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// Ibtissem Djaafri 1,2,* , Farah Khayati 1,2,3,* , Suzanne Menashi 4 , Jorg Tost 5,6 , Marie-Pierre Podgorniak 3 , Aurelie Sadoux 1,3 , Antoine Daunay 6 , Luis Teixeira 7 , Jean Soulier 2,8 , Ahmed Idbaih 9,10 , Niclas Setterblad 1,2 , Francoise Fauvel 1,2 , Fabien Calvo 1,2 , Anne Janin 2,11,12 , Celeste Lebbe 2,13,14 and Samia Mourah 1,2 1 Inserm UMR-S 940 Paris, France 2 Institute of Hematology (IUH), U niversite Paris-Diderot, Sorbonne Paris Cite, Paris, France 3 AP-HP, Hopital Saint-Louis, Laboratoire de Pharmacologie-Genetique, Paris, France 4 Universite Paris Est Creteil, CNRS-UMR; Creteil, France 5 Laboratory for Epigenetics, Centre National de Genotypage, CEA-Institut de Genomique, Evry, France 6 Laboratory for Functional Genomics, Fondation Jean Dausset - CEPH, Paris, France 7 AP-HP ; Hopital Saint-Louis, Service d’oncologie medicale, Paris, France 8 Hematology Laboratory APHP, Saint-Louis Hospital, Paris, France 9 AP-HP, Groupe Hospitalier Pitie-Salpetriere, Service de Neurologie 2-Mazarin, Paris, France 10 Inserm U 975, Paris, 75013 France, CNRS, UMR, Paris, France 11 Inserm, U728, Paris, France 12 AP-HP, Hopital Saint-Louis, Laboratoire de Pathologie, Paris, France 13 AP-HP, Hopital Saint-Louis, Departement de Dermatologie, Paris, France 14 Inserm U976, Paris, France * These Authors contributed equally to this work Correspondence: Samia Mourah, email: // Keywords : Tumor suppressor gene, Kindlin-3, Invasion/Migration, metastasis, Integrins Received : May 26, 2014 Accepted : June 18, 2014 Published : June 20, 2014 Abstract Kindlin-3 (FERMT-3) is known to be central in hemostasis and thrombosis control and its deficiency disrupts platelet aggregation and causes Leukocyte Adhesion Deficiency disease. Here we report that Kindlin-3 has a tumor suppressive role in solid cancer. Our present genetic and functional data show that Kindlin-3 is downregulated in several solid tumors by a mechanism involving gene hypermethylation and deletions. In vivo experiments demonstrated that Kindlin-3 knockdown in 2 tumor cell models (breast cancer and melanoma) markedly increases metastasis formation, in accord with the in vitro increase of tumor cell malignant properties. The metastatic phenotype was supported by a mechanism involving alteration in β3-integrin activation including decreased phosphorylation, interaction with talin and the internalization of its active form leading to less cell attachment and more migration/invasion. These data uncover a novel and unexpected tumor suppressor role of Kindin-3 which can influence integrins targeted therapies development.

International audience; Type III collagen binding protein (TIIICBP) was previously described as a platelet membrane protein that recognizes the KOGEOGPK peptide sequence within type III collagen. In order to better characterize this protein, we performed different approaches including mass spectrometry sequencing and functional experiments. This study leads to identify high biochemical and functional similarities between TIIICBP and kindlin-3, a member of a family of focal adhesion proteins. Indeed, mass spectrometry surveys indicated that TIIICBP contains several peptides identical to kindlin-3, covering 41% of the amino acid sequence. Polyclonal antibodies raised against a kindlin-3 specific N-terminal sequence, recognized and immunoprecipitated TIIICBP from platelet lysates. Electron microscopy and flow cytometry experiments showed that kindlin-3, as well as TIIICBP, were present associated to platelet membrane and a trans-location of cytosolic kindlin-3 to the platelet membrane was observed after platelet activation. Similarly to anti-TIIICBP antibodies and the KOGEOGPK peptide, anti-kindlin-3 antibodies inhibited platelet interactions with type III collagen under flow conditions and slowed down platelet aggregation induced by glycoprotein VI agonists; e.g. collagen-related peptides and convulxin. In addition, the anti-kindlin-3 antibody inhibited platelet aggregation induced by low e but not high e doses of ADP or thrombin which depends on a IIb b 3 integrin function. In conclusion, our results show that the peptides identified by mass spectrometry from purified TIIICBP correspond to the kindlin-3 protein and demonstrate biochemical and functional similarities between TIIICBP and kindlin-3, strengthening a key role for TIIICBP/kindlin-3 in platelet interactions with collagen by cooperating with glycoprotein VI activation and integrin clustering in focal adhesion complexes.

