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Advanced colorectal adenocarcinoma (CRAC), featured by distinctive histopathological appearance, distant organ metastasis, acquired chemoresistance, and tumorigenic stemness is a group of heterogeneous cancers with unique genetic signatures and malignant phenotypes. Treatment of CRAC is a daunting task for oncologists. Currently, various strategies including molecular targeting using therapeutic monoclonal antibodies, small molecule kinase inhibitors and immunoregulatory checkpoint therapy have been applied to combat this deadly disease. However, these therapeutic modalities and approaches achieve only limited success. Thus, there is a pharmaceutical need to discover new targets and develop novel therapeutics for CRAC therapy. MET and RON receptor tyrosine kinases have been implicated in CRAC pathogenesis. Clinical studies have revealed that aberrant MET and/or RON expression and signaling are critical in regulating CRAC progression and malignant phenotypes. Increased MET and/or RON expression also has prognostic value for CRAC progression and patient survival. These features provide the rationale to target MET and RON for clinical CRAC intervention. At present, the use of small molecule kinase inhibitors targeting MET for CRAC treatment has achieved significant progress with several approvals for clinical application. Nevertheless, antibody-based biotherapeutics, although under clinical trials for more than 8 years, have made very little progress. In this review, we discuss the importance of MET and/or RON in CRAC tumorigenesis and development of anti-MET, anti-RON, and MET and RON-dual targeting antibody-drug conjugates for clinical application. The findings from both preclinical studies and clinical trials highlight the potential of this novel type of biotherapeutics for CRAC therapy in the future.

Treatment of triple-negative breast cancer (TNBC) is a challenge to oncologists. Currently, the lack of effective therapy has fostered a major effort to discover new targets and therapeutics to combat this disease. The recepteur d’origine nantais (RON) receptor has been implicated in the pathogenesis of TNBC. Clinical studies have revealed that aberrant RON expression is crucial in regulating TNBC malignant phenotypes. Increased RON expression also has prognostic value for breast cancer progress. These features provide the rationale to target RON for TNBC treatment. In this review, we discuss the importance of RON in TNBC tumorigenesis and the development of anti-RON antibody–drug conjugates (ADCs) for clinical application. The findings from preclinical studies lay the foundation for clinical trials of this novel biotherapeutic for TNBC therapy.

Triple-negative breast cancer (TNBC) is a highly diverse group of malignant neoplasia with poor outcome. Currently, the lack of effective therapy has fostered a major effort to discover new targets to treat this malignant cancer. Here we identified the RON receptor tyrosine kinase as a therapeutic target for potential TNBC treatment. We analyzed RON expression in 168 primary TNBC samples via tissue microarray using anti-RON IHC staining and demonstrated that RON was widely expressed in 76.8% TNBC samples with overexpression in 76 cases (45.2%). These results provide the molecular basis to target RON for TNBC therapy. To this end, anti-RON monoclonal antibody Zt/g4-drug monomethyl auristatin E conjugate (Zt/g4-MMAE) was developed with a drug to antibody ratio of 3.29 and tested in a panel of TNBC cell lines with different phenotypes. In vitro, Zt/g4-MMAE rapidly induced RON internalization, resulted in cell-cycle arrest followed by massive cell death. The calculated IC50 values ranged from 0.06 to 3.46 μg/mL dependent on individual TNBC cell lines tested. Zt/g4-MMAE also effectively killed TNBC stem-like cells with RON+/CD44+/CD24− phenotypes and RON-negative TNBC cells through the bystander effect. In vivo, Zt/g4-MMAE at 10 mg/kg in a Q12 × 2 regimen completely eradicated TNBC xenografts without the regrowth of xenograft tumors. In conclusion, increased RON expression is a pathogenic feature in primary TNBC samples. Zt/g4-MMAE is highly effective in eradicating TNBC xenografts in preclinical models. These findings lay the foundation for using anti-RON Zt/g4-MMAE in clinical trials as a novel strategy for TNBC treatment.

Aberrant expression of the RON receptor tyrosine kinase, a cell surface protein, is a pathogenic feature in pancreatic cancer, which renders it a drug target for targeted therapy. Nevertheless, development of therapeutics targeting RON for pancreatic cancer therapy is hampered due to the lack of full addiction by pancreatic cancer cells to RON signaling for growth and survival. Here we describe a novel strategy using anti-RON antibody-directed drug delivery in the form of an antibody-drug conjugate for inhibition and/or eradication of pancreatic cancers. Monoclonal antibody Zt/g4 specific to the RON Sema domain was selected as the drug carrier based on its ability to induce robust RON internalization. Conjugation of Zt/g4 with monomethyl auristatin E, designated as Zt/g4-MMAE, was achieved through a protease-sensitive dipeptide linker to reach a drug to antibody ratio of 3.29:1. Zt/g4-MMAE was stable in human plasma with a dissociation rate less than 4% within a 10 day period. In vitro, Zt/g4-MMAE rapidly induced RON internalization, resulting in cell cycle arrest followed by massive cell death. The maximal effect was seen in pancreatic cancer cells with more than 10 000 receptor molecules per cell. Zt/g4-MMAE also synergized in vitro with chemotherapeutics including gemcitabine, 5-fluorouracil, and oxaliplatin to further reduce PDAC cell viability. In vivo, Zt/g4-MMAE exerts a long-lasting activity, which not only inhibited but also eradicated pancreatic xenograft tumors. These finding indicate that Zt/g4-directed drug delivery is highly effective for eradicating pancreatic tumors. Thus, Zt/g4-MMAE is a novel biotherapeutic with potential for therapy of RON-expressing pancreatic malignancies.

Purpose Triple-negative breast cancer (TNBC) is highly malignant and has poor prognosis and a high mortality rate. The lack of effective therapy has spurred our investigation of new targets for treating this malignant cancer. Here, we identified RON (macrophage-stimulating 1 receptor) and MET (MET proto-oncogene, receptor tyrosine kinase) as a prognostic biomarker and therapeutic targets for potential TNBC treatment.Materials and MethodsWe analyzed RON and MET expression in 187 primary TNBC clinical samples with immunohistochemistry. We validated the targeted therapeutic effects of RON and MET in TNBC using three tyrosine kinase inhibitors (TKIs): BMS-777607, INCB28060, and tivantinib. The preclinical therapeutic efficacy of the TKIs was mainly estimated using a TNBC xenograft model.ResultsPatients with TNBC had widespread, abnormal expression of RON and MET. There was RON overexpression, MET overexpression, and RON and MET co-overexpression in 63 (33.7%), 63 (33.7%), and 43 cases (23.0%), respectively, which had poor prognosis and short survival. In vivo, the TKI targeting RON ant MET inhibited the activation of the downstream signaling molecules, inhibited TNBC cell migration and proliferation, and increased TNBC cell apoptosis; in the xenograft model, they significantly inhibited tumor growth and shrank tumor volumes. The TKI targeting RON and Met, such as BMS-777607 and tivantinib, yielded stronger anti-tumor effects than INCB28060.ConclusionRON and MET co-overexpression can be significant pathological characteristics in TNBC for poor prognosis. TKIs targeting RON and MET have stronger drug development potential for treating TNBC.

