Small ArfGAP1 (stromal
membrane-associated protein 1, SMAP1), a
GTPase-activating protein specific for
ADP-ribosylation factor 6 (Arf6), which is a
small GTPase acting on membrane trafficking and actin remodeling, is frequently mutated in various
tumors displaying
microsatellite instability (MSI), notably in MSI
colorectal cancers (CRC). Genotyping of 93 MSI
CRCs (40 stage II, 32 stage III and 21 stage IV) allowed us to underscore that SMAP1 mutation frequency was inversely correlated with disease stage (P=0.01). Analysis of 46
cancer cell lines showed that SMAP1 mutations occurred only in MSI
tumors, and consisted exclusively in short insertion or deletion in the coding 10-adenine repeat, generating a
premature termination codon located downstream the ArfGAP domain. SMAP1 transcript levels were significant decreased (P=0.006), and truncated SMAP1
protein could not be detected in cells displaying biallelic SMAP1 mutations, owing to its sensitivity to
proteasome degradation. To investigate the role of SMAP1 mutations, we used the SMAP1-null HCT116 cell line and we established three isogenic SMAP1-complemented clones. Cell proliferation was first assessed in vivo using subcutaneous xenografts into immunodeficient mice.
Tumors developed in all animals regardless of the cell line injected, but
tumor volumes were significantly smaller for both SMAP1-complemented clones compared with HCT116 (P<0.0001, at the time of killing). In vitro, SMAP1 mutations also increased cell clonogenicity (P=0.02-0.04), cell proliferation (P=0.008) by shortening the G2/M phase and decreased cell invasiveness (P=0.03-0.003). In keeping, SMAP1-complemented HCT116 gained several mesenchymal markers (Snail, Slug and
vimentin) considered as a hallmark of epithelial-to-mesenchymal transition. These observations are reminiscent of some clinical characteristics of MSI
CRCs, notably their larger size and lower rate of
metastasis. Our observations suggest that SMAP1 loss-of-function mutations in MSI CRC may contribute to the emerging oncogenic pathway involving abnormal Arf6 regulation.