RON Receptor Research Articles

The Ron Receptor Tyrosine Kinase Regulates Macrophage Heterogeneity and Plays a Protective Role in Diet-Induced Obesity, Atherosclerosis, and Hepatosteatosis.

Obesity is a chronic inflammatory disease mediated in large part by the activation of inflammatory macrophages. This chronic inflammation underlies a whole host of diseases including atherosclerosis, hepatic steatosis, insulin resistance, type 2 diabetes, and cancer, among others. Macrophages are generally classified as either inflammatory or alternatively activated. Some tissue-resident macrophages are derived from yolk sac erythromyeloid progenitors and fetal liver progenitors that seed tissues during embryogenesis and have the ability to repopulate through local proliferation. These macrophages tend to be anti-inflammatory in nature and are generally involved in tissue remodeling, repair, and homeostasis. Alternatively, during chronic inflammation induced by obesity, bone marrow monocyte-derived macrophages are recruited to inflamed tissues, where they produce proinflammatory cytokines and exacerbate inflammation. The extent to which these two populations of macrophages are plastic in their phenotype remains controversial. We have demonstrated previously that the Ron receptor tyrosine kinase is expressed on tissue-resident macrophages, where it limits inflammatory macrophage activation and promotes a repair phenotype. In this study, we demonstrate that Ron is expressed in a subpopulation of macrophages during chronic inflammation induced by obesity that exhibit a repair phenotype as determined by the expression of arginase 1. In addition, we demonstrate that the Ron receptor plays a protective role in the progression of diet-induced obesity, hepatosteatosis, and atherosclerosis. These results suggest that altering macrophage heterogeneity in vivo could have the potential to alleviate obesity-associated diseases.

The Ron receptor tyrosine kinase regulates inflammatory response in an experimental model of multiple sclerosis

Abstract In the experimental autoimmune encephalitis (EAE) murine model of multiple sclerosis (MS), both central nervous system (CNS)-resident macrophages (microglia) and infiltrating monocyte-derived macrophages contribute to disease progression. The Ron receptor tyrosine kinase is expressed on tissue resident macrophages including microglia and is involved in regulating inflammatory responses. An in vivo deletion of Ron (Ron KO) promotes inflammatory macrophage activation (M1) and limits a reparative macrophage phenotype (M2). Herein we investigated whether Ron expression plays a critical role in regulating disease progression in EAE. Ron KO mice exhibit delayed onset of EAE (day 14) compared to Wild-Type (WT) (day 10), however Ron KO mice exhibit greater peak severity at onset. Ron KO mice display T-cell mediated peripheral inflammation, as demonstrated by the significant increase in the secretion of interferon gamma (IFNγ) but not IL-17 and IL-10. At day 14, cultured lymph node cells from Ron KO mice exhibit increased expression of M1-macrophage mediated biomarkers iNOS, COX-2, IL-6, IL12B, IL1β and TNF-α. A likely cause of this increased inflammatory response can in part be attributed to a T-cell mediated increase in IFNγ. Furthermore, CNS tissues from Ron KO mice at day 14 have increased gene expression of hallmark inflammatory biomarkers iNOS, IL12B and COX-2. The results indicate an interplay between the innate and adaptive immune system in fostering an M1-macrophage mediated inflammatory state, as the underlying factor for the observed increase in disease severity in Ron KO mice. Maintenance of Ron expressing macrophages could then be a potential therapeutic approach to treating MS symptoms.

Ron receptor tyrosine kinase plays a protective role in diet induced obesity and associated steatohepatitis

Abstract Obesity is one of the leading causes of preventable death in the U.S and a well-established risk factor for several pathological conditions including steatohepatitis. The activation of inflammatory tissue resident macrophages has proven to play a key role in the progression of the metabolic syndrome. Consequently, mechanisms that manipulate macrophage phenotype has become of great interest for developing therapeutic strategies for combating obesity and its associated diseases. Our lab has shown that Ron tyrosine kinase, a receptor expressed on tissue resident macrophages, limits an inflammatory response while promoting tissue repair. In this study, we first investigate the impact of Ron signaling on metabolic features preceding steatohepatitis using high fat diet (HFD) fed wild type (WT) and Ron deficient (Ron KO) mice. Ron KO mice demonstrated more severe events associated with obesity such as increased adiposity, exacerbated hyperglycemia and trend toward increased liver weights when compared to WT animals. Altered Ron signaling resulted in a shift in the plasma lipoprotein distribution from primarily VLDL and LDL to predominantly HDL in the WT control mice. To then establish the role of Ron signaling in steatohepatitis, high cholesterol diet (HCD) fed ApoE KO and Ron KO ApoE KO mice (DKO) were evaluated. We showed that DKO mice experience more severe steatohepatitis which is attributed to an increased prevalence of activated inflammatory Kupffer cells. In vitro studies ascertain that Ron manipulation of macrophage phenotype is in part, a result of downstream AMPK activation. These data indicate the role of Ron in attenuating obesity and its associated diseases and identify a novel downstream pathway involved in this process.

