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Pharmaceutical innovation in the development of novel antibody-based biotherapeutics with increased therapeutic indexes makes MET-targeted cancer therapy a clinical reality.

The dysfunction of bone marrow mesenchymal stem cells (BMSCs) in balancing osteogenesis and adipogenesis plays an important role in the occurrence and development of osteoporosis. It's still unknown that whether ATP-binding cassette g1 (Abcg1), a well-proved regulation gene of adipogenesis, regulates osteogenesis. In our previous study, we identified 30 differentially expressed genes in osteogenesis and found the expression level ofAbcg1 negatively related to osteogenesis among these genes. Abcg1 is a well-proven adipogenesis regulator and cholesterol transporter, but it's role in osteogenesis remained unknown. In this study we found it may control osteogenesis, further elucidating the exact role of Abcg1 in regulating osteoblast differentiation would help propose new strategies to prevent and treat osteoporosis. Therefore, we established Abcg1 up- or down-expressed C3H10T1/2 and C2C12 cell lines and verified that Abcg1 knockdown enhanced expression of osteogenic factors runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP), while Abcg1 overexpression reversed the promoting effect. Furthermore, we confirmed that Abcg1 modulated osteogenesis via the Wnt/β-catenin and AMPK signaling pathways. taken together, these results suggest that Abcg1 may have an important role in regulating osteogenesis via Wnt/β-catenin and AMPK signaling pathways, and expect to be a potential therapeutic target for osteoporosis.

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.

Advanced epithelial cancers such as gastric, lung, and pancreatic tumors are featured by invasive proliferation, distant metastasis, acquired chemoresistance, and tumorigenic stemness. For the last decade, molecular-targeted therapies using therapeutic antibodies, small molecule kinase inhibitors and immune-checkpoint blockades have been applied for these diseases with significant clinical benefits. Nevertheless, there is still a large gap to achieve curative outcomes. MET (mesenchymal-epithelial transition protein), a receptor tyrosine kinase, is a tumorigenic determinant that regulates epithelial cancer initiation, progression, and malignancy. Increased MET expression also has prognostic value for cancer progression and patient survival. These features provide the rationale to target MET for cancer treatment. In this review, we discuss the importance of MET in epithelial tumorigenesis and the development of antibody-based biotherapeutics, including bispecific antibodies and antibody-drug conjugates, for clinical application. The findings from both preclinical and clinical studies highlight the potential of MET-targeted biotherapeutics for cancer therapy in the future.

The recepteur d’origine nantais (RON) receptor tyrosine kinase, belonging to the mesenchymal-to-epithelial transition proto-oncogene family, has been implicated in the pathogenesis of cancers derived from the colon, lung, breast, and pancreas. These findings lay the foundation for targeting RON for cancer treatment. However, development of RON-targeted therapeutics has not gained sufficient attention for the last decade. Although therapeutic monoclonal antibodies (TMABs) targeting RON have been validated in preclinical studies, results from clinical trials have met with limited success. This outcome diminishes pharmaceutical enthusiasm for further development of RON-targeted therapeutics. Recently, antibody–drug conjugates (ADCs) targeting RON have drawn special attention owing to their increased therapeutic activity. The rationale for developing anti-RON ADCs is based on the observation that cancer cells are not sufficiently addicted to RON signaling for survival. Thus, TMAB-mediated inhibition of RON signaling is ineffective for clinical application. In contrast, anti-RON ADCs combine a target-specific antibody with potent cytotoxins for cancer cell killing. This approach not only overcomes the shortcomings in TMAB-targeted therapies but also holds the promise for advancing anti-RON ADCs into clinical trials. In this review, we discuss the latest advancements in the development of anti-RON ADCs for targeted cancer therapy including drug conjugation profile, pharmacokinetic properties, cytotoxic effect in vitro, efficacy in tumor models, and toxicological activities in primates.

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.

