Intratumoral heterogeneity of c-KIT mutations in a feline splenic mast cell tumor and their functional effects on cell proliferation

Abstract

A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of c-KIT was detected in the masses from skin and spleen by a commercial laboratory test. Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.

Figure 1

Schematic representation of the structure of feline c-KIT gene and c-KIT protein. Boxes show the exon structure of feline c-KIT cDNA, and the numbers in each box indicate the exon number. The number above and below each box indicates the numbers of the first nucleotide and amino acid in each exon, respectively. Protein structure is shown below the DNA structure in gray line and gray boxes, as predicted from the amino acid sequence of feline c-KIT by InterPro (https://www.ebi.ac.uk/interpro/). Numbers below the protein structure indicate the amino acid numbers of each domain. D1–D5 refer to immunoglobulin domains. TM, transmembrane domain; JM, juxtamembrane domain; TK, tyrosine kinase domain. M537R and W854R indicate the point mutation in the TM domain and TK2 domain, respectively. Dotted boxes below the protein structure represent the mutations in exons 8 and 9. WT, wild type; Ins8, the insertional mutation in exon 8; Δ9, the deleted mutation in exon 9.

Figure 2

Cell proliferation rate of Ba/F3 cell lines expressing mutant c-KIT proteins. (A) Schematic representation of mutation patterns of c-KIT clones used for this assay. Asterisk indicates the point mutation in exon 10 (M537R) or the point mutation in exons 18 (W854R). The gray box and the blank between exons 9 and 10 indicate the duplicated mutation in exon 8 and the four amino acids deletion in exon 9, respectively. (B) Cell proliferation rate of Ba/F3 cell lines expressing each mutant c-KIT protein. Each mutant cell line was cultured for 48 h in the medium, without (mock, white bar) or with feline SCF (fSCF; gray bar). After 48 h, CCK-8 was added, and the cell proliferation rate was measured by plate reader. Bar graph indicates the mean from four independent biological replicates, and open dots indicate the values obtained from each experiment. p-values were calculated for the comparison among all cell lines using two-tailed one-way ANOVA with a post hoc Tukey–Kramer test. Dagger indicates a significant difference (p < 0.0001) between Mock and fSCF-treated each cell line.

Figure 3

Phosphorylation status of c-KIT in Ba/F3 cells expressing the mutant c-KIT. Ba/F3 cell lines stably expressing mutant forms of c-KIT protein were cultured in plain RPMI for 2 h and stimulated with fSCF (100 ng/ml) for 5 min. After stimulation, cells were collected and the extracted whole cell lysates were conducted for SDS-PAGE, followed by western blotting with anti-phospho-c-KIT (Tyr719), anti-Flag (M2), and anti-β-actin antibodies. A representative result of three independent experiments is shown. Full image of western blotting is shown in Supplementary Figure.

Figure 4

The effects of toceranib on cell proliferation rate of Ba/F3 cell lines expressing mutant c-KIT proteins. Cell proliferation rate of Ba/F3 cell lines expressing each mutant c-KIT protein was examined with the different amounts of toceranib (0, 0.1, 1.0 and 10 µM). Each mutant cell line was cultured for 48 h in the medium (mock, white bar) or with fSCF (gray bar). After 48 h, CCK-8 was added, and the cell proliferation rate was measured by plate reader. Bar graph indicates the mean from three independent biological replicates, and open dots indicate the values obtained from each experiment. p-values were calculated for the comparison among all cell lines using two-tailed one-way ANOVA with a post hoc Tukey–Kramer test. Dagger indicates a significant difference (p < 0.05) between with or without fSCF, within each cell line. Double dagger indicates the significance (p < 0.05) between the different dosages within fSCF-treated each cell line.