Biological evaluation of antibody-maytansinoid conjugates as a strategy of RON targeted drug delivery for treatment of non-small cell lung cancer

BackgroundAberrant expression of the RON receptor tyrosine kinase, a member of the MET proto-oncogene family, in breast cancer and non-small cell lung cancer (NSCLC) has therapeutic implication. Here we evaluated the efficacy of a novel anti-RON antibody-drug maytansinoid conjugate Zt/g4-DM1 for treatment of breast and NSCLC xenograft tumors in mouse models and explored a treatment strategy by combination of Zt/g4-DM1 with chemotherapeutics to achieve the maximal therapeutic activity.MethodsMouse monoclonal antibody Zt/g4 (IgG1a/κ) specific to human RON was conjugated to DM1 via thioether linkage to form Zt/g4-DM1 with a drug-antibody ratio of 4:1. Several breast cancer and NSCLC cell lines, expressing different levels of RON, were used as the model. Immunofluorescence was used to determine Zt/g4-induced RON internalization. Flow cytometric analysis and cell viability assay were used to determine the effect of Zt-g4-DM1 on cell cycle and death. Mouse xenograft NSCLC models were used in vivo to determine the therapeutic efficacy of Zt/g4-DM1 alone or in combination with chemotherapeutics.ResultsIn vitro, Zt/g4 treatment of breast cancer and NSCLC cells rapidly induced cell surface RON internalization, which results in intracellular delivery of DM1 sufficient to arrest cell cycle at G2/M phase, reduce cell viability, and cause massive cell death. In mouse tumor xenograft models, Zt/g4-DM1 at 20 mg/kg in a Q12 × 2 regimen effectively blocked breast cancer and NSCLC cell- mediated tumor growth. More than 95 % inhibition of tumor growth among three tumor xenograft models tested was achieved according to the measured tumor volume. The minimal dose to balance the tumor growth and inhibition (tumoristatic concentration) was established at 2.02 mg/kg for H2228, 1.94 mg/kg for H358 cell, and 6.25 mg/kg for T-47D cell-mediated xenograft tumors.ConclusionZt/g4 is highly effective in RON-directed drug delivery for targeted inhibition of NSCLC cell-derived tumor growth in mouse xenograft models. This work provides the basis for clinical development of humanized Zt/g4-DM1 for potential cancer therapy in the future.

Glycomic and sialoproteomic data of gastric carcinoma cells overexpressing ST3GAL4.

Gastric carcinoma MKN45 cells stably transfected with the full-length ST3GAL4 gene were characterised by glycomic and sialoproteomic analysis. Complementary strategies were applied to assess the glycomic alterations induced by ST3GAL4 overexpression. The N- and O-glycome data were generated in two parallel structural analyzes, based on PGC-ESI-MS/MS. Data on glycan structure identification and relative abundance in ST3GAL4 overexpressing cells and respective mock control are presented. The sialoproteomic analysis based on titanium-dioxide enrichment of sialopeptides with subsequent LC-MS/MS identification was performed. This analysis identified 47 proteins with significantly increased sialylation. The data in this article is associated with the research article published in Biochim Biophys Acta “Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer” [1].

HnRNP A1-mediated translational regulation of the G quadruplex-containing RON receptor tyrosine kinase mRNA linked to tumor progression.