RON (recepteur d'origine nantais) and MET (hepatocyte growth factor receptor) are tyrosine kinase receptors. Various cancers have aberrant RON and MET expression and activation, which contribute to cancer cell proliferation, invasiveness, and metastasis. Here, we explored RON and MET expression in pancreatic cancer and their relationship with overall survival (OS) time, and evaluated their significance as therapeutic targets of tyrosine kinase inhibitors in pancreatic cancer. We enrolled 227 patients with pancreatic cancer in the study. RON and MET expression was analyzed by immunohistochemical staining. Four human pancreatic cancer cell lines expressing variable levels of RON or MET and four MET superfamily inhibitors (BMS777607, PHA665752, INCB28060, Tivantinib) were used. The effect of the four tyrosine kinase inhibitors on cell viability, migration, and apoptosis were determined using cell viability, scratch wound healing, and Caspase-Glo 3/7 assays. Cellular signaling was analyzed by immunoprecipitation and western blotting. The therapeutic efficacy of the tyrosine kinase inhibitors was determined with mouse xenograft pancreatic cancer models in vivo. There was wide aberrant RON and MET expression in the cancer tissues. In 227 pancreatic cancer samples, 33% had RON overexpression, 41% had MET overexpression, and 15.4% had RON and MET co-overexpression. RON and MET expression were highly correlated. RON and MET expression levels were significantly related to OS. Patients with RON and MET co-overexpression had poorer OS. BMS777607 and PHA665752 inhibited pancreatic cancer cell viability and migration, and promoted apoptosis by inhibiting RON and MET phosphorylation and further inhibiting the downstream signaling pathways in vitro. They also inhibited tumor growth and further inhibited phosphorylated (phosphor)-RON and phospho-MET expression in the mouse xenograft models in vivo effectively. INCB28060, which inhibits the MET signaling pathway alone, was not effective. RON and MET can be important indicators of prognosis in pancreatic cancer. Tyrosine kinase inhibitors targeting RON and MET in pancreatic cancer are a novel and potential approach for pancreatic cancer therapy.

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.

Podoplanin, lymphatic vessel endothelial hyaluronic acid receptor-1, prospero-related homeobox-1 and vascular endothelial growth factor receptor 3 have been demonstrated to have crucial roles in the development of the lymphatic system and lymphangiogenesis process by combining with their corresponding receptors. Thus, the four markers have been widely used in labelling lymphatic vessels for the detection of lymphangiogenesis and lymphatic vessel invasion. Numerous authors have aimed to identify the roles of these four markers in the lymphatic system and the mechanisms have been partly clarified at the molecular level. The aim of the present review was to comprehensively clarify the characteristics and latent action modes of the four markers in order to determine which is the best one for the detection of lymphangiogenesis and lymphatic vessel invasion.

Ribosomes are essential components of the protein synthesis machinery. The process of ribosome biogenesis is well organized and tightly regulated. Recent studies have shown that ribosomal proteins (RPs) have extraribosomal functions that are involved in cell proliferation, differentiation, apoptosis, DNA repair, and other cellular processes. The dysfunction of RPs has been linked to the development and progression of hematological, metabolic, and cardiovascular diseases and cancer. Perturbation of ribosome biogenesis results in ribosomal stress, which triggers activation of the p53 signaling pathway through RPs-MDM2 interactions, resulting in p53-dependent cell cycle arrest and apoptosis. RPs also regulate cellular functions through p53-independent mechanisms. We herein review the recent advances in several forefronts of RP research, including the understanding of their biological features and roles in regulating cellular functions, maintaining cell homeostasis, and their involvement in the pathogenesis of human diseases. We also highlight the translational potential of this research for the identification of molecular biomarkers, and in the discovery and development of novel treatments for human diseases.

