Agonistic monoclonal antibodies potentiate tumorigenic and invasive activities of splicing variant of the RON receptor tyrosine kinase

Abstract

Ligand-dependent or independent activation of the RON receptor tyrosine kinase is essential in transducing invasive signals leading to increased tumorigenic activities. In this study, we characterized two monoclonal antibodies (mAbs) specific to the extracellular domains of human RON and studied their agonistic effect on tumorigenic activities mediated by oncogenic variant RON∆160. The mAb Zt/g4 and Zt/c1 are specific to human RON. They bind to RON with high affinities and recognized different epitopes on the RON extracellular domain. Because of their reactivity with native RON, Zt/g4 and Zt/c1 are useful in various applications such as immunoprecipitation, immunofluorescent analysis, and immunohistochemical staining. Functional studies revealed that Zt/g4 and Zt/c1 are capable of inducing RON phosphorylation which activates signaling proteins such as Erk1/2 and Akt. In NIH3T3 cells expressing RON∆160, both mAbs significantly enhanced RON∆160-mediated tumorigenic activities including cell proliferation, focus formation, and anchorage-independent growth. Cell shape changes with increased motile and invasive activities were also observed. Studies in vivo further demonstrated that Zt/g4 and Zt/c1 increase RON∆160-mediated tumor growth in nude mice with a shortened time of onset and enlarged tumor volume. Thus, by recognizing specific epitopes on the RON extracellular domains, Zt/g4 and Zt/c1 have abilities to elicit a full array of RON-mediated responses. These mAbs will be useful in studying mechanisms underlying RON activation which lead to increased tumorigenic activities.

Synergistic Activities of MET/RON Inhibitor BMS-777607 and mTOR Inhibitor AZD8055 to Polyploid Cells Derived from Pancreatic Cancer and Cancer Stem Cells

“…Proteins were separated in an 8% of SDS-PAGE under reduced conditions. Phosphorylated RON and other signaling proteins were detected by Western blotting using PY-100, R5029, and other antibodies and visualized using enhanced chemiluminescent reagents (21). Membranes also were reprobed with rabbit IgG antibody to b-actin to ensure equal sample loading.…”

“…These methods were performed as previously described (21). For inducting RON phosphorylation, cells (3 Â 10 6 cells) were stimulated at 37 C with 1 nmol/L MSP for 15 minutes.…”

“…L3.6pl-derived CD24 þ CD44 þ ESA þ triple-positive cancer stem cells [designated as cancer stem cells (CSC) þ24/44/ESA ] were generated by us from L3.6pl spheroids by sequential magnetic cell sorting methods as previously described (13). Human macrophage-stimulating protein (MSP), mouse mAb Zt/g4, and rabbit polyclonal IgG antibody R5029 to RON were used as previously described (20,21). Mouse or rabbit IgG antibodies specific to phospho-tyrosine (clone PY-100), p85/70S6K, phospho-p85/70S6K (Thr 421 /Ser 424 ), AKT, and phospho-AKT (Ser 473 ) were from Cell Signaling Technology.…”

Small-Molecule Inhibitor BMS-777607 Induces Breast Cancer Cell Polyploidy with Increased Resistance to Cytotoxic Chemotherapy Agents

“…Mouse mAb Zt/g4 and rabbit polyclonal IgG antibody R5029 specific to RON were used as previously described (5,15,23,24). Mouse or rabbit IgG antibodies specific to MET, a-tubulin, g-tubulin, and other signaling proteins were from Cell Signaling.…”

“…These methods were conducted as previously described (23,24). For immunoprecipitation, cellular proteins (250 mg per sample) were mixed with 1.5 mg/mL anti-RON mAb Zt/g4 coupled to protein G Sepharose beads.…”