1. The Tumor Suppressor SASH1 Interacts With the Signal Adaptor CRKL to Inhibit Epithelial–Mesenchymal Transition and Metastasis in Colorectal Cancer
- Author
-
Solenne Chardonnet, Eduardo Domínguez Medina, Fabian Christoph Franke, Johannes Müller, Ulrich Nitsche, Klaus-Peter Janssen, Miguel Abal, Ewa Ninio, Henri Weidmann, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), Universidade de Santiago de Compostela [Spain] (USC ), Sorbonne Université (SU), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Plateforme Post-génomique de la Pitié-Salpêtrière (P3S), UMS omique (OMIQUE), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases [IHU ICAN], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut de Cardiométabolisme et Nutrition = Institute of Cardiometabolism and Nutrition [CHU Pitié Salpêtrière] (IHU ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), and Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)
- Subjects
0301 basic medicine ,DMEM, Dulbecco's modified Eagle medium ,Colorectal cancer ,Amino Acid Motifs ,Metastasis ,0302 clinical medicine ,GTPase, guanosine triphosphatase ,RNA interference ,Neoplasm Metastasis ,TGF, transforming growth factor ,Original Research ,GFP, green fluorescent protein ,CRKL, V-Crk avian sarcoma virus CT10 oncogene homolog-like ,TNF, tumor necrosis factor ,SRC-Kinase ,ZEB, zinc-finger δEF1 family ,Gastroenterology ,EMT ,Nuclear Proteins ,gRNA, guide RNA ,mRNA, messenger RNA ,3. Good health ,ddc ,CRISPR/Cas9, Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 ,Phenotype ,030211 gastroenterology & hepatology ,EMT, epithelial-mesenchymal transition ,Colorectal Neoplasms ,Tumor Suppressor ,Chemoresistance ,Protein Binding ,Signal Transduction ,Proto-oncogene tyrosine-protein kinase Src ,Epithelial-Mesenchymal Transition ,PBS, phosphate-buffered saline ,SH3N, N-terminal Src-homology 3 domain ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,Biology ,SH2, Src-homology 2 domain ,src Homology Domains ,cDNA, complementary DNA ,03 medical and health sciences ,medicine ,Humans ,Neoplasm Invasiveness ,Epithelial–mesenchymal transition ,lcsh:RC799-869 ,Adaptor Proteins, Signal Transducing ,Hepatology ,Oncogene ,Tumor Suppressor Proteins ,[SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology ,HCT116 Cells ,medicine.disease ,qRT-PCR, quantitative reverse-transcription polymerase chain reaction ,CRKL ,HEK293 Cells ,030104 developmental biology ,MS, mass spectrometry ,SH3, Src-homology 3 domain ,NLS, nuclear localization signal ,Cell culture ,Cancer research ,lcsh:Diseases of the digestive system. Gastroenterology ,BSA, bovine serum albumin ,CRISPR-Cas Systems ,SASH1, sterile α motif– and Src-homology 3–domain containing 1 - Abstract
Background & Aims The tumor-suppressor sterile α motif– and Src-homology 3–domain containing 1 (SASH1) has clinical relevance in colorectal carcinoma and is associated specifically with metachronous metastasis. We sought to identify the molecular mechanisms linking decreased SASH1 expression with distant metastasis formation. Methods SASH1-deficient, SASH1-depleted, or SASH1-overexpressing HCT116 colon cancer cells were generated by the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9-method, RNA interference, and transient plasmid transfection, respectively. Epithelial-mesenchymal transition (EMT) was analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblotting, immunofluorescence microscopy, migration/invasion assays, and 3-dimensional cell culture. Yeast 2-hybrid assays and co-immunoprecipitation/mass-spectrometry showed V-Crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) as a novel interaction partner of SASH1, further confirmed by domain mapping, site-directed mutagenesis, co-immunoprecipitation, and dynamic mass redistribution assays. CRKL-deficient cells were generated in parental or SASH1-deficient cells. Metastatic capacity was analyzed with an orthotopic mouse model. Expression and significance of SASH1 and CRKL for survival and response to chemotherapy was assessed in patient samples from our department and The Cancer Genome Atlas data set. Results SASH1 expression is down-regulated during cytokine-induced EMT in cell lines from colorectal, pancreatic, or hepatocellular cancer, mediated by the putative SASH1 promoter. Deficiency or knock-down of SASH1 induces EMT, leading to an aggressive, invasive phenotype with increased chemoresistance. SASH1 counteracts EMT through interaction with the oncoprotein CRKL, inhibiting CRKL-mediated activation of SRC kinase, which is crucially required for EMT. SASH1-deficient cells form significantly more metastases in vivo, depending entirely on CRKL. Patient tumor samples show significantly decreased SASH1 and increased CRKL expression, associated with significantly decreased overall survival. Patients with increased CRKL expression show significantly worse response to adjuvant chemotherapy. Conclusions We propose SASH1 as an inhibitor of CRKL-mediated SRC signaling, introducing a potentially druggable mechanism counteracting chemoresistance and metastasis formation., Graphical abstract
- Published
- 2019