The expression and role of RNA binding proteins (RBPs) controlling mRNA translation during tumor progression remains largely uncharacterized. Analysis by immunohistochemistry of the expression of hnRNP A1, hnRNPH, RBM9/FOX2, SRSF1/ASF/SF2, SRSF2/SC35, SRSF3/SRp20, SRSF7/9G8 in breast tumors shows that the expression of hnRNP A1, but not the other tested RBPs, is associated with metastatic relapse. Strikingly, hnRNP A1, a nuclear splicing regulator, is also present in the cytoplasm of tumor cells of a subset of patients displaying exceedingly worse prognosis. Expression of a cytoplasmic mutant of hnRNP A1 leads to increased translation of the mRNA encoding the tyrosine kinase receptor RON/MTS1R, known for its function in tumor dissemination, and increases cell migration in vitro. hnRNP A1 directly binds to the 5′ untranslated region of the RON mRNA and activates its translation through G-quadruplex RNA secondary structures. The correlation between hnRNP A1 and RON tumoral expression suggests that these findings hold clinical relevance.

Crosstalk between RON and androgen receptor signaling in the development of castration resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) is the fatal form of prostate cancer. Although reactivation of androgen receptor (AR) occurs following androgen deprivation, the precise mechanism involved is unclear. Here we show that the receptor tyrosine kinase, RON alters mechanical properties of cells to influence epithelial to mesenchymal transition and functions as a transcription factor to differentially regulate AR signaling. RON inhibits AR activation and subset of AR-regulated transcripts in androgen responsive LNCaP cells. However in C4-2B, a castrate-resistant sub-line of LNCaP and AR-negative androgen independent DU145 cells, RON activates subset of AR-regulated transcripts. Expression of AR in PC-3 cells leads to activation of RON under androgen deprivation but not under androgen proficient conditions implicating a role for RON in androgen independence. Consistently, RON expression is significantly elevated in castrate resistant prostate tumors. Taken together our results suggest that RON activation could aid in promoting androgen independence and that inhibition of RON in combination with AR antagonist(s) merits serious consideration as a therapeutic option during hormone deprivation therapy.

Abstract B41: HGFL-Ron signaling enhances breast cancer stem cell populations

Abstract The Ron receptor tyrosine kinase is overexpressed in a high fraction of human breast cancers (BCs). Ron and its ligand, the Hepatocyte Growth Factor-Like protein (HGFL), play important roles during BC initiation, progression, and therapeutic resistance, with overexpression of this signaling cascade associated with increased BC metastasis and poor prognosis in humans. Reports demonstrate that a small subpopulation of cancer cells, called the breast cancer stem cells (BCSCs), are essential for the development and maintenance of BC, leading to tumor formation and recurrence. Despite the role of HGFL-Ron signaling as a key regulator of BC, nothing is known about this signaling pathway in BCSC populations. To investigate the function of HGFL-Ron signaling in BCSCs, the expression levels of Ron and HGFL were analyzed in BCSCs. mRNA analyses show increased levels of Ron and HGFL in BCSC-enriched Mammospheres (MSs) compared to parental BC epithelial cells. Next, the association between Ron signaling and the BCSC population was examined. Aldefluor assays of several Ron replete and depleted human and murine BC epithelial cell lines show that Ron overexpression increases the BCSC population. In vivo, flow cytometry analyses of breast tumors for BCSC markers show that Ron signaling is associated with increases in the BCSC population. MS formation assays using several Ron replete and depleted human and murine BC cell lines also demonstrate that cells expressing high levels of Ron have increased MS formation. Ex vivo studies of sorted BCSCs further support that Ron signaling enhances the MS formation ability of BCSCs. Interestingly, our data suggest that both genetic ablation and chemical inhibition of Ron signaling is sufficient to decrease BCSC MS formation ability. Mammosphere culture over several passages also suggest that Ron signaling BCSCs have higher self-renewal capabilities than Ron deficient BCSCs, highlighting the role of HGFL-Ron signaling in regulating BCSC function. Recent preliminary studies correlate Ron signaling in BCSCs with increased β-catenin and NF-κB pathways, suggesting these as the potential molecular mechanisms through which Ron signaling promotes BCSC function in BC. Overall, our data provide (i) the first evidence demonstrating both HGFL and Ron as critical factors regulating the BCSC population and (ii) that this regulation might occur in a β-catenin and NF-κB pathway dependent manner. These studies suggest both Ron and HGFL as potential therapeutic targets to eradicate BCSCs and improve the outcome of BC patients. Citation Format: Sasha J. Ruiz-Torres, Susan E. Waltz. HGFL-Ron signaling enhances breast cancer stem cell populations. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B41.

Abstract A20: Short-form Ron kinase as a novel therapeutic target in ovarian cancer.

