Blocking tumorigenic activities of colorectal cancer cells by a splicing RON receptor variant defective in the tyrosine kinase domain

Abstract

Altered expression of the RON receptor tyrosine kinase, accompanied by generation of splicing variants, contributes to the pathogenesis of epithelial cancers such as invasive growth of colorectal caners. In this study, we have studied a novel RON variant (designated as RONdelta170) that regulates tumorigenic activities of colorectal cancer cells by blocking RON-mediated tumorigenic signals. RONdelta170 is a splicing variant with a deletion of exon 19 that encodes 46 amino acids in the catalytic kinase domain. This deletion also causes a reading-frame shift and creates a new stop codon, which effectively eliminates the multi-functional docking site and truncates the RON C-terminus. As a RON variant without kinase activities and the C-terminal docking domain, RONdelta170 acts as a variant receptor that negatively regulates biochemical and biological activities mediated by RON or its oncogenic variant RONdelta160. In NIH3T3 expressing RONdelta160, RONdelta170 formed a complex with RONdelta160 and prevented RONdelta160-mediated activation of signaling proteins such as Erk1/2 and AKT. These effects resulted in decreased cell proliferation, reduced colony formation, and diminished cell migration. These negative activities were also observed in colorectal cancer cells naturally expressing RON or RONdelta160 including HT-29, HCT116 and SW620. Introduction of RONdelta170 into HCT116 cells blocked MSP-induced Erkl/2 and AKT phosphorylation, reduced cytoplasmic beta-catenin accumulation, restored glycogen synthase kinase-beta activity, and attenuated various tumorigenic activities. Moreover, RONdelta170 expression significantly reduced SW620 cell-mediated tumor growth in vivo. Thus, RONdelta170 is a naturally occurring variant with dominant negative activities and has potential for inhibiting RON-mediated tumorigenic activities in colorectal cancer cells.

Making alternative splicing decisions during epithelial-to-mesenchymal transition (EMT)

“…With the exception of RonD170, over-expression of any of these isoforms increases the motile and invasive phenotypes of tumor cells, while only RonD160 and RonD155 have the ability to transform rodent fibroblasts in vitro and cause tumor growth in athymic nude mice [45]. Interestingly, RonD170 acts as dominant negative and has a great therapeutic potential being capable of inhibiting tumorigenesis of epithelial cancers with altered Ron expression [46].…”

Pro-metastatic splicing of Ron proto-oncogene mRNA can be reversed: Therapeutic potential of bifunctional oligonucleotides and indole derivatives

“…27 Recently, the dominant negative kinase-defective RonΔ170 variant has been shown to have a pharmacological potential and to inhibit Ron-mediated tumorigenic activities. 30 Moreover, a monoclonal antibody against Ron was used to inhibit tumor growth in vivo. 31 However, where Ron is activated in a ligand-independent manner these therapeutic interventions are unlikely to be effective.…”

Inhibition of MSP-RON signaling pathway in cancer cells by a novel soluble form of RON comprising the entire sema sequence

“…These methods were performed as previously described (22). Individual samples (350 μg protein/sample) were immunoprecipitated by respective antibodies (2 μg/sample) coupled to protein G Sepharose beads.…”