Background Antibody-drug conjugates (ADCs) targeting the RON receptor, a tumorigenic factor contributing to cancer malignancy, has been considered as a novel strategy for cancer therapy. Here we describe a humanized antibody recognizing the RON plexin-semaphorin-integrin (PSI) domain with increased drug delivery capability for potential clinical application. Method Monoclonal antibody PCM5B14 specific to the human and monkey RON PSI domain was generated and characterized by various immunological methods. Humanized antibody H5B14 was created by grafting PCM5B14 complementarity-determining regions into human IgG1/κ acceptor frameworks and conjugated with monomethyl auristatin E and duocarmycin to form two H5B14-based ADCs. Stability of H5B14-based ADCs in human plasma was measured using hydrophobic interaction chromatography. Various biochemical and biological assays were used to determine ADC- regulated RON internalization, cell viability, spheroid formation, and death of cancer stem-like cells. Efficacies of H5B14-based ADCs in vivo were validated using tumor xenograft models. Maximal tolerated doses of H5B14-based ADCs were established in mice. Results H5B14 was highly specific to the human RON PSI domain and superior over other anti-RON ADCs in induction of RON internalization in various cancer cell lines tested. H5B14-based ADCS had a drug to antibody ratio of ~ 3.70:1 and were stable in human plasma with a minimal dissociation within a 10-day period. Functionally, H5B14-mediated drug delivery decreased cell viability at early stages with an average IC50 at ~ 20 nM in multiple cancer cell lines examined. H5B14-based ADCs also inhibited spheroid formation and caused death of cancer stem-like cells with RON+/CD44+/ESA+ phenotypes. In vivo, H5B14-based ADCs in a single injection inhibited tumor xenograft growth mediated by multiple cancer cell lines. Tumoristatic concentrations calculated from xenograft tumor models were in the range of 0.63 to 2.0 mg/kg bodyweight. Significantly, H5B14-based ADCs were capable of eradicating tumors at variable levels across multiple xenograft models regardless their malignant statuses. Toxicologically, H5B14-based ADCs were well tolerated in mice up to 60 mg/kg. Conclusion H5B14-based ADCs targeting the RON PSI domain are superior in inducing RON internalization, leading to robust drug delivery and overall inhibition and eradication of tumors in multiple xenograft models. These findings warrant H5B14-based ADCs for clinical trials in the future.

Aberrant expression of the RON receptor tyrosine kinase is a pathogenic feature and a validated drug target in various types of cancers. Currently, therapeutic antibodies targeting RON for cancer therapy are under intensive evaluation. Here we report the development and validation of a novel humanized anti-RON antibody-drug conjugate for cancer therapy.Antibody humanization was achieved by grafting sequences of complementarity-determining regions from mouse monoclonal antibody Zt/g4 into human IgG1/κ acceptor frameworks. The selected humanized Zt/g4 subclone H1L3 was conjugated with monomethyl auristatin E using a dipeptide linker to form H-Zt/g4-MMAE. Pharmacokinetic analysis of H-Zt/g4-MMAE was determined using hydrophobic interaction chromatography and a MMAE ADC ELISA kit. Biochemical and biological assays were used for measuring RON expression, internalization, cell viability and death. Therapeutic efficacies of H-Zt/g4-MMAE were validated in vivo using three pancreatic cancer xenograft models. Toxicological activities of H-Zt/g4-MMAE were determined in mouse and cynomolgus monkey.H-Zt/g4-MMAE had a drug to antibody ratio of 3.77:1 and was highly stable in human plasma with a dissociation rate less than 5% within a 20 day period. H-Zt/g4-MMAE displayed a favorable pharmacokinetic profile in both mouse and cynomolgus monkey. In vitro, H-Zt/g4-MMAE induced RON internalization, which results in killing of pancreatic cancer cells with ICH-Zt/g4-MMAE is superior in eradication of pancreatic cancer xenografts with favorable pharmacokinetic profiles and manageable toxicological activities. These findings warrant the transition of H-Zt/g4-MMAE into clinical trials in the future.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with poor prognosis and high mortality. Molecular aberrations associated with PDAC pathogenesis and progression have been extensively investigated. Nevertheless, these findings have not been translated into clinical practice. Lack of therapeutics for PDAC treatment is another challenge. Recent application of molecularly targeted and immunoregulatory therapies appears to be disappointing. Thus, discovery of new targets and therapeutics is urgently needed to combat this malignant disease. The RON receptor tyrosine kinase is a tumorigenic determinant in PDAC malignancy, which provides the rationale to target RON for PDAC treatment. In this review, we summarize the latest evidence of RON in PDAC pathogenesis and the development of anti-RON antibody-drug conjugates for potential PDAC therapy. The finding that anti-RON antibody-drug conjugates show efficacy in preclinical animal models highlights the potential of this novel class of anti-cancer biotherapeutics in future clinical trials.

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/κ) 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 G2–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. ©2014 AACR.

Aberrant expression of the RON receptor tyrosine kinase contributes to breast cancer malignancy. Although clinical trials of RON targeting are underway, the intriguing issue is the diversity of RON expression as evident by cancer cells expressing different variants including oncogenic RON160. The current study determines aberrant RON160 expression in breast cancer and its potential as a target for breast cancer therapy. Using mouse monoclonal antibody Zt/h12 in immunohistochemical staining of breast cancer tissue microarray, we observed that RON160 was expressed in high frequency in primary invasive ductal (77.2%, 61/79 cases), lobular (42.5%, 34/80 cases), and lymph node-involved (63.9%, 26/36 cases) breast cancer samples. Moreover, RON160 overexpression was predominantly observed in invasive ductal (26.6%, 21/79 cases) and lymph node-involved (33.3%, 12/36) cases. Among a panel of breast cancer cell lines analyzed, Du4475 cells naturally expressed RON160. Silencing RON160 expression by siRNA reduced Du4475 cell viability. Inhibition of RON160 signaling by tyrosine kinase inhibitor PHA665752 also suppressed Du4475 cell anchorage-independent growth and induced apoptotic cell death. Studies in vivo revealed that PHA665752 inhibited 3T3- RON160 and Du4475 cell-mediated tumor growth in mouse mammary fat pad. A 60% reduction in tumor volume compared to controls was achieved after a 13-day treatment. We conclude from these studies that RON160 is highly expressed in breast cancer and its signaling is integrated into cellular signaling network for tumor cell growth and survival. Experimental treatment by PHA665752 in Du4475 breast cancer xenograft model highlights the significance of RON160 as a drug target in molecular-targeted breast cancer therapy.