Abstract Background: Although 70–80% of women respond to standard platinum-based chemotherapy, a majority of patients will develop recurrent platinum-resistant disease. Novel treatment approaches directed against key oncogenic drivers and/or the use of novel drug combinations to prevent/overcome platinum resistance are promising opportunities to combat this disease. Our lab has developed a strong interest in short-form Ron (sfRon) in the context of ovarian cancer pathogenesis. sfRon is an understudied, alternative isoform of the full-length Ron receptor tyrosine kinase, the latter of which has been investigated in cancer for several years. Unlike full-length Ron, the sfRon protein lacks the extracellular ligand-binding domain, but organizes into a constitutively-active transmembrane protein. Our in vitro and in vivo studies indicate that sfRon expression has more potent oncogenic activity than full-length Ron, presumably because of the constitutive kinase activity of the sfRon protein. Such data carries additional importance because sfRon does not require ligand binding for activity, appears to function in a manner distinct from full length Ron, and may not be targetable using standard approaches such as antibody inhibitors (e.g. RON8, which is now being tested in clinical trials). Instead, our data suggest that inhibition of Ron kinase activity, which would block both Ron and sfRon, might have great therapeutic benefit in ovarian tumors that express sfRon. Results: Our lab has recently made the striking and unexpected discovery that sfRon expression is necessary for development of carcinogen-driven ovarian tumors in mice. Our study involved treating wild type mice vs. mice with a specific genetic deletion of sfRon (ΔsfRon) with DMBA, a known carcinogen. The study revealed that lack of sfRon completely protected the mice from ovarian cancer. The frequency of ovarian tumors was 25% in wild-type mice vs. 0% in ΔsfRon mice (p<0.0011). To our knowledge, this study is the first to show the importance of sfRon expression in development of ovarian tumors. An increased understanding of how sfRon contributes to ovarian tumor development and progression is critical. Based on these findings supporting the role of sfRon in ovarian cancer, we aimed to investigate the expression of sfRon in primary ovarian samples. Our studies using ovarian tumors from patients revealed that sfRon becomes overexpressed in several different subtypes of ovarian cancer, with no expression in healthy ovarian tissue. Our studies revealed that introduction of sfRon into OVCAR-3 cells resulted in increased proliferation of tumor cells in vitro, and accelerated tumor growth in vivo as compared with parental OVCAR-3 cells. Our further studies aimed to investigate the potential clinical relevance of total Ron expression (Ron and sfRon) in a large cohort of high-grade serous ovarian cancer (HG-SOC) patients, with a particular focus on response to platinum drugs. Our data demonstrated that in the population of HG-SOC patients, Ron and/or sfRon are expressed in 86/126 (68%) of all cases, and high expression of Ron and/or sfRon was detected in 27/126 (21%) of all cases. Importantly, we also observed that significantly more patients in the cisplatin refractory/resistant group express high levels of Ron receptors (Ron and sfRon), compared to patients in the cisplatin sensitive group. Conclusion: Our data implicate sfRon in ovarian cancer pathogenesis, and indicate that Ron kinase inhibitors targeting both Ron and sfRon may not only suppress ovarian tumor progression, but also have potential to overcome resistance to chemotherapy. These data could have significant impact on future clinical trials for both Ron kinase inhibitors and platinum compounds, which has high potential to impact patients in the short-term. Citation Format: Magdalena Bieniasz, Alana L. Welm. Short-form Ron kinase as a novel therapeutic target in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A20.

RON Signaling Is a Key Mediator of Tumor Progression in Many Human Cancers

With an increasing body of literature covering RON receptor tyrosine kinase function in different types of human cancers, it is becoming clear that RON has prominent roles in both cancer cells and in the tumor-associated microenvironment. RON not only activates several oncogenic signaling pathways in cancer cells, leading to more aggressive behavior, but also promotes an immunosuppressive, alternatively activated phenotype in macrophages and limits the antitumor immune response. These two unique functions of this oncogene, the strong correlation between RON expression and poor outcomes in cancer, and the high tolerability of a new RON inhibitor make it an exciting therapeutic target, the blocking of which offers an advantage toward improving the survival of cancer patients. Here, we discuss recent findings on the role of RON signaling in cancer progression and its potential in cancer therapy.

Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer

BackgroundTerminal α2-3 and α2-6 sialylation of glycans precludes further chain elongation, leading to the biosynthesis of cancer relevant epitopes such as sialyl-Lewis X (SLeX). SLeX overexpression is associated with tumor aggressive phenotype and patients' poor prognosis. MethodsMKN45 gastric carcinoma cells transfected with the sialyltransferase ST3GAL4 were established as a model overexpressing sialylated terminal glycans. We have evaluated at the structural level the glycome and the sialoproteome of this gastric cancer cell line applying liquid chromatography and mass spectrometry. We further validated an identified target expression by proximity ligation assay in gastric tumors. ResultsOur results showed that ST3GAL4 overexpression leads to several glycosylation alterations, including reduced O-glycan extension and decreased bisected and increased branched N-glycans. A shift from α2-6 towards α2-3 linked sialylated N-glycans was also observed. Sialoproteomic analysis further identified 47 proteins with significantly increased sialylated N-glycans. These included integrins, insulin receptor, carcinoembryonic antigens and RON receptor tyrosine kinase, which are proteins known to be key players in malignancy. Further analysis of RON confirmed its modification with SLeX and the concomitant activation. SLeX and RON co-expression was validated in gastric tumors. ConclusionThe overexpression of ST3GAL4 interferes with the overall glycophenotype of cancer cells affecting a multitude of key proteins involved in malignancy. Aberrant glycosylation of the RON receptor was shown as an alternative mechanism of oncogenic activation. General significanceThis study provides novel targets and points to an integrative tumor glycomic/proteomic-profiling for gastric cancer patients' stratification. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells.

The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. The splice variant is detected in 73% of pancreatic adenocarcinoma patient derived xenografts and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirms translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing (HPNE) cells activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target.

Abstract 789: CT413 is a novel dual Axl/Mer inhibitor that potently inhibited the growth of Axl overexpressed tumors

Abstract Receptor tyrosine kinases (RTKs), which play an important role in cell signal transduction, are proven therapeutic targets for the treatment of human cancers. Axl and Mer are members of the TAM family which also includes Tyro-3. Axl has been established as a strong drug target candidate for therapeutic inhibition of cancer invasion and dissemination. As metastatic disease is the most frequent cause of cancer patient mortality, therapeutics specifically aimed at inhibiting metastatic dissemination and colonization would be important additions to the arsenal of drugs in clinical use. Some FDA-approved or in clinical trial molecules block Axl and Mer activity, however, there is no inhibitor clearly designed to inhibit Axl and Mer on the market. As a potential new therapy against malignant tumor, the development of Axl and Mer dual inhibitors is of great significance. CT413 is a novel, orally bioavailable Axl/Mer inhibitor that is currently in our preclinical development pipeline. In biochemical assay, CT413 potently inhibit Axl and Mer with respective IC50 values of 4 and < 1 nM. Among 97 kinase screened, CT413 inhibited Met and Ron receptor tyrosine kinases with IC50 values of 3 and 9 nM, respectively. Furthermore, CT413 blocked the Axl, Met and Ron autophosphorylation in cells with IC50 values of 13, 34 and 540 nM, respectively, demonstrating high kinase selectivity in both biochemical and cell-based assays. CT413 potently suppressed tumor growth in BALB/cA-nu/nu mice bearing subcutaneous human tumor xenografts. For example, in A549 (NSCLC), SK-OV-3 (ovarian) and K562 (CML) tumor models, CT413 inhibited tumor growth with TGIs of 84%, 110% and 66% dosed orally at 30 mg/kg QD, as compared to the vehicle-treated group. CT413 did not inhibit CYP450 subtype 3A4, 2D6, 1A2, 2C9 and 2C19 (IC50 > 10 μM) and showed no induction effect on CYP450 subtype 1A2, 2B6 and 3A4. Furthermore, the IC50 of CT413 at hERG sodium ion channel was determined to be greater than 30 μM, indicating its minimal cardio-liability. CT413 exhibited good metabolic stability when incubated with human or rat microsome. Pharmacokinetic studies performed in rodents and large animals showed that CT413 had low clearance and high oral exposures across animal species. The oral bioavailability for mice, rats, dogs and monkeys are 133%, 85%, 83% and 68% respectively. The non-GLP 28-day toxicity studies in rats and dogs indicated that CT413 has an excellent therapeutic window. Based on these promising results, CT413 is advanced in our project pipeline as a clinical candidate for cancer therapy. Citation Format: Ning Xi, Tingjin Wang, Yanjun Wu, Min Liao, Yanming Feng, Ning Kang, Zhaohe Wang, Yingjun Zhang. CT413 is a novel dual Axl/Mer inhibitor that potently inhibited the growth of Axl overexpressed tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2015-789

Design, synthesis and RON receptor tyrosine kinase inhibitory activity of new head groups analogs of LCRF-0004.

