The tumor suppressor SASH1 interacts with the signal adaptor CRKL to inhibit epithelial-mesenchymal transition and metastasis in colorectal cancer

Fabian Christoph Franke, Johannes Müller, Miguel Abal, Eduardo Domínguez Medina, Ulrich Nitsche, Henri Weidmann, Solenne Chardonnet, Ewa Ninio, Klaus-Peter Janssen
2018 Cellular and Molecular Gastroenterology and Hepatology  
This study showed a metastasis-suppressive function of sterile a motif-and Src-homology 3-domain containing 1 (SASH1) in vivo. Furthermore, SASH1 antagonizes epithelial-mesenchymal transition, tumor aggressiveness, and chemoresistance in colon cancer. Mechanistically, SASH1 directly inhibits the oncoprotein V-Crk avian sarcoma virus CT10 oncogene homolog-like, introduced as its new interaction partner. BACKGROUND & AIMS: The tumor-suppressor sterile a motifand Src-homology 3-domain containing 1
more » ... 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 SASH1overexpressing 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. Epithelialmesenchymal 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, Abbreviations used in this paper: BSA, bovine serum albumin; cDNA, complementary DNA; CRISPR/Cas9, Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9; CRKL, V-Crk avian sarcoma virus CT10 oncogene homolog-like; DMEM, Dulbecco's modified Eagle medium; EMT, epithelial-mesenchymal transition; GFP, green fluorescent protein; gRNA, guide RNA; GTPase, guanosine triphosphatase; mRNA, messenger RNA; MS, mass spectrometry; NLS, nuclear localization signal; PBS, phosphate-buffered saline; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; SASH1, sterile a motif-and Src-homology 3-domain containing 1; SH2, Srchomology 2 domain; SH3, Src-homology 3 domain; SH3N, N-terminal Src-homology 3 domain; TGF, transforming growth factor; TNF, tumor necrosis factor; ZEB, zinc-finger dEF1 family. Most current article
doi:10.1016/j.jcmgh.2018.08.007 fatcat:ulfpnibmrbde5bcpvgjwcwwpsu