Tyrosine kinase inhibitor BMS-777067 is an inhibitor of RON/MET receptor tyrosine kinases currently under clinical trials. Here, we report the synergistic activity of BMS-777607 in combination with mTOR inhibitor AZD8055 in killing chemoresistant pancreatic cancer and cancer stem cells. Treatment of pancreatic cancer L3.6pl cells with BMS-777607 alone inhibited clonogenic growth and moderately induced apoptotic death. However, BMS-777607 caused extensive polyploidy in L3.6pl cells through inhibition of aurora kinase B activity, independent of RON expression. In contrast, L3.6pl-derived cancer stem cells were highly resistant to BMS-777607–induced growth inhibition and apoptosis. The effect of BMS-777607 on induction of cancer stem cell polyploidy was also weak. BMS-777607–induced polyploidy features a predominant cell population with 8N chromosome content in both L3.6pl and cancer stem cells. These cells also showed decreased sensitivity toward chemotherapeutics by increased survival of IC50 values in response to doxorubicin, cisplatin, methotrexate, 5-fluorouracial, and gemcitabine. Among a panel of chemical inhibitors that target different signaling proteins, we found that BMS-777607 in combination with mTOR inhibitor AZD8055 exerted synergistic effects on L3.6pl and cancer stem cells. More than 70% of L3.6pl and cancer stem cells lost their viability when both inhibitors were used. Specifically, BMS-777607 in combination with inhibition of mTORC2, but not mTORC1, was responsible for the observed synergism. Our findings demonstrate that BMS-777607 at therapeutic doses exerts inhibitory activities on pancreatic cancer cells but also induces polyploidy insensitive to chemotherapeutics. Combination of BMS-777607 with AZD8055 achieves the maximal cytotoxic effect on pancreatic cancer and cancer stem cells. Mol Cancer Ther; 13(1); 37–48. ©2013 AACR.

Identification and validation of molecular targets are considered as key elements in new drug discovery and development. We have recently demonstrated that a novel synthetic iminoquinone analog, termed [7-(benzylamino)- 1,3,4,8-tetrahydropyrrolo [4,3, 2-de]quinolin-8(1H)-one] (BA-TPQ), has significant anti-breast cancer activity both in vitro and in vivo, but the underlying molecular mechanisms are not fully understood. Herein, we report the molecular studies for BA-TPQ's effects on JNK and its upstream and downstream signaling pathways. The compound up-regulates the JNK protein levels by increasing its phosphorylation and decreasing its polyubiquitination-mediated degradation. It activates ZAK at the MAPKKK level and MKK4 at the MAPKK level. It also up-regulates the TGFβ2 mRNA level, which can be abolished by the JNK-specific inhibitor SP600125, but not TGFβ pathway-specific inhibitor SD-208, indicating that both JNK and TGFβ signaling pathways are activated by BA-TPQ and that the JNK pathway activation precedes TGFβ activation. The pro-apoptotic and anti-growth effects of BA-TPQ are significantly blocked by both the JNK and TGFβ pathway inhibitors. In addition, BA-TPQ activates the ZAK-MKK4-JNK pathway in MCF7 cells, but not normal MCF10A cells, demonstrating its cancer-specific activities. In conclusion, our results demonstrate that BA-TPQ activates the ZAK-MKK4-JNK-TGFβ signaling cascade as a molecular target for its anticancer activity.

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.

There is an increasing interest in determining the role of ribosomal proteins (RPs) in the regulation of MDM2-p53 pathway in coordinating cellular response to stress. Herein, we report a novel regulatory role of ribosomal protein S25 (RPS25) in MDM2-mediated p53 degradation and a feedback regulation of S25 by p53. We demonstrated that S25 interacted with MDM2 and inhibited its E3 ligase activity, resulting in the reduction of MDM2-mediated p53 ubiquitination and the stabilization and activation of p53. S25, MDM2 and p53 formed a ternary complex following ribosomal stress. The nucleolar localization and MDM2-binding domains of S25 were critical for its role in MDM2-mediated p53 regulation. Knockdown of S25 by siRNA attenuated the induction and activation of p53 following ribosomal stress. S25 stabilized and cooperated with MDMX to regulate MDM2 E3 ligase activity. Furthermore, S25 was identified to be a transcriptional target of p53; p53 directly bound to S25 promoter and suppressed S25 expression. Our results suggest that there is a S25-MDM2-p53 regulatory feedback loop, which may have an important role in cancer development and progression.

We have recently designed and synthesized a novel iminoquinone anticancer agent, 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ) and initiated its preclinical development. Herein we investigated its efficacy, safety, and pharmacokinetics in in vitro and in vivo models of human pancreatic cancer. Our results demonstrated that FBA-TPQ inhibited pancreatic cancer cell growth, induced apoptosis, and caused cell cycle arrest in vitro. It inhibited the growth of xenograft tumors with minimal host toxicity. To facilitate future preclinical and clinical development of the agent, we also developed and validated a Rapid Resolution Liquid Chromatography (RRLC) method for quantitative analysis of FBA-TPQ in plasma and tissue samples. The method was found to be precise, accurate, and specific. Using this method, we carried out in vitro and in vivo evaluations of the pharmacological properties of FBA-TPQ, including stability in plasma, plasma protein binding, metabolism by S9 enzymes, plasma pharmacokinetics, and tissue distribution. Our results indicate that FBA-TPQ is a potential therapeutic agent for pancreatic cancer, providing a basis for future preclinical and clinical development.