Products of proto-oncogenes c-MET and RON belong to a subfamily of receptor tyrosine kinases that contribute significantly to tumorigenic progression. In primary tumors, altered c-MET/RON expression transduces signals regulating invasive growth that is characterized by cell migration and matrix invasion. These pathogenic features provide the basis for targeting c-MET/RON in cancer therapy. In the last decade, various approaches have been investigated to suppress c-MET/RON-transduced oncogenesis. Among the therapeutics developed, monoclonal antibodies (mAbs) and small-molecule inhibitors (SMIs) have emerged as promising candidates. The mechanism of these therapeutic candidates is the disruption of tumor dependency on c-MET/RON signals for survival. The mAbs specific to hepatocyte growth factor (AMG102) and c-MET (MetMAb) are both humanized and able to block c-MET signaling, leading to inhibition of tumor cell proliferation in vitro and inhibition of tumor growth in xenograft models. The mAb AMG102 neutralizes hepatocyte growth factor and enhances the cytotoxicity of various chemotherapeutics to tumors in vivo. AMG102 is currently in phase II clinical trials for patients with advanced solid tumors. IMC-41A40 and Zt/f2 are RON-specific mAbs that down-regulate RON expression and inhibit ligand-induced phosphorylation. Both mAbs inhibit tumor growth in mice mediated by colon and pancreatic cancer cells. SMIs specific to c-MET (ARQ107 and PF-02341066) are in various phases of clinical trials. Therapeutic efficacy has also been observed with dual inhibitors such as Compound I, which is specific to c-MET/RON. However, a potential issue is the emergence of acquired resistance to these inhibitors. Clearly, development of c-MET/RON therapeutics provides opportunities and challenges for combating cancer in the future.

Overexpression of the RON receptor tyrosine kinase exists in various cancers and contributes to malignant progression. To validate RON as a targeting moiety for delivery of chemoagents for enhanced tumor cytotoxicity, immunoliposomes (IL) loaded with doxorubicin (Dox) were formulated followed by postinsertion of monoclonal antibodies Zt/g4, Zt/c1, or their Fab fragments specific to the RON extracellular domains. Flow cytometry analysis showed that Zt/g4 or Zt/c1-IL binds to cancer cells and causes RON internalization as evident in confocal analysis of intracellular fluorescence intensity. The antibody-directed IL uptake by cancer cells is in both dose and time-dependent manners. Studies of cytotoxicity of individual IL in vitro against colon or breast cancer cell lines revealed that Zt/g4 directed Dox-IL displayed increased cytotoxic activities with a significant reduction of IC(50) values. An average of 8-fold increases in cytotoxic efficiency was achieved among four cell lines tested. Moreover, Zt/g4 directed Dox-IL also displayed the effective killing of cancer cells that are insensitive to pegylated liposomal doxorubicin. The effect of Zt/c1-Dox-IL was not as strong as Zt/g4-Dox-IL, and only moderate activities were observed. IL coupled with the Fab fragments of Zt/g4 or Zt/c1 show moderate activities against cancer cells. The ineffectiveness seemed to be related to the weak activities of the Fab fragments in the induction of RON internalization, which resulted in reduced drug uptakes. We conclude that anti-RON antibody-directed drug delivery is effective for increased uptake of cytotoxic drugs. Antibody-based RON targeting could be developed into a potential therapeutic for treatment of malignant cancers.

Claudins are integral membrane proteins essential in tight junction formation and function. Altered expression of claudins has been implicated in epithelial malignant transformation. We report here that activation of recepteur d'origine nantais (RON) differentially regulates tight junction function and claudin expression. In Martin-Darby canine kidney (MDCK) cells, macrophage-stimulating protein-induced RON activation or expression of constitutively active variant RON160 significantly disrupted cellular tight junctions and reduced transepithelial electrical resistance. These changes were featured by diminished claudin-1 expression and redistribution of claudin-3 and -4 into cytoplasmic compartments. The inhibition of claudin-1 was also seen in breast cancer T-47D cells. By analyzing the signaling events, we found that activation of the extracellular signal-regulated kinase 1/2 pathway is required for RON-mediated inhibition of claudin-1 expression and redistribution of claudin-3 and -4. Results from luciferase reporter assays showed that inhibition is acted at the transcriptional levels because RON activation decreases claudin-1 promoter activities and increases transcriptional repressor Snail-1 expression. Functional analysis further revealed that reduced claudin-1 expression is linked to increased motilities of MDCK and T-47D cells as evident in cell migration and wound-healing assays. Forced expression of claudin-1 prevented RON-mediated cell migration and restored cell morphologies to their original epithelial appearance. In conclusion, RON activation differentially regulates claudin expression in epithelial cells. Inhibition of claudin-1 expression may represent a novel mechanism that contributes to RON-mediated invasive activities, leading to increased tumor malignancy.

The beta-catenin pathway plays a critical role in the pathogenesis of certain types of cancers. To gain insight into mechanisms by which altered receptor tyrosine kinases regulate cytoplasmic beta-catenin accumulation, the effect of an oncogenic receptor originated from Nantes (RON) variant on beta-catenin accumulation and the role of beta-catenin in RON-mediated tumorigenic activities were studied. In NIH3T3 cells harboring oncogenic variant RONDelta160, increased beta-catenin accumulation with tyrosine phosphorylation and nuclear translocation was observed. Overexpression of RONDelta160 also resulted in increased expression of beta-catenin target genes c-myc and cyclin D1. By analyzing cellular proteins that regulate beta-catenin stabilities, it was found that RONDelta160 activates the protein disheveled (DVL) and inactivates glycogen synthase kinase-3beta by Ser-9 residue phosphorylation. These effects were channeled by RONDelta160-activated PI 3-kinase-AKT pathways that are sensitive to specific inhibitors, such as wortmannin, but not to other chemical inhibitors. Silencing RONDelta160 expression by specific small interfering RNA blocked not only beta-catenin expression but also c-myc and cyclin D1 expression, suggesting that RON expression is required for the activation of the beta-catenin signaling pathway. Moreover, it was found that knockdown of the beta-catenin gene expression by small interfering RNA techniques reduces significantly the RONDelta160-mediated NIH3T3 cell proliferation, focus-forming activities and anchorage-independent growth. Thus, the oncogenic RON variant regulates beta-catenin stabilities through activation of DVL and inactivation of glycogen synthase kinase-3beta. The activated beta-catenin cascade is one of the pathways involved in tumorigenic activities mediated by the oncogenic RON variant.

The RON (recepteur d'origine nantais) receptor belongs to the MET proto-oncogene family that is implicated in the oncogenesis of the gastrointestinal epithelium. The present study aimed to determine the role of RON in regulating epithelial phenotypes in response to transforming growth factor (TGF)-beta1. Expression and activation of RON in SV40-immortalized mouse intestinal epithelial MODE-K cells result in reduction of cellular sensitivities towards apoptotic signals elicited by TGF-beta1. This effect is dependent on RON expression and phosphorylation that inhibit the TGF-beta1-induced activation of caspase-3 and truncation of BAD. Among cellular signaling components, the activation of MAP kinase is critical in the RON-mediated inhibitory effect. PD98059, a specific MAP kinase inhibitor, prevented RON-mediated anti-apoptotic activities. PD98059 also prevented the inhibitory effect of RON on TGF-beta1-induced cleavage of caspase-3 and BAD. By protecting cells from apoptotic death, activated RON collaborates with TGF-beta1 in the induction of cell morphological changes with decreased E-cadherin expression and increased migration and morphogenesis. Thus, RON expression and activation modulate phenotypes of gastrointestinal epithelial cells in response to TGF-beta1 with reduced sensitivity to apoptosis and increased migration. These activities might represent a mechanism by which RON activation increases tumorigenic activities and facilitates the progression of transformed epithelial cells towards malignancy.

