ABSTRACT
Stemness was recently depicted as a dynamic condition in normal and tumor cells. We found that the embryonic protein Cripto-1 (CR1) was expressed by normal stem cells at the bottom of colonic crypts and by cancer stem cells (CSCs) in colorectal tumor tissues. CR1-positive populations isolated from patient-derived tumor spheroids exhibited increased clonogenic capacity and expression of stem-cell-related genes. CR1 expression in tumor spheroids was variable over time, being subject to a complex regulation of the intracellular, surface and secreted protein, which was related to changes of the clonogenic capacity at the population level. CR1 silencing induced CSC growth arrest in vitro with a concomitant decrease of Src/Akt signaling, while in vivo it inhibited the growth of CSC-derived tumor xenografts and reduced CSC numbers. Importantly, CR1 silencing in established xenografts through an inducible expression system decreased CSC growth in both primary and metastatic tumors, indicating an essential role of CR1 in the regulation the CSC compartment. These results point to CR1 as a novel and dynamically regulated effector of stem cell functions in colorectal cancer.
Subject(s)
Colorectal Neoplasms/metabolism , GPI-Linked Proteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/metabolism , Animals , Colorectal Neoplasms/physiopathology , Female , GPI-Linked Proteins/genetics , GPI-Linked Proteins/physiology , Gene Expression Regulation, Neoplastic , Genes, src , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/physiology , Mice , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Neoplastic Stem Cells/physiology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Spheroids, Cellular , Tumor Cells, CulturedABSTRACT
Numerous stem cell niches are present in the different tissues and organs of the adult human body. Among these tissues, dental pulp, entrapped within the 'sealed niche' of the pulp chamber, is an extremely rich site for collecting stem cells. In this study, we demonstrate that the isolation of human dental pulp stem cells by the explants culture method (hD-DPSCs) allows the recovery of a population of dental mesenchymal stem cells that exhibit an elevated proliferation potential. Moreover, we highlight that hD-DPSCs are not only capable of differentiating into osteoblasts and chondrocytes but are also able to switch their genetic programme when co-cultured with murine myoblasts. High levels of MyoD expression were detected, indicating that muscle-specific genes in dental pulp cells can be turned on through myogenic fusion, confirming thus their multipotency. A perivascular niche may be the potential source of hD-DPSCs, as suggested by the consistent Ca(2+) release from these cells in response to endothelin-1 (ET-1) treatment, which is also able to significantly increase cell proliferation. Moreover, response to ET-1 has been found to be superior in hD-DPSCs than in DPSCs, probably due to the isolation method that promotes release of stem/progenitor cells from perivascular structures. The ability to isolate, expand and direct the differentiation of hD-DPSCs into several lineages, mainly towards myogenesis, offers an opportunity for the study of events associated with cell commitment and differentiation. Therefore, hD-DPSCs display enhanced differentiation abilities when compared to DPSCs, and this might be of relevance for their use in therapy.
Subject(s)
Cell Culture Techniques/methods , Cell Differentiation , Dental Pulp/cytology , Stem Cells/cytology , Adult , Calcium Signaling/drug effects , Cell Differentiation/drug effects , Cell Lineage/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Chondrogenesis/drug effects , Endothelin-1/pharmacology , Humans , Multipotent Stem Cells/cytology , Multipotent Stem Cells/drug effects , Multipotent Stem Cells/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/physiology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Osteogenesis/drug effects , Phenotype , Regeneration/drug effects , Stem Cell Transplantation , Stem Cells/drug effects , Stem Cells/metabolism , Young AdultABSTRACT
Beta-dystrobrevin, a member of the dystrobrevin protein family, is a dystrophin-related and -associated protein restricted to non-muscle tissues and is highly expressed in kidney, liver and brain. Dystrobrevins are now thought to play an important role in intracellular signal transduction, in addition to providing a membrane scaffold in muscle, but the precise role of beta-dystrobrevin has not yet been determined. To study beta-dystrobrevin's function in brain, we used the yeast two-hybrid approach to look for interacting proteins. Four overlapping clones were identified that encoded Kif5A, a neuronal member of the Kif5 family of proteins that consists of the heavy chains of conventional kinesin. A direct interaction of beta-dystrobrevin with Kif5A was confirmed by in vitro and in vivo association assays. Co-immunoprecipitation with a monoclonal kinesin heavy chain antibody precipitated both alpha- and beta-dystrobrevin, indicating that this interaction is not restricted to the beta-dystrobrevin isoform. The site for Kif5A binding to beta-dystrobrevin was localized in a carboxyl-terminal region that seems to be important in heavy chain-mediated kinesin interactions and is highly homologous in all three Kif5 isoforms, Kif5A, Kif5B and Kif5C. Pull-down and immunofluorescence experiments also showed a direct interaction between beta-dystrobrevin and Kif5B. Our findings suggest a novel function for dystrobrevin as a motor protein receptor that might play a major role in the transport of components of the dystrophin-associated protein complex to specific sites in the cell.