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.

Therapeutics targeting known oncoproteins have been applied for pancreatic cancer treatment but clinical outcomes are not promising. Hence, there is an urgent need to identify novel targets and develop effective drugs to improve pancreatic cancer therapeutic index. Antibody-drug conjugates (ADC) represent a promising class of drugs for targeted cancer therapy. Here we developed a novel ADC targeting RON receptor tyrosine kinase for potential pancreatic cancer treatment. To this end, we have synthesized a bis-alkylating linker (BL), attached to a lysosomal protease-cleavable dipeptide with payload Monomethyl auristatin E (MMAE). The BL-MMAE was then conjugated to Zt/g4 (anti-RON mAb) through cysteine bridging technology to produce Zt/g4-BL-MMAE with a homogeneous conjugation profile and an antibody to drug ratio of 1:4. Zt/g4-BL-MMAE showed significant improvement in drug conjugation homogeneity and serum stability over conventional ADCs prepared through maleimide based linkers. In pancreatic cancer cell lines overexpressing RON, Zt/g4-BL-MMAE specifically targeted RON-expressing tumor cells and was effective in rapid induction of cell surface RON endocytosis. Functional analysis revealed that Zt/g4-BL-MMAE caused cell cycle arrest at G2/M phase, reduction of cell viability and subsequently resulted in massive cell death. The calculated IC50 is in the range of 1 to 2 µg/ml. We conclude that Zt/g4-BL-MMAE is a novel anti-RON ADC with excellent conjugation profile, serum stability, and selective cytotoxicity for pancreatic cancer cells. This work provides a pharmaceutical opportunity for evaluating potentials of RON-targeted ADCs in pancreatic cancer treatment in the future. Citation Format: Sreedhar Reddy Suthe, Hang-Ping Yao, Paul C. Trippier, Ming-Hai Wang. Development of a novel ron receptor targeted antibody-drug conjugates using cysteine bridging technology for potential treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 745.

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.

Ring1 and YY1 binding protein (RYBP) is a member of the Polycomb group (PcG) proteins and regulates cell growth through both PcG-dependent and -independent mechanisms. Our initial study indicated that RYBP is down-regulated in human non-small cell lung cancer (NSCLC) tissues. The present study determined the molecular role of RYBP in the development of NSCLC. We systemically investigated the association between the RYBP expression and the survival of patients with NSCLC. We also carried out in vitro and in vivo studies to explore the molecular basis for the tumor suppressor role of RYBP in NSCLC. Our clinical results demonstrated that the RYBP mRNA and protein expressions were significantly down-regulated in NSCLC and significantly linked to the poor prognosis in NSCLC patients. The enforced expression of RYBP inhibited cell survival, induced apoptosis, and increased chemosensitivity in NSCLC cells; knockdown of RYBP showed the opposite effects. Moreover, adenoviral delivery of RYBP sensitized the NSCLC cells to chemotherapy in vivo. In addition, RYBP expression was induced by paclitaxel, the first-line chemotherapeutic agent for NSCLC. Our results reveal that RYBP inhibits the aggressiveness of NSCLC cells and downregulation of RYBP is associated with poor prognosis, suggesting that RYBP could be developed as a biomarker and a novel target for therapy in patients with lung cancer.

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 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.

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.

A requirement for Mouse Double Minute 2 (MDM2) oncogene activation has been suggested to be associated with cancer progression and metastasis, including breast cancer. To date, most MDM2 inhibitors have been designed to block the MDM2-p53-binding interphase, and have low or no efficacy against advanced breast cancer with mutant or deficient p53. Here we use a high-throughput screening and computer-aided, structure-based rational drug design, and identify a lead compound, SP-141, which can directly bind to MDM2, inhibit MDM2 expression and induce its autoubiquitination and proteasomal degradation. SP-141 has strong in vitro and in vivo antibreast cancer activity, with no apparent host toxicity. While further investigation is needed, our data indicate that SP-141 is a novel targeted therapeutic agent that may especially benefit patients with advanced disease.