Epithelial to mesenchymal transition (EMT) is a process occurring during embryonic development and cancer progression. Using recepteur d'origine nantais (RON)-expressing epithelial cells as a model, we showed that RON activation causes spindle-shaped morphology with increased cell motilities. These activities resemble those observed in EMT induced by transforming growth factor (TGF)-beta1 or by Ras-Raf signaling. By immunofluorescent and Western blot analyses, we found that constitutive RON expression results in diminished expression of E-cadherin, redistribution of beta-catenin, reorganization of actin cytoskeleton, and increased expression of vimentin, a mesenchymal filament. RON expression is also essential for TGF-beta1-induced expression of alpha-smooth muscle actin (alpha-SMA), a specialized mesenchymal marker. In the study of signaling pathways responsible for RON-mediated EMT, it was found that PD98059, a MAP kinase inhibitor, blocks the collaborative activities of RON and TGF-beta1 in induction of alpha-SMA expression and restores epithelial cells to their original morphology. Moreover, we showed that RON expression increases Smad2 gene promoter activities and protein expression, which significantly lowers TGF-beta1 threshold for EMT induction. These results suggest that persistent RON expression and activation cause the loss of epithelial phenotypes. These changes, collaborating with TGF-beta1 signaling, could play a critical role in epithelial transdifferentiation towards invasiveness and metastasis of certain cancers.

The RON receptor tyrosine kinase is activated by macrophage-stimulating protein, which regulates macrophage migration, phagocytosis, and nitric oxide production. We report here the inhibitory effect of RON on lipopolysaccharide (LPS)-induced cyclooxygenase (Cox)-2 expression in mouse macrophages. In RON-expressing macrophages treated with macrophage stimulating protein, LPS-induced prostaglandin E(2) (PGE(2)) production was significantly reduced. The inhibition was accompanied by reduction of Cox-2 protein and mRNA expression. Transcriptional studies indicated that RON activation inhibits LPS-induced luciferase activity driven by the Cox-2 gene promoter. To determine whether RON activation affects LPS-induced NF-kappa B pathway, which is important for Cox-2 expression. Western blot analyses were performed showing that RON activation inhibits LPS-induced I kappa B alpha degradation. The decreased I kappa B alpha degradation was due to reduced I kappa B alpha phosphorylation at Ser-32 as determined by I kappa B alpha (Ser-32) phosphor-antibody. Moreover, we found that LPS-induced IKK beta activity, an enzyme responsible for phosphorylation of I kappa B alpha, was inhibited upon RON activation. Interestingly, these inhibitory effects were not regulated by RON-mediated phosphatidylinositol-3 kinase. These results suggest that RON activation inhibits LPS-induced macrophage Cox-2 expression. The inhibitory effect is mediated by impairing LPS-activated cascade enzymes that activate NF-kappa B. The inhibition of Cox-2 expression might represent a novel mechanism for the inhibitory functions of RON in vivo against LPS-induced inflammation and septic shock.

RON is a receptor tyrosine kinase activated by macrophage-stimulating protein. We demonstrate here that RON activation inhibits LPS-induced apoptosis of mouse peritoneal macrophages and Raw264.7 cells expressing RON or a constitutively active RON mutant. The antiapoptotic effect of RON was accompanied with the inhibition of LPS-induced production of nitric oxide (NO), a molecule responsible for LPS-induced cell apoptosis. This conclusion is supported by experiments using a chemical NO donor GSNO, in which RON activation directly blocked GSNO-induced apoptotic death of Raw264.7 cells and inhibited LPS-induced p53 accumulation. Furthermore, we showed that treatment of cells with wortmannin, which inhibits phosphatidylinositol (PI)-3 kinase, prevents the inhibitory effect of RON on LPS-induced macrophage apoptosis. These results were confirmed further by expression of a dominant inhibitory PI-3 kinase p85 subunit. These data suggest that by activating PI-3 kinase and inhibiting p53 accumulation, RON protects macrophage from apoptosis induced by LPS and NO. The antiapoptotic effect of RON might represent a novel mechanism for the survival of activated macrophages during inflammation.

Targeting receptor tyrosine kinases by therapeutic monoclonal antibodies and antibody-drug conjugates has met with tremendous success in clinical oncology. Currently, numerous therapeutic monoclonal antibodies are under preclinical development. The potential for moving candidate antibodies into clinical trials relies heavily on therapeutic efficacy validated by human tumor xenografts in mice. Here we describe methods used to determine therapeutic efficacy of monoclonal antibodies or antibody-drug conjugates specific to human receptor tyrosine kinase using human tumor xenografts in mice as the model. The end point of the study is to determine whether treatment of tumor-bearing mice with a monoclonal antibody or antibody-drug conjugates results in significant delay of tumor growth.

Ron receptor activation induces numerous cellular responses in vitro, including proliferation, dissociation, and migration. Ron is thought to be involved in blood cell development in vivo, as well as in many aspects of the immune response including macrophage activation, antigen presentation, and nitric oxide regulation. In previous studies to determine the function of Ron in vivo, mice were generated with a targeted deletion of the extracellular and transmembrane regions of this gene. Mice homologous for this deletion appear to die early during embryonic development. To ascertain the in vivo function of Ron in more detail, we have generated mice with a germline ablation of the tyrosine kinase domain. Strikingly, our studies indicate that this domain of Ron, and therefore Ron cytoplasmic signaling, is not essential for embryonic development. While mice deficient in this domain are overtly normal, mice lacking Ron signaling have an altered ability to regulate nitric oxide levels and, in addition, have enhanced tissue damage following acute and cell-mediated inflammatory responses.

Previously, we identified macrophage-stimulating protein (MSP) as being expressed during hamster lung injury induced by nitrosamine carcinogens. Transient, generalized epithelial-cell hyperplasia during the preneoplastic period, and eventually nonneuroendocrine (non-NE) lung tumors, are known to develop in these nitrosamine-treated hamsters. We wished to test the hypothesis that MSP and its tyrosine kinase receptor, RON, might represent an autocrine/paracrine system involved in the pathogenesis of human nonneuroendocrine lung tumors, the non-small-cell carcinomas (NSCLCs). We found that this occurred in a paracrine fashion in three of eight primary human NSCLCs that expressed messenger RNA (mRNA) for MSP at high levels in histologically normal lung adjacent to the tumor, but not in the primary tumor, together with mRNA for RON in both normal and tumor tissue. MSP and RON could also constitute an autocrine/paracrine system in human NSCLC cell lines: five of 16 cell lines (squamous and adenosquamous) expressed both MSP and RON; and an additional five of 16 cell lines expressed RON without detectable MSP. Although three cases of primary squamous-cell carcinomas expressed MSP (two of three in the tumor and one of three in nonneoplastic lung), mRNA for RON was not detectable in these cases. RON was functional in all tested RON mRNA-positive cell lines, with exogenous MSP inducing RON-mediated tyrosine phosphorylation. Treatment of a RON-positive adenosquamous carcinoma cell line with MSP additionally resulted in increased motility in a cell-migration assay, suggesting that MSP might promote cell migration of some NSCLCs. In conclusion, MSP and RON might represent an autocrine/paracrine system involved in the pathogenesis of lung cancer, although the nature of the biologic responses in different cell types might vary considerably.

