growth and migration in vitro. Stable transfectants overexpressing FGFR4^arg or FGFR4^gly in SW480, HCT116, or HT29 cells were used to assess growth and malignant characteristics in vitro. Stable transfectants with the pcDNA3 vector were used as controls. A, 100 and 200 cells were seeded/6-well, medium was changed after 24 hours, and the number of colonies counted after 10 days of growth. B, five thousand cells each were suspended in soft agar medium and plated in 6-well plates. The number of colonies was counted at a magnification of 10-fold after 3 weeks of growth. C, cell migration was determined by filter migration assay from 2 × 10⁴ cells/24-well. Photographs of cultures show representative results from SW480 transfectants. Quantitative results from all cell lines were calculated as % of control, pooled from at least 3 independent experiments, and presented as mean ± SD. *, **, *** indicate an increase as compared with the control at P < 0.05, 0.01, and 0.001, respectively. ## indicates a decrease as compared with control at P < 0.01. &, &&, and &&& indicate a difference between the FGFR4^gly and FGFR4^arg groups at P < 0.05, 0.01, and 0.001, respectively.

Figure 2

Impact of FGFR4 knockdown on clonogenicity and migration in vitro. siRNA oligonucleotides were introduced by lipofection into HCT116 and HT29 cells. Controls were transfected with scrambled control siRNA. A, five days after knockdown, cell viability was measured by MTT assay. B, 24 hours after knockdown, proliferation was determined by ³H-thymidine uptake. From parallel cultures, cells were harvested 24 hours after transfection and plated at 100 and 200 cells/6-well for colony formation assays (C) and 2 × 10⁴ cells/filter for migration assays (D). Results were pooled from at least 3 independent experiments and presented as mean ± SD. # and ## indicate a decrease as compared with control at P < 0.05 and 0.01, respectively.

Figure 3

FGFR4-dependent downstream signaling. A and B, semiconfluent cultures of SW480 transfectants were starved and cell membranes were prepared and extracted. Protein amount and phosphorylation of PLCγ and FRS2α was analyzed by Western blot analysis. C and D, from parallel cultures, protein lysates were prepared after 24 hours for analysis of signaling molecules using phospho-specific antibodies. Photographs of Western blot analysis show results of representative gel runs. Quantification was conducted from 2 independent experiments using duplicate samples.

Figure 4

Impact of FGFR4 overexpression on tumor growth and metastasis in vivo. 10⁶ cells each of SW480^gly, SW480^arg, and SW480^co cells were injected s.c. into SCID mice. A and B, tumor growth was monitored and the mice sacrificed when tumor size reached 5 cm³ or after 9 weeks, whichever came first. The tumors were analyzed by immunohistochemistry using antibodies recognizing cytokeratin 20 and Ki67. C and D, lungs were isolated from tumor-bearing mice and fixed with formalin. Serial sections were stained using antibodies recognizing Ki67 to detect tumor cells that were scored according to the criteria given in Supplementary Materials.