ABSTRACT
The tetraspanin and tumor suppressor KAI1 is downregulated or lost in many cancers which correlates with poor prognosis. KAI1 acts via physical/functional crosstalk with other membrane receptors. Also, a splice variant of KAI1 (KAI1-SP) has been identified indicative of poor prognosis. We here characterized differential effects of the two KAI1 variants on tumor biological events involving integrin (αvß3) and/or epidermal growth factor receptor (EGF-R). In MDA-MB-231 and -435 breast cancer cells, differential effects were documented on the expression levels of the tumor biologically relevant integrin αvß3 which colocalized with KAI1-WT but not with KAI1-SP. Cellular motility was assessed by video image processing, including motion detection and vector analysis for the quantification and visualization of cell motion parameters. In MDA-MB-231 cells, KAI1-SP provoked a quicker wound gap closure and higher closure rates than KAI1-WT, also reflected by different velocities and average motion amplitudes of singular cells. KAI1-SP induced highest cell motion adjacent to the wound gap borders, whereas in MDA-MB-435 cells a comparable induction of both KAI1 variants was noticed. Moreover, while KAI1-WT reduced cell growth, KAI1-SP significantly increased it going along with a pronounced EGF-R upregulation. KAI1-SP-induced cell migration and proliferation was accompanied by the activation of the focal adhesion and Src kinase. Our findings suggest that splicing of KAI1 does not only abrogate its tumor suppressive functions, but even more, promotes tumor biological effects in favor of cancer progression and metastasis.
ABSTRACT
Heterodimeric integrin receptors are mediators of cell adhesion, motility, invasion, proliferation, and survival. By this, they are crucially involved in (tumor) cell biological behavior. Integrins trigger signals bidirectionally across cell membranes: by outside-in, following binding of protein ligands of the extracellular matrix, and by inside-out, where proteins are recruited to ß-integrin cytoplasmic tails resulting in conformational changes leading to increased integrin binding affinity and integrin activation. Computational modeling and experimental/mutational approaches imply that associations of integrin transmembrane domains stabilize the low-affinity integrin state. Moreover, a cytoplasmic interchain salt bridge is discussed to contribute to a tight clasp of the α/ß-membrane-proximal regions; however, its existence and physiological relevance for integrin activation are still a controversial issue. In order to further elucidate the functional role of salt bridge formation, we designed mutants of the tumor biologically relevant integrin αvß3 by mutually exchanging the salt bridge forming amino acid residues on each chain (αvR995D and ß3D723R). Following transfection of human ovarian cancer cells with different combinations of wild type and mutated integrin chains, we showed that loss of salt bridge formation strengthened αvß3-mediated adhesion to vitronectin, provoked recruitment of cytoskeletal proteins, such as talin, and induced integrin signaling, ultimately resulting in enhanced cell migration, proliferation, and activation of integrin-related signaling molecules. These data support the notion of a functional relevance of integrin cytoplasmic salt bridge disruption during integrin activation.