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.

The RON receptor tyrosine kinase is a therapeutic target for cancer treatment. Here, we report therapeutic effect and phenotypic change of breast cancer cells in response to BMS-777607, a RON tyrosine kinase inhibitor. Treatment of breast cancer cells with BMS-777607 at therapeutic doses inhibited cancerous clonogenic growth but had only minimal effect on cell apoptosis. Significantly, BMS-777607 induced extensive polyploidy with multiple sets of chromosomes in cancer cells. This effect is independent of RON expression. Knockdown of RON in T-47D and ZR-75-1 cells by specific siRNA did not prevent polyploid formation. Immunofluorescent analysis of α-tubulin and γ-tubulin expression in polyploid cells revealed that BMS-777607 disrupts bipolar spindle formation and causes multipolar-like microtubule assembly. Also, both metaphase equatorial alignment and chromosomal segregation were absent in polyploid cells. These results suggest that cellular mitosis arrests at prophase/pro-metaphase and fails to undergo cytokinesis. By analyzing kinase-inhibitory profiles, aurora kinase B was identified as the target molecule inhibited by BMS-777607. In BMS-777607–treated cells, aurora kinase B was inhibited followed by protein degradation. Moreover, BMS-777607 inhibited Ser10 phosphorylation of histone H3, a substrate of aurora kinase B. Chemosensitivity analysis indicated the resistance of polyploid cells toward chemotherapeutics. Treatment with doxorubicin, bleomycin, methotrexate, and paclitaxel significantly increased cellular IC50 values. These findings highlight the theory that BMS-777607 acts as a multikinase inhibitor at therapeutic doses and is capable of inducing polyploidy by inhibiting aurora kinase B. Increased resistance of polyploid cells to cytotoxic chemotherapeutics could have a negative impact on targeted cancer therapy using BMS-777607. Mol Cancer Ther; 12(5); 725–36. ©2013 AACR.

Macrophage stimulating protein (MSP) is a chemotactic factor for murine peritoneal macrophages. The receptor for human MSP was recently identified as the ron gene product, a transmembrane protein tyrosine kinase cloned from a human keratinocyte cDNA library. Here we report that MSP induced proliferation of murine primary keratinocytes and established keratinocyte cell lines in a concentration-dependent manner. The growth efficacy of MSP was comparable to that of epidermal growth factor and keratinocyte growth factor. In three of four cell lines tested in a chemotaxis chamber, MSP also stimulated migration of keratinocytes on a collagen type IV substratum. The action of MSP was mediated by specific binding of MSP to the STK gene product, a murine homologue of the RON MSP receptor. Binding of MSP to keratinocyte STK induced phosphorylation of the 150 kDa STK beta chain. Herbimycin A, a protein tyrosine kinase inhibitor, blocked MSP-mediated phosphorylation of the STK receptor as well as proliferation of keratinocytes, suggesting the importance of tyrosine kinase activity for transduction of the message delivered by MSP. Previously, the only known target cell for MSP was the resident peritoneal macrophage. These studies establish the keratinocyte as a new target cell for MSP. The action of MSP on keratinocytes may have implications for tissue repair, wound healing, and tumor growth.

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.

Overexpression of the RON receptor tyrosine kinase contributes to pathogenesis of epithelial cancers and disruption of RON signals has potential for therapeutic intervention. Here, we report the inhibitory effects of monoclonal antibodies (Zt/g4, Zt/f2 and Zt/c9) on RON expression and tumorigenic activities in colon cancer cells. Persistent treatment of colon SW620 or other cells with Zt/g4 dramatically down-regulated RON expression as evident by Western blot and cell surface fluorescent analyses. The effect was both concentration and time-dependent and specific to RON but not to structure-related MET or -unrelated EGFR. The cause of reduction was antibody-induced receptor internalization followed by protein degradation through lysosome and proteasome-mediated pathways. Down-regulation of RON impaired intracellular signaling events. Phosphorylation of Erk1/2 and AKT was dramatically reduced after Zt/g4 treatment. Zt/g4 treatment also affects activities of DVL and GSK-3beta, which results in diminished beta-catenin nuclear translocation. Functional studies revealed that Zt/g4 treatment changes cellular morphology and affects colony formation in soft agar. It also increased the sensitivity of SW620 cells in response to gemcitabine-induced cytotoxicity. In this case, the death of SW620 cells was significantly increased when Zt/g4 was used in combination with gemcitabine. We conclude that persistent treatment of cancer cells with antibodies specific to RON extracellular domains results in down-regulation of RON expression. The reduced RON expression is accompanied with impaired signaling events, diminished tumorigenic activities and enhanced sensitivity towards cytotoxic drugs. Thus, Zt/g4-directed targeting could have therapeutic implication for controlling tumorigenic phenotypes of cancer cells.

The RON receptor tyrosine kinase and its ligand macrophage stimulating protein (MSP) play a role in epithelial tumorigenesis. We report here a novel RON variant that antagonizes the RON-MSP pathway in various cancer cells. The variant is an 85 kDa soluble protein from an mRNA transcript with an insertion of 49 nucleotides between exons 5 and 6. The insertion created a stop codon leading to the formation of a RON variant consisting of the entire 35 kDa alpha-chain and a 45 kDa partial extracellular beta-chain. The protein was featured by a sema domain, a hinge motif and a portion of the first IPT unit (designated as RONDelta85). RONDelta85 binds directly to MSP, forms MSP-RONDelta85 complex, and inhibits RON phosphorylation. RONDelta85 disrupts RON or RONDelta160 dimerization, prevents their phosphorylation, and attenuates downstream signaling events. The action of RONDelta85 is specific to RON and has no effect on MET and EGFR. In colon and pancreatic cancer cells, RONDelta85 inhibits spontaneous or MSP-induced Erk1/2 and AKT phosphorylation, which results in impaired cell proliferation and colony formation. RONDelta85 also inhibits spontaneous and MSP-induced cell migration. We conclude that RONDelta85 is an antagonist to the MSP-RON pathway, which has potential for regulating RON/RON160-mediated tumorigenic activities.

Hypoxia contributes to acquired drug resistance in various cancer cells. The underlying mechanism is cellular insensitivity regulated by hypoxia-inducible factors (HIF), which impairs drug uptake, transport, and metabolism. The current study determines anti-RON antibody-directed cytotoxicity of doxorubicin (Dox)-immunoliposomes (IL) in hypoxic colon cancer cells.Cells were cultured under hypoxia (1% O(2), 5% CO(2), and 96% N(2)) for 24 h. Dox-loaded IL were formulated followed by post-insertion of monoclonal antibody Zt/g4 specific to RON. Western blotting was used to detect HIF-1α and RON expression. Cellular uptake of Zt/g4-conjugated IL was determined by confocal and internalization assays. Cell viability was assessed by the MTT assay.RON and HIF-1α expression were observed in hypoxic colon HCT116 and SW620 cells. Resistance to Dox-induced cytotoxicity was acquired in hypoxic cells with increased IC(50) values. However, acquired resistance was attenuated by Zt/g4-directed Dox-IL, which displays increased cytotoxic activities. IL binding and uptake revealed that hypoxic RON expression is functional, which mediates high levels of Zt/g4-Dox-IL binding and cytoplasmic internalization. Zt/g4-Dox-IL is effective in killing hypoxic HCT116 and SW620 cells with reduced IC(50) values compared to Dox and pegylated-liposomal Dox. These effects were dependent on hypoxic RON expression. HCC1937 cells with diminished RON expression under hypoxia were insensitive to Zt/g4-Dox-IL-induced cytotoxic effect.RON expressed by hypoxic colon cancer cells is thus a potential targeting molecule for delivery of chemotherapeutics. The ability of anti-RON mAb to direct Dox-IL cytotoxicity could be developed for attenuating hypoxia-acquired drug resistance in various cancer cells.