Targeted inhibition of MET/RON signaling by tyrosine kinase inhibitor BMS-777607 for cancer treatment is currently under clinical trials. We have previously shown that BMS-777607 induces chemoresistance in vitro by causing polyploidy, which hampers therapeutic efficacy. Here, we studied polyploidy-associated senescence induced by BMS-777607 in breast cancer cells and its prevention by mTOR inhibitor AZD8055, leading to increased chemosensitivity. In breast cancer T-47D and ZR-75-1 cells, BMS-777607 induced phenotypic changes including enlarged cellular size, flattened morphology, increased DNA content, and activity of senescence-associated β-galactosidase. These changes were accompanied by increased p21/WAF1 expression and decreased Retinoblastoma Ser(780) phosphorylation, indicating that BMS-777607 induces not only polyploidy but also senescence. The appearance of senescence was associated with polyploidy in which β-galactosidase is exclusively expressed in polyploid cells. Survivin expression was increased in polyploid/senescent cells as analyzed by Western blotting. Increased survivin accumulated both in the nucleus and cytoplasm and dissociated with condensed DNA and mitotic spindle at the metaphase. Abnormal accumulation of survivin also rendered polyploid/senescent cells insensitive to cytotoxic activities of YM155, a DNA damaging agent with a suppressive effect on survivin gene transcription. AZD8055, a specific mTOR inhibitor, effectively prevented BMS-777607-induced polyploidy and senescence and restored survivin expression and its nuclear localization to normal levels. Although a synergism was not observed, BMS-777607 plus AZD8055 increased cancer cell sensitivity toward different cytotoxic chemotherapeutics. In conclusion, BMS-777607-induced chemoresistance is associated with cell polyploidy and senescence. Inhibition of mTOR signaling by AZD8055 prevents BMS-777607-induced polyploidy/senescence and increases breast cancer cell chemosensitivity.

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.

The oncogene MDM2, which encodes an E3 ubiquitin ligase, is overexpressed in pancreatic cancers and is therefore a therapeutic target. Current inhibitors of MDM2 target the interaction between MDM2 and P53; these would have no effect on cancer cells that do not express full-length P53, including many pancreatic cancer cells. We searched for a compound that specifically inhibits MDM2 itself.We performed a virtual screen and structure-based design to identify specific inhibitors of MDM2. We tested the activities of compounds identified on viability, proliferation, and protein levels of HPAC, Panc-1, AsPC-1, and Mia-Paca-2 pancreatic cancer cell lines. We tested whether intraperitoneal injections of one of the compounds identified affected growth of xenograft tumors from Panc-1 cells, or orthotopic tumors from Panc-1 and AsPC-1 cells (injected into pancreata), in nude mice.We identified a compound, called SP141, which bound directly to MDM2, promoting its auto-ubiquitination and degradation by the proteasome. The compound reduced levels of MDM2 in pancreatic cancer cell lines, as well as their proliferation, with 50% inhibitory concentrations0.5 μM (0.38-0.50 μM). Increasing concentrations of SP141 induced increasing levels of apoptosis and G2-M-phase arrest of pancreatic cancer cell lines, whether or not they expressed functional P53. Injection of nude mice with SP141 (40 mg/kg/d) inhibited growth of xenograft tumors (by 75% compared with control mice), and led to regression of orthotopic tumors.In a screen for specific inhibitors of MDM2, we identified a compound called SP141 that reduces levels of MDM2 in pancreatic cancer cell lines, as well as their proliferation and ability to form tumors in nude mice. SP141 is a new class of MDM2 inhibitor that promotes MDM2 auto-ubiquitination and degradation. It might be further developed as a therapeutic agent for pancreatic cancer.

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.

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.

