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Involvement of small ArfGAP1 (SMAP1), a novel Arf6-specific GTPase-activating protein, in microsatellite instability oncogenesis

Oncogene volume 33, pages 2758–2767 (2014)Cite this article

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Abstract

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.

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Acknowledgements

We thank Drs Mathieu Boissan, Philippe Chavrier, Marie-Lise Lacombe and Guillaume Montagnac for their constant support and helpful discussions, as well as Dr Peggy Cuilliere-Dartigues for sharing some data. We are grateful to Martine Mergey, Anne-Marie Faussat and Philippe Fontanges for their invaluable help in RT-PCRq analyses, cytometry and confocal microscopy, respectively. We further acknowledge the expert technical assistance of Fatiha Mérabtene in performing immunohistochemical analyses of mouse and human tumor samples. We are indebted to the members of INSERM UMR_S 903 for sharing the ImageJ plugin. Fatiha Sangar received a 3-year fellowship from ‘Ministère de l’Enseignement Supérieur et de la Recherche’ followed by a 6-month funding from ‘Fondation ARC pour la Recherche sur le Cancer’. This work has been supported by grants from the Association pour la Recherche sur le Cancer (#5091) and from the Ligue Nationale Contre le Cancer (RS10/75-72 and RS11/75-89).

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Authors and Affiliations

  1. INSERM, UMR_S 938, Saint-Antoine Research Center, Paris, France

    F Sangar, A-S Schreurs, C Umaña-Diaz, A Clapéron, C Desbois-Mouthon, C Calmel, O Mauger, A Zaanan, J-F Fléjou & F Praz

  2. UPMC Univ Paris 06, UMR_S 938, Saint-Antoine Research Center, Paris, France

    F Sangar, A-S Schreurs, C Umaña-Diaz, A Clapéron, C Desbois-Mouthon, C Calmel, O Mauger, A Zaanan, J-F Fléjou & F Praz

  3. Department of Pathology, Sainte-Anne Hospital, University Paris Descartes, Paris, France

    C Miquel

  4. Department of Pathology, Saint-Antoine Hospital, AP-HP, Paris, France

    J-F Fléjou

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Correspondence to F Praz.

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Sangar, F., Schreurs, AS., Umaña-Diaz, C. et al. Involvement of small ArfGAP1 (SMAP1), a novel Arf6-specific GTPase-activating protein, in microsatellite instability oncogenesis. Oncogene 33, 2758–2767 (2014). https://doi.org/10.1038/onc.2013.211

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