Cell migration followed by matrix invasion is a critical step required for epithelial cell differentiation, growth and survival. This study determined the RON receptor in regulation of motile-invasive phenotypes of epithelial cells. Two RON variants, RON165.e11p and RON165, with alterations in a defined extracellular domain were used as the model. RON165 is a splicing variant generated by an mRNA transcript with an in-frame deletion of 49 amino acids encoded by exon 11. In contrast, RON165.e11p was produced by a partial deletion of exon 11 with the elimination of the first 40 amino acids. Thus, RON165.e11p differs from RON165 with nine amino acids retained in the fourth immunoglobulin-plexin-transcription (IPT) domain. Biochemically, both RON165 and RON165.e11p exist as a single-chain protein, residing in the cytoplasm, and failed to mature into the two-chain receptor. Both RON165 and RON165.e11p spontaneously formed oligomers in vivo leading to constitutive phosphorylation and the activation of downstream signaling proteins. Although lacking cell-transforming activities, RON165 and RON165.e11p mediated the epithelial to mesenchymal transition with spindle-like cell morphologies, diminished E-cadherin expression, and increased N-cadherin and vimentin expression. These changes facilitated epithelial cell migration and invasion as modeled in Martin-Darby canine kidney (MDCK) cells. Moreover, expression of RON165 or RON165.e11p in breast epithelial MCF-7 cells diminished epithelial cell phenotypes and increased motile and invasive activities. Thus, alterations in the defined extracellular region result in two unique RON variants with similar biological properties. The ability of RON165 or RON165.e11p to promote motile-invasive phenotypes may represent a mechanism by which RON regulates epithelial cell phenotypes and biological activities.

The RON receptor tyrosine kinase regulates epithelial cell homeostasis and tumorigenesis by transducing multiple signals through its functional domains. The present study was to determine the significance of the entire C-terminus in RON or its variant RON160-mediated activities related to cell motility and tumorigenesis. Analysis of protein phosphorylation revealed that elimination of the entire C-terminus significantly impairs the ligand-dependent or independent RON or RON160 phosphorylation and dimerization. Phosphorylation of downstream signaling proteins such as Erk1/2, AKT, and p38 MAP kinase was also diminished in cells expressing the C-terminus-free RON or RON160. These dysfunctional activities were accompanied with the inability of truncated RON or RON160 to mediate cytoplasmic β-catenin accumulation. Functional analysis further demonstrated that truncation of the C-terminus significantly impairs RON or RON160-mediated cell proliferation, morphological changes, and cellular migration. Significantly, oncogenic RON160-mediated tumor growth in athymic nude mice was lost after the deletion of the C-terminus. Thus, the C-terminus is a critical component of the RON receptor. The entire C-terminus is required for RON or RON160-mediated intracellular signaling events leading to various cellular activities.

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.

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 RONDelta160. 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 RONDelta160, both mAbs significantly enhanced RONDelta160-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 RONDelta160-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.

Altered expression of receptor tyrosine kinases contributes to tumorigenic behaviors of epithelial cancers. In this study, the pathogenic roles of receptor tyrosine kinase RON (recepteur d'origine nantais) in regulating oncogenic phenotypes in colorectal epithelial cells were studied. Increased expression of RON and its variants resulted in colony formation and motile activities of colonic epithelial AA/C1 cells as evident in soft-agar and migration assays, respectively. These results suggest that overexpression of wild-type RON mediates the transformed phenotypes in immortalized colon epithelial cells. In colorectal cancer cells (HT-29, HCT116, and SW620) that naturally express RON, the RON gene expression was silenced by RNA interference. The introduction of RON-specific small interfering (si) RNA significantly affected cancer cell proliferation, motility, and led to increased apoptotic cell death. Focus-forming activities and anchorage-independent growth of colon cancer cells were also dramatically reduced. Moreover, it was demonstrated in tumor growth assays that silencing RON gene expression significantly reduces tumorigenic activities of SW620 cells in vivo. By analysing signaling proteins involved in colon carcinogenesis, we found that the effect of RON-specific siRNA is associated with diminished expression of beta-catenin, a critical component in the Wnt signaling pathway. Taken together, our results demonstrate that altered expression of RON in colon cancer cells is required to maintain tumorigenic phenotypes. Thus, silencing RON gene expression could have potential to reverse malignant activities of colon tumors in vivo.

The product of the RON (recepteur d'origine nantais) gene belongs to the MET proto-oncogene family, a distinct subfamily of receptor tyrosine kinases. The ligand of RON was identified as macrophage-stimulating protein (MSP), a member of the plasminogen-related growth factor family. RON is mainly expressed in cells of epithelial origin and is required for embryonic development. In vitro RON activation results in epithelial cell dissociation, migration and matrix invasion, suggesting that RON might be involved in the pathogenesis of certain epithelial cancers in vivo. Indeed, recent studies have shown that RON expression is significantly altered in several primary human cancers, including those of the breast and colon. Truncation of the RON protein has also been found in primary tumors from the gastrointestinal tract. These alterations lead to constitutive activation of RON that causes cell transformation in vitro, induces neoplasm formation in athymic nude mice, and promotes tumor metastasis into the lung. Studies employing transgenic models further demonstrated that over-expression of RON in lung epithelial cells results in multiple tumor formation with features of large cell undifferentiated carcinoma. The oncogenic activities of RON are mediated by RON-transduced signals that promote unbalanced cell growth and transformation leading to tumor development. Thus, abnormal accumulation and activation of RON could play a critical role in vivo in the progression of certain malignant human epithelial cancers.

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 RONdelta165, RONdelta160, and RONdelta155 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 beta chain. Functional studies showed that expression of RONdelta160 or RONdelta155 in Martin-Darby canine kidney cells resulted in increased cell dissociation (scatter-like activity). RON variants, RONdelta160 and RONdelta155, also exerted the ability to induce multiple focus formation and sustain anchorage-independent growth of transfected NIH3T3 cells. Moreover, NIH3T3 cells expressing RONdelta160 or RONdelta155 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.

Macrophage-stimulating protein (MSP) is a member of the hepatocyte growth factor-scatter factor (HGF-SF) family. Labeled MSP bound to Madin-Darby canine kidney (MDCK) cells transfected with complementary DNA encoding Ron, a cell membrane protein tyrosine kinase. Cross-linking of 125I-labeled MSP to transfected cells (MDCK-RE7 cells) and immunoprecipitation by antibodies to Ron revealed a 220-kilodalton complex, a size consistent with that of MSP (80 kilodaltons) cross-linked to the beta chain of Ron (150 kilodaltons). The binding of 125I-labeled MSP to MDCK-RE7 cells was inhibited by unlabeled MSP, but not by HGF-SF. MSP caused phosphorylation of the beta chain of Ron and induced migration of MDCK-RE7 cells. These results establish the ron gene product as a specific cell-surface receptor for MSP.