Although ginseng and related herbs have a long history of utility for various health benefits, their application in cancer therapy and underlying mechanisms of action are not fully understood. Our recent work has shown that 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH(3)-PPD), a newly identified ginsenoside from Panax notoginseng, exerts activities against a variety of cancer cells in vitro and in vivo. This study was designed to investigate its anti-breast cancer activity and the underlying mechanisms of action. We observed that 25-OCH(3)-PPD decreased the survival of breast cancer cells by induction of apoptosis and G1 phase arrest and inhibited the growth of breast cancer xenografts in vivo. We further demonstrated that, in a dose- and time-dependent manner, 25-OCH(3)-PPD inhibited MDM2 expression at both transcriptional and post-translational levels in human breast cancer cells with various p53 statuses (wild type and mutant). Moreover, 25-OCH(3)-PPD inhibited in vitro cell migration, reduced the expression of epithelial-to-mesenchymal transition (EMT) markers, and prevented in vivo metastasis of breast cancer. In summary, 25-OCH(3)-PPD is a potential therapeutic and anti-metastatic agent for human breast cancer through down-regulating MDM2. Further preclinical and clinical development of this agent is warranted.

JKA97, a benzylidene analog of harmine, has been found to be a promising drug candidate for human cancer therapy, although the underlying molecular mechanisms have not been fully demonstrated. In this study, we evaluated the effects of JKA97 on human breast cancer cells in vitro and in vivo. JKA97 inhibited the growth and proliferation of MCF7 (p53 wild-type), MCF7 (p53 knockdown), and MDA-MB-468 (p53 mutant) cells in a dose-dependent manner. Treatment with JKA97 arrested breast cancer cells in G1 phase and induced apoptosis. JKA97 also significantly suppressed the growth of MCF7 and MDA-MB-468 xenograft tumors. It regulated the expression levels of G1 phase regulators, such as p21, p27, cyclinE, and cylinD1. JKA97 activated p21 transcription, independent of p53, but had little effect on p21 protein stability/degradation. In summary, our results suggest that JKA97 inhibits human breast cancer cell growth through activating p21, independent of p53, which provides a basis for developing this compound as a novel drug for human breast cancer therapy.

Promacrophage-stimulating protein (MSP) is an 80-kDa protein that acquires biological activity after cleavage at an Arg-Val bond to a disulfide-linked alpha beta heterodimer by serine proteases of the intrinsic coagulation cascade. These proteases, which include serum kallikrein, factor XIIa and factor XIa, are members of the trypsin family of serine proteases. We now report that two other members of the family, nerve growth factor-gamma (NGF-gamma) and epidermal growth factor-binding protein (EGF-BP), cleave and activate pro-MSP to the disulfide-linked alpha beta heterodimer. Cleavage of 1.5 nM pro-MSP by 1 nM NGF-gamma or EGF-BP at 37 degrees C was almost complete within 30 min. These concentrations of enzyme are about 2 orders of magnitude less than is required for cleavage by serum kallikrein or factor XIIa. Cleavage of pro-MSP to MSP was associated with a conformational change in the protein, because the cleaved product, but not pro-MSP, was detected by a sandwich enzyme-linked immunoassay. Cleavage caused the appearance of biological activity, as measured by chemotactic activity of MSP for resident peritoneal macrophages, by MSP-induced macrophage shape change, and by stimulation of macrophage ingestion of C3bi-coated erythrocytes. These findings suggest the possibility of cooperative interactions between NGF-gamma or EGF-BP and pro-MSP in inflammation and wound healing.

Human serum macrophage-stimulating protein (MSP) is a disulfide-linked heterodimer that induces motile and phagocytic activity of mouse resident peritoneal macrophages. In this work, we found that MSP blocked the increase in macrophage nitric oxide synthase mRNA, as well as the associated increase in nitric oxide production, that occurred in response to several stimuli. These included bacterial products and mammalian cytokines: endotoxin, and interferon-gamma plus endotoxin, interleukin-2, or tumor necrosis factor-alpha. The inhibition by MSP of induction of nitric oxide synthase mRNA and nitric oxide secretion was concentration-dependent. The concentration of MSP that caused maximal inhibition of nitric oxide production was comparable with the optimum for stimulation of macrophage motile and phagocytic activity. Time course studies showed that nitrite was first detected in culture fluid about 8 h after endotoxin stimulation, and it accumulated at a linear rate during the ensuing 16 h. Inhibition by MSP occurred during the 8-h lipopolysaccharide (LPS) induction period; inhibition was maximal when MSP and LPS were added together and decreased progressively to no inhibition as the interval between LPS and MSP addition increased to 11 h.

