The RON receptor tyrosine kinase is a member of the MET proto-oncogene family that has been implicated in regulating motile-invasive phenotypes in certain types of epithelial cancers. The purpose of this study was to determine if RON expression is altered in primary human colorectal adenocarcinomas. Results from immunohistochemical staining showed that RON is highly expressed in the majority of colorectal adenocarcinomas (29/49 cases). Accumulated RON is also constitutively active with autophosphorylation in tyrosine residues. Moreover, three splicing variants of RON, namely ROND165, ROND160, and ROND155 were detected and cloned from two primary colon cancer samples. These RON variants were generated by deletions in different regions in extracellular domains of the RON b chain. Functional studies showed that expression of ROND160 or ROND155 in MartinDarby canine kidney cells resulted in increased cell dissociation (scatter-like activity). RON variants, ROND160 and ROND155, also exerted the ability to induce multiple focus formation and sustain anchorageindependent growth of transfected NIH3T3 cells. Moreover, NIH3T3 cells expressing ROND160 or ROND155 formed tumors in athymic nude mice and colonized in the lungs. These data suggest that RON expression is altered in certain primary colon cancers. Abnormal accumulation of RON variants may play a role in the progression of certain colorectal cancers in vivo.
Since the discovery of MSP (macrophage-stimulating protein; also known as MST1 and hepatocyte growth factor-like (HGFL)) as the ligand for the receptor tyrosine kinase RON (also known as MST1R) in the early 1990s, the roles of this signalling axis in cancer pathogenesis has been extensively studied in various model systems. Both in vitro and in vivo evidence has revealed that MSP-RON signalling is important for the invasive growth of different types of cancers. Currently, small-molecule inhibitors and antibodies blocking RON signalling are under investigation. Substantial responses have been achieved in human tumour xenograft models, laying the foundation for clinical validation. In this Review, we discuss recent advances that demonstrate the importance of MSP-RON signalling in cancer and its potential as a therapeutic target.
Purpose: The receptor tyrosine kinase RON is critical in epithelial tumorigenesis and a drug target for cancer therapy. Here, we report the development and therapeutic efficacy of a novel anti-RON antibody Zt/g4-maytansinoid (DM1) conjugates for targeted colorectal cancer (CRC) therapy.Experimental Design: Zt/g4 (IgG1a/k) was conjugated to DM1 via thioether linkage to form Zt/g4-DM1 with a drug-antibody ratio of 4:1. CRC cell lines expressing different levels of RON were tested in vitro to determine Zt/g4-DM1-induced RON endocytosis, cell-cycle arrest, and cytotoxicity. Efficacy of Zt/g4-DM1 in vivo was evaluated in mouse xenograft CRC tumor model.Results: Zt/g4-DM1 rapidly induced RON endocytosis, arrested cell cycle at G 2 -M phase, reduced cell viability, and caused massive cell death within 72 hours. In mouse xenograft CRC models, Zt/g4-DM1 at a single dose of 20 mg/kg body weight effectively delayed CRC cell-mediated tumor growth up to 20 days. In a multiple dose-ranging study with a five injection regimen, Zt/g4-DM1 inhibited more than 90% tumor growth at doses of 7, 10, and 15 mg/kg body weight. The minimal dose achieving 50% of tumor inhibition was approximately 5.0 mg/kg. The prepared Zt/g4-DM1 is stable at 37 C for up to 30 days. At 60 mg/kg, Zt/g4-DM1 had a moderate toxicity in vivo with an average of 12% reduction in mouse body weight. Conclusion: Zt/g4-DM1 is highly effective in targeted inhibition of CRC cell-derived tumor growth in mouse xenograft models. This work provides the basis for development of humanized Zt/g4-DM1 for RON-targeted CRC therapy in the future. Clin Cancer Res; 20(23); 6045-58. Ó2014 AACR.