The receptor tyrosine kinase RON (recepteur d'origine nantais), a member of the MET proto-oncogene family, has been implicated in the pathogenesis of certain epithelial cancers including lung adenocarcinomas. To determine the oncogenic potential of RON, transgenic mice were generated using the surfactant protein C promoter to express human wild-type RON in the distal lung epithelial cells. The mice were born normal without morphological defects in the lung, however, multiple lung adenomas with distinct morphology and growth pattern were observed. Tumors appeared as a single mass in the lung around 2 months of age and gradually developed into multiple nodules throughout the lung. Most of the tumors were characterized as cuboidal epithelial cells with type II cell phenotypes. They grew along the alveolar walls and projected into the alveolar septa. A transition from pre-malignant adenomas to adenocarcinomas was observed. The RON transgene is highly expressed and constitutively activated in the tumors as evident by immunohistochemical staining and western blot analyses. Moreover, we found that Ras expression was dramatically increased in the majority of tumors. However, no mutation in the 'hot spots' of the K-Ras or p53 gene was observed, although limited genomic instability occurs in individual tumors. Taken together, this is a mouse lung tumor model with unique biological characteristics. The model may provide an opportunity to study the role of RON in lung tumors and to elucidate the mechanisms underlying this distinct lung tumor.

RON, a member of the MET proto-oncogene family, has been implicated in the progression of certain epithelial cancers. The purpose of this study was to determine the oncogenic potential of RON in vivo in lung epithelial cells. Transgenic mice were established using surfactant protein C promoter to express human RON in the distal lung epithelial cells. These mice were born normal but developed multiple lung tumors with distinct morphology and growth patterns. Tumors appeared as a single mass in the lung around 2 months of age and gradually developed into multiple nodules located mostly in the peripheral portions of the lung. A transition from early adenomas to later adenocarcinomas was observed. Morphologically, tumors were characterized as cuboidal epithelial cells with a type II cell phenotype, grew along the alveolar walls, and projected into the alveolar septa. RON was highly expressed and constitutively activated in tumors. These results indicate that overexpression of human wild-type RON causes the formation of lung tumors with unique biological characteristics in vivo. This model provides opportunities to study the role of RON in the pathogenesis of lung tumors and to elucidate the mechanisms underlying this distinct lung tumor.

RON is a receptor tyrosine kinase belonging to the MET proto-oncogene family. The purposes of this study are to determine the expression and activation of RON in a panel of human colon carcinoma cell lines. Western blotting showed that RON is barely detectable in normal and SV-40-transformed colon epithelial cells, but highly expressed and constitutively activated in several colon carcinoma cell lines including Colo201, HT-29, HCT116, and SW837. Moreover, a novel RON variant with a molecular mass of 160 kDa (RONΔ160) was identified from HT-29 cells. The cDNA encoding RONΔ160 has an in-frame deletion of 109 amino acids in the extracellular domain of the RON β chain, which is caused by splicing out of two exons in the RON mRNA. No mutations were found in the kinase domain of the RON gene in five carcinoma cell lines screened. By expressing RON in colon epithelial cells, we found that RON activation increases cell motile-invasive activities and protects cells against apoptotic death. These data suggest that RON expression and activation are deregulated in colon carcinoma cell lines. By abnormal activation of RON, this receptor and its variant may regulate motile-invasive phenotypes of certain colon carcinoma cells in vivo.

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-γ, and TNF-α regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-α plus IFN-γ abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-α plus IFN-γ induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (±)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-β prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-α plus IFN-γ are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.

To delineate the functional protein domains necessary for the biological activity of hepatocyte growth factor-like protein (HGFL), we created various site-directed and deletion mutated cDNAs coding for this protein. Wild-type and mutated versions of HGFL were produced after transfection of the corresponding cDNAs into tissue culture cells. The biological importance of the domains within HGFL was then examined by addition of recombinant wild-type or mutant forms of HGFL to assays aimed at elucidating regions involved in the stimulation of DNA synthesis, the induction of shape changes in macrophages, and the ability to stimulate cell scattering. Mutant proteins lacking the serine protease-like domain (light chain) were not biologically active in any of the assays tested and could not compete with wild-type HGFL in cell scattering experiments. These data, in addition to direct enzyme-linked immunosorbent assay analyses, suggest that the light chain may play an important role in the interaction of HGFL with its receptor, Ron. Elimination of the proposed protease cleavage site between the heavy and light chains (by mutation of Arg-483 to Glu) produced a protein with activity comparable to wild-type HGFL. Further studies with this mutated protein uncovered an additional proteolytic cleavage site that produces biologically active protein. Deletion of the various kringle domains or the amino-terminal hairpin loop had various effects in the multiple assays. These data suggest that the heavy chain may play a pivotal role in determining the functional aspects of HGFL.

Macrophage stimulating protein (MSP) is a 78-kDa disulfide-linked heterodimer belonging to the plasminogen-related kringle protein family. MSP activates the RON receptor protein-tyrosine kinase, which results in cell migration, shape change, or proliferation. A structure-activity study of MSP was performed using pro-MSP, MSP, MSP alpha and beta chains, and a complex including the first two kringles and IgG Fc (MSP-NK2). Radioiodinated MSP and MSP beta chain both bound specifically to RON. The Kd of 1.4 nM for MSP beta chain is higher than the reported Kd range of 0.6-0.8 nM for MSP. Pro-MSP, MSP alpha chain, and MSP-NK2 did not bind. Only MSP stimulated RON autophosphorylation. Although the beta chain bound to RON and partially inhibited MSP-induced RON phosphorylation in kidney 293 cells, it did not induce RON phosphorylation. Pro-MSP, MSP alpha chain, or MSP-NK2 failed to activate RON, consistent with their inability to bind to the RON receptor. Functional studies showed that only MSP induced cell migration, and shape change in resident macrophages, and growth of murine keratinocytes. Our data indicate that the primary receptor binding domain is located in a region of the MSP beta chain, in contrast to structurally similar hepatocyte growth factor, in which the receptor binding site is in the alpha chain. However, full activation of RON requires binding of the complete MSP disulfide-linked alphabeta chain heterodimer.

Transient pulmonary neuroendocrine cell hyperplasia and non-neuroendocrine lung tumors develop in nitrosaminetreated hamsters, which we hypothesized might modulate epithelial cell phenotype by expressing gene(s) homologous to human chromosome 3p gene(s) deleted in small cell carcinoma of the lung (SCLC). We differentially screened a chromosome 3 library using nitrosamine-treated versus normal hamster lung cDNAs and identified hepatocyte growth factor-like/macrophage-stimulating protein (HGFL/MSP) in injured lung. HGFL/MSP mRNA is low to undetectable in human SCLC and carcinoid tumors, but the HGFL/MSP tyrosine kinase receptor, RON, is present and functional on many of these neuroendocrine tumors. In H835, a pulmonary carcinoid cell line, and H187, a SCLC cell line, HGFL/ MSP induced adhesion/flattening and apoptosis. Using viable cell counts to assess proliferation after 14 d of treatment with HGFL/MSP, there is growth inhibition of H835 but not H187. Nitrosamine-treated hamsters also demonstrate pulmonary neuroendocrine cell apoptosis in situ during the same time period as expression of the endogenous HGFL/ MSP gene, immediately preceding the spontaneous regression of neuroendocrine cell hyperplasia. These observations suggest that HGFL/MSP might regulate neuroendocrine cell survival during preneoplastic lung injury, which could influence the ultimate tumor cell phenotype.