Human serum macrophage stimulating protein (MSP) is a disulfide-linked heterodimer that induces motile and phagocytic activity of mouse resident peritoneal macrophages. It is a member of the family of kringle proteins, which typically exist in extracellular fluid as single chain precursors that are activated by proteolytic cleavage. In this work, we expressed [35S]cysteine-labeled recombinant pro-MSP in MSP cDNA-transfected Chinese hamster ovary cells and studied proteolytic processing of pro-MSP and the requirement of cleavage for biological activity. In media containing heat-inactivated fetal bovine serum, the protein was secreted as single chain pro-MSP, which was cleaved over a period of hours to the mature heterodimer. Cleavage was prevented by serine protease inhibitors such as leupeptin or aprotinin; it did not occur if cells were cultured in serum-free medium. Nanomolar concentrations of coagulation proteases kallikrein, factor XIIa or factor XIa cleaved pro-MSP to MSP within 30 min. Pro-MSP had no biological activity. After cleavage by kallikrein, biological activity was quantitatively comparable to that of natural MSP isolated from human plasma. These results support our hypothesis that MSP circulates as the biologically inactive precursor and can be activated by enzymes of the intrinsic coagulation cascade.

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.

A human hepatoma (HepG2) cell line library was screened with an oligonucleotide probe for macrophage stimulating protein (MSP) to clone an MSP cDNA. Deduced sequences of isolated clones were compared with peptide fragment sequences of MSP. MSP9 cDNA encoded most of the known sequence of MSP except for a small segment of the 5' end of the open reading frame. Consequently, a hybrid 2300-base pair cDNA that encoded the complete MSP amino acid sequence was constructed from 2 clones. Culture fluid from COS-7 cells transfected with this full-length MSP cDNA had MSP biological activity, and the expressed MSP was detected by immunoprecipitation with antibody against native MSP. The deduced amino acid sequence of MSP includes 4 kringle domains, which have been found in hepatocyte growth factor and several proteins of the blood coagulation system. Among them, MSP has the highest sequence similarity to hepatocyte growth factor (45% identity). The MSP cDNA hybridized strongly to mRNA from liver, and to a lesser extent to mRNA from kidney and pancreas, suggesting that a cell type in the liver is the source of MSP. Several cloned and sequenced MSP cDNAs had insertions or deletions, suggesting that alternatively spliced MSP mRNAs may occur. This was reflected in Northern blots probed with an MSP cDNA, which showed more than one mRNA species. Furthermore, although the gene coding for MSP is on chromosome 3, the sequence of one of the cDNAs was identical with a unique sequence in chromosome 1, indicating that there may be a family of MSP genes, located on chromosomes 3 and 1.

We previously reported that a derivative of the interleukin-6 (IL-6)-dependent B9 B-cell hybridoma (B9/LPNU1L) constitutively expressing an interleukin-1 alpha (IL-1 alpha) gene introduced by retrovirus-mediated gene transfer preferentially metastasized to bone marrow following intravenous injection into unirradiated syngeneic BALB/c mice. B9/LPNU1L cells recovered from the femoral marrow of a recipient with hind limb paralysis (denoted B9/BM1) retained their IL-6-dependency yet displayed enhanced metastatic capacity during serial transplantation in vivo. In contrast, autonomously-growing B9 variants spontaneously arising in vitro or IL-6-independent B9 derivatives created by infection with recombinant IL-6 retroviruses rarely gave rise to experimental metastases in syngeneic BALB/c or nude mice. Examination of cell adhesion molecule profiles by immunofluorescence flow cytometry has revealed high levels of CD44, moderate levels of VLA-4 and low levels of LFA-1 on all B9-series cells. By comparison, ICAM-1 expression was significantly elevated on B9/BM1 cells, with independent isolates stably expressing about 4-fold higher levels which were paralleled by corresponding increases in the steady-state levels of ICAM-1 mRNA. L-Selectin was not expressed by any of the cell lines. Despite higher ICAM-1 levels, cell aggregation assays revealed that LFA-1-ICAM-1 adhesive interactions were not involved in the homotypic adhesion of B9/BM1 cells but rather that binding of CD44 to endogenously-synthesized hyaluronan was responsible. Furthermore, B9/BM1 cells expressing high levels of ICAM-1 were found to be less susceptible to cytolysis by natural killer (NK) cells than their weakly metastatic or nonmetastatic counterparts.