BackgroundAberrant expression of the RON receptor tyrosine kinase, a member of the MET proto-oncogene family, in breast cancer and non-small cell lung cancer (NSCLC) has therapeutic implication. Here we evaluated the efficacy of a novel anti-RON antibody-drug maytansinoid conjugate Zt/g4-DM1 for treatment of breast and NSCLC xenograft tumors in mouse models and explored a treatment strategy by combination of Zt/g4-DM1 with chemotherapeutics to achieve the maximal therapeutic activity.MethodsMouse monoclonal antibody Zt/g4 (IgG1a/κ) specific to human RON was conjugated to DM1 via thioether linkage to form Zt/g4-DM1 with a drug-antibody ratio of 4:1. Several breast cancer and NSCLC cell lines, expressing different levels of RON, were used as the model. Immunofluorescence was used to determine Zt/g4-induced RON internalization. Flow cytometric analysis and cell viability assay were used to determine the effect of Zt-g4-DM1 on cell cycle and death. Mouse xenograft NSCLC models were used in vivo to determine the therapeutic efficacy of Zt/g4-DM1 alone or in combination with chemotherapeutics.ResultsIn vitro, Zt/g4 treatment of breast cancer and NSCLC cells rapidly induced cell surface RON internalization, which results in intracellular delivery of DM1 sufficient to arrest cell cycle at G2/M phase, reduce cell viability, and cause massive cell death. In mouse tumor xenograft models, Zt/g4-DM1 at 20 mg/kg in a Q12 × 2 regimen effectively blocked breast cancer and NSCLC cell- mediated tumor growth. More than 95 % inhibition of tumor growth among three tumor xenograft models tested was achieved according to the measured tumor volume. The minimal dose to balance the tumor growth and inhibition (tumoristatic concentration) was established at 2.02 mg/kg for H2228, 1.94 mg/kg for H358 cell, and 6.25 mg/kg for T-47D cell-mediated xenograft tumors.ConclusionZt/g4 is highly effective in RON-directed drug delivery for targeted inhibition of NSCLC cell-derived tumor growth in mouse xenograft models. This work provides the basis for clinical development of humanized Zt/g4-DM1 for potential cancer therapy in the future.
Cancer stem cells (CSCs) contribute to pancreatic cancer tumorigenesis through tumor initiation, drug resistance, and metastasis. Currently, therapeutics targeting pancreatic CSCs are under intensive investigation. This study tested a novel strategy that utilizes the RON receptor as a drug delivery moiety for increased therapeutic activity against pancreatic CSCs. CD24(+)CD44(+)ESA(+) triple-positive pancreatic CSCs (CSCs(+24/44/ESA)) were obtained from spheroids of pancreatic L3.6pl cancer cells by sequential magnetic cell sorting methods. These cells displayed a spherical growth pattern, expressed the unique self-renewal marker Bmi-1, redifferentiated into an epithelial phenotype, acquired an epithelial to mesenchymal phenotype, and caused tumor formation in animal models. Among several receptor tyrosine kinases examined, RON was highly expressed and sustained by CSCs(+24/44/ESA). This feature provided the cellular basis for validating the therapeutic effectiveness of anti-RON antibody Zt/c9-directing doxorubicin-immunoliposomes (Zt/c9-Dox-IL). Zt/c9-Dox-IL specifically interacted with CSCs(+24/44/ESA) and rapidly caused RON internalization, which led to the uptake of liposome-coated Dox. Moreover, Zt/c9-Dox-IL was effective in reducing viability of L3.6pl cells and CSCs(+24/44/ESA). The IC(50) values between free Dox (62.0 ± 3.1 μM) and Zt/c9-Dox-IL (95.0 ± 6.1 μM) treated CSCs(+24/44/ESA) were at relatively comparable levels. In addition, Zt/c9-Dox-IL in combination with small molecule inhibitors lapatinib, sunitinib, or dasatinib further reduced the viability of CSCs(+24/44/ESA). In conclusion, RON expression by CSCs(+24/44/ESA) is a suitable molecule for the targeted delivery of chemoagents. The anti-RON antibody-directed delivery of chemotherapeutics is effective in reducing viability of pancreatic CSCs.
Recepteur d'origine nantais (RON) belongs to a subfamily of receptor tyrosine kinases (RTK) with unique expression patterns and biological activities. RON is activated by a serum-derived growth factor macrophage stimulating protein (MSP). The RON gene transcription is essential for embryonic development and critical in regulating certain physiological processes. Recent studies have indicated that altered RON expression contributes significantly to cancer progression and malignancy. In primary tumors, such as colon and breast cancers, overexpression of RON exists in large numbers and is often accompanied by the generation of different splicing variants. These RON variants direct a unique program that controls cell transformation, growth, migration, and invasion, indicating that altered RON expression has the ability to regulate motile/invasive phenotypes. These activities were also seen in transgenic mice, in which targeted expression of RON in lung epithelial cells resulted in numerous tumors with pathological features of human bronchioloalveolar carcinoma. Thus, abnormal RON activation is a pathogenic factor that transduces oncogenic signals leading to uncontrolled cell growth and subsequent malignant transformation. Considering these facts, RON and its variants can be considered as potential targets for therapeutic intervention. Experiments using small interfering RNA and neutralizing monoclonal antibodies demonstrated that suppressing RON expression and activation decreases cancer cell proliferation, increases apoptotic death, prevents tumor formation in nude mice, and reduces malignant phenotypes. Thus, blocking RON expression and activation has clinical significance in reversing malignant phenotypes and controlling tumor growth.
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