STK, a new member of the hepatocyte growth factor receptor family, is the receptor for macrophage-stimulating protein (MSP), which acts on murine resident peritoneal macrophages. We established polyclonal and monoclonal antibodies against STK and characterized the structure of STK protein and STK expression on cells of the mononuclear phagocyte system. Western blotting showed that the STK transcript is translated into a single-chain precursor and then cleaved into a 165-kD disulfide- linked heterodimer composed of a 35-kD alpha-chain and a 144-kD beta- chain. Western blotting detected STK protein on resident peritoneal macrophages, a target of MSP, and showed that it was autophosphorylated in cells stimulated by MSP. By flow cytometric analysis using a monoclonal anti-STK antibody, we showed that STK protein is expressed on restricted macrophage populations such as resident peritoneal macrophages, but not on exudate peritoneal macrophages or mononuclear phagocytes of the bone marrow, peripheral blood, spleen, or alveoli. Resident peritoneal macrophages were classified into two fractions according to their reactivity with an anti-STK antibody and a marker antibody for macrophages: STKhigh-F4/80high cells and STKnegative- F4/80low cells. Acute exudative macrophages were all STKnegative- F4/80low, but they gradually became predominantly STKhigh-F4/80high several days after entrance into the peritoneal cavity. These results showed that after monocytes migrate into the peritoneal cavity, they undergo terminal differentiation in the peritoneal microenvironment. This is the first evidence of tissue-specific terminal differentiation of peritoneal macrophages, and this terminal differentiation can be characterized by the expression of STK receptor tyrosine kinase.

Background Overexpression of the RON receptor tyrosine kinase contributes to epithelial cell transformation, malignant progression, and acquired drug resistance. RON also has been considered as a potential target for therapeutic intervention. This study determines biochemical features and inhibitory activity of a mouse monoclonal antibody (mAb) Zt/f2 in experimental cancer therapy. Results Zt/f2 is a mouse IgG2a mAb that is highly specific and sensitive to human RON and its oncogenic variants such as RON160 (ED50 = 2.3 nmol/L). Receptor binding studies revealed that Zt/f2 interacts with an epitope(s) located in a 49 amino acid sequence coded by exon 11 in the RON β-chain extracellular sequences. This sequence is critical in regulating RON maturation and phosphorylation. Zt/f2 did not compete with ligand macrophage-stimulating protein for binding to RON; however, its engagement effectively induced RON internalization, which diminishes RON expression and impairs downstream signaling activation. These biochemical features provide the cellular basis for the use of Zt/f2 to inhibit tumor growth in animal model. Repeated administration of Zt/f2 as a single agent into Balb/c mice results in partial inhibition of tumor growth caused by transformed NIH-3T3 cells expressing oncogenic RON160. Colon cancer HT-29 cell-mediated tumor growth in athymic nude mice also was attenuated following Zt/f2 treatment. In both cases, ~50% inhibition of tumor growth as measured by tumor volume was achieved. Moreover, Zt/f2 in combination with 5-fluorouracil showed an enhanced inhibition effect of ~80% on HT-29 cell-mediated tumor growth in vivo. Conclusions Zt/f2 is a potential therapeutic mAb capable of inhibiting RON-mediated oncogenesis by colon cancer cells in animal models. The inhibitory effect of Zt/f2 in vivo in combination with chemoagent 5-fluorouracil could represent a novel strategy for future colon cancer therapy.

Background Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP) has been implicated in cellular EMT program; however, the major signaling determinant(s) responsible for MSP-induced EMT is unknown. Results The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration. Conclusions MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

Macrophage stimulating protein (MSP) exists in blood as inactive pro-MSP. Cleavage yields active MSP, the ligand for a membrane receptor (RON) that is expressed on keratinocytes as well as macrophages. Because both cells have roles in tissue injury, we looked for active MSP and expressed RON in wounds. Concentration of pro-MSP + MSP in wound exudates was in the range for optimal activity. Western blot showed that MSP comprised about half the total, in contrast to less than 10% of the total in blood plasma. The presence of MSP was attributed to an exudate pro-MSP convertase that had an inhibitor profile consistent with a trypsin-like serine protease. Exudate evoked morphologic changes in macrophages in vitro like that of MSP. Removal of this activity by an anti-MSP column shows that exudate stimulation of macrophages is due to MSP. RON was infrequently detected in normal skin. RON protein was markedly upregulated in burn wound epidermis and accessory structures, in proliferating cells or differentiated cells, or both. RON was also detected on macrophages and capillaries. Tissue injury leads to cleavage of pro-MSP to MSP, which has potential to act on keratinocytes, macrophages, and capillaries, all components of the wound healing response.

Background Aberrant 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. Methods Mouse 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. Results In 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. Conclusion Zt/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.

Background Activation of the RON receptor tyrosine kinase, a member of the c-MET family, regulates tumorigenic phenotypes. The RON extracellular domains are critical in regulating these activities. The objective of this study was to determine the role of the first IPT domain in regulating RON-mediated tumorigenic activities and the underlying mechanisms. Results Two RON variants, RON160 and RONE5/6in with deletion and insertion in the first IPT domain, respectively, were molecularly cloned. RON160 was a splicing variant generated by deletion of 109 amino acids encoded by exons 5 and 6. In contrast, RONE5/6in was derived from a transcript with an insertion of 20 amino acids between exons 5 and 6. Both RON160 and RONE5/6in were proteolytically matured into two-chain receptor and expressed on the cell surface. RON160 was constitutively active with tyrosine phosphorylation. However, activation of RONE5/6in required ligand stimulation. Deletion resulted in the resistance of RON160 to proteolytic digestion by cell associated trypsin-like enzymes. RON160 also resisted anti-RON antibody-induced receptor internalization. These features contributed to sustained intracellular signaling cascades. On the other hand, RONE5/6in was highly susceptible to protease digestion, which led to formation of a truncated variant known as RONp110. RONE5/6in also underwent rapid internalization upon anti-RON antibody treatment, which led to signaling attenuation. Although ligand-induced activation of RONE5/6in partially caused epithelial to mesenchymal transition (EMT), it was RON160 that showed cell-transforming activities in cell focus formation and anchorage-independent growth. RON160-mediated EMT is also associated with increased motile/invasive activity. Conclusions Alterations in the first IPT domain in extracellular region differentially regulate RON mediated tumorigenic activities. Deletion of the first IPT results in formation of oncogenic variant RON160. Enhanced degradation and internalization with attenuated signaling cascades could be the mechanisms underlying non-tumorigenic features of RONE5/6in.