Kindlin-3 (FERMT-3) is a key integrin activating protein belonging to the Kindlin family which includes three members Kindlin-1, 2 and 3. Kindlins can directly bind to various classes of integrins and participate in their activation, thus playing a key role in the regulation of cell-matrix adhesions. Kindlin-3 has been known to be central in the control of hemostasis and thrombosis as its deficiency disrupts platelet aggregation and causes Leukocyte Adhesion Deficiency disease. Its expression has so far been only described in hematopoietic cells, and more recently in endothelial cells. The current manuscript highlights key findings from our recent research describing a novel role of Kindlin-3 as a tumor suppressor in cancer. We present data showing that Kindlin-3 is expressed more ubiquitously than previously thought, and that through gene inactivation mechanisms, its expression is significantly reduced in several human solid cancer lesions. The increase in the malignant properties of tumor cells when Kindlin-3 expression is silenced and the inverse expression of Kindlin-3 with the tumor promoter EMMPRIN/CD147 corroborate the tumor suppressor role of Kindlin-3. A schematic model based on our research data describing Kindlin-3 mode of action in tumor cells is presented.

This work aimed to investigate the role of the disintegrin domain of the human ADAM9 (ADAM9D) on the adhesion of breast tumor cells and platelets to collagen I, in a dynamic flow assay to simulate in vivo shear conditions. Recombinant ADAM9D was able to support tumor cell adhesion through binding to the beta1 integrin subunit and also to inhibit the invasion through matrigel in vitro. In a dynamic flow assay ADAM9D inhibited about 75% and 65% of MDA-MB-231 tumor cells and platelet adhesion to collagen I, respectively. In addition, it was demonstrated that alphaVbeta3 integrin is new interacting partner for ADAM9D. In conclusion, these results suggest a role for the disintegrin domain of ADAM9 in the metastatic process. Also, ADAM9D may be a tool for investigating the role of ADAMs in metastasis and cancer progression and for the design of selective inhibitors against the adhesion and extravasation of cancer cells.

International audience; EMMPRIN is known to promote tumor invasion through extracellular matrix (ECM) degradation. Here we report that EMMPRIN can regulate melanoma cell adhesion to the ECM through an interaction with β1 integrin involving kindlin-3. In this study, EMMPRIN knockdown in the human melanoma cell line M10 using siRNA decreased cell invasion and significantly increased cell adhesion and spreading. A morphological change from a round to a spread shape was observed associated with enhanced phalloidin-labelled actin staining. In situ proximity ligation assay and co-immunoprecipitation revealed that EMMPRIN silencing increased the interaction of β1 integrin with kindlin-3, a focal adhesion protein. This was associated with an increase in β1 integrin activation and a decrease in the phosphorylation of the downstream integrin kinase FAK. Moreover, the expression at both the transcript and protein level of kindlin-3 and of β1 integrin was inversely regulated by EMMPRIN. EMMPRIN did not regulate either talin expression or its interaction with β1 integrin. These results are consistent with our in vivo demonstration that EMMPRIN inhibition increased β1 integrin activation and its interaction with kindlin-3. To conclude, these findings reveal a new role of EMMPRIN in tumor cell migration through ß1 integrin/kindlin-3-mediated adhesion pathway.