New heteroarylcarboxamide head groups substituted with two aromatic rings analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Potent inhibitors of RON tyrosine kinase with various level of selectivity for c-Met RTK were obtained.

Vitamin D3-dependent VDR signaling delays ron-mediated breast tumorigenesis through suppression of β-catenin activity.

The Ron receptor is upregulated in human breast cancers and correlates with enhanced metastasis and reduced patient survival. Ron overexpression drives mammary tumorigenesis through direct β-catenin activation and augmented tumor cell proliferation, migration and invasion. Ron and β-catenin are also coordinately elevated in breast cancers. The vitamin D receptor (VDR) antagonizes β-catenin signaling. Herein, we examined mammary tumor onset and progression using a Ron-driven murine model of breast tumorigenesis crossed with VDR deficient mice. VDR ablation accelerated mammary tumor onset and led to tumors that exhibited a desmoplastic phenotype and enhanced metastases. Tumor levels of active β-catenin were markedly increased in the absence of VDR. In vitro, VDR activation in breast cancer cells reduced β-catenin activation and transcriptional activity leading to elevated expression of the extracellular Wnt inhibitor dickkopf-related protein 1, and a reduction in the interaction of β-catenin with the cyclin D1 promoter. Expression of a stabilized form or β-catenin ablated the protective effects of VDR activation.Collectively, these studies delineate a protective role for VDR signaling in Ron-induced mammary tumorigenesis through disruption of β-catenin activation.

Design and synthesis of close analogs of LCRF-0004, a potent and selective RON receptor tyrosine kinase inhibitor.

New carboxamide head group analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Potent and selective inhibitors of RON enzyme versus c-Met RTK were obtained.

HGFL supports mammary tumorigenesis by enhancing tumor cell intrinsic survival and influencing macrophage and T-cell responses.

The Ron receptor is overexpressed in human breast cancers and is associated with heightened metastasis and poor survival. Ron overexpression in the mammary epithelium of mice is sufficient to induce aggressive mammary tumors with a high degree of metastasis. Despite the well-documented role of Ron in breast cancer, few studies have examined the necessity of the endogenous Ron ligand, hepatocyte growth factor-like protein (HGFL) in mammary tumorigenesis. Herein, mammary tumor growth and metastasis were examined in mice overexpressing Ron in the mammary epithelium with or without HGFL. HGFL ablation decreased oncogenic Ron activation and delayed mammary tumor initiation. HGFL was important for tumor cell proliferation and survival. HGFL loss resulted in increased numbers of macrophages and T-cells within the tumor. T-cell proliferation and cytotoxicity dramatically increased in HGFL deficient mice. Biochemical analysis of HGFL proficient tumors showed increased local HGFL production, with HGFL loss decreasing β-catenin expression and NF-κB activation. Re-expression of HGFL in HGFL deficient tumor cells stimulated cell migration and invasion with coordinate activation of NF-κB and reduced apoptosis. Together, these results demonstrate critical in vivo functions for HGFL in promoting breast tumorigenesis and suggest that targeting HGFL may inhibit tumor growth and reactivate anti-tumor immune responses.

Loss of Ron receptor signaling leads to reduced obesity, diabetic phenotypes and hepatic steatosis in response to high-fat diet in mice.

The Ron receptor tyrosine kinase is a heterodimeric, membrane-spanning glycoprotein that participates in divergent processes, including proliferation, motility, and modulation of inflammatory responses. We observed male C57BL/6 mice with a global deletion of the Ron tyrosine kinase signaling domain (TK(-/-)) to be leaner compared with control (TK(+/+)) mice under a standard diet. When fed a high-fat diet (HFD), TK(-/-) mice gained 50% less weight and were more insulin sensitive and glucose tolerant than controls. Livers from HFD TK(-/-) mice were considerably less steatotic and weighed significantly less than TK(+/+) livers. Serum cytokine levels of HFD TK(-/-) mice were also significantly altered compared with TK(+/+) mice. Fewer and smaller adipocytes were present in the TK(-/-) mice on both control and HFD and were accompanied by diminished adiponectin and peroxisome proliferator-activated receptor-γ expression. In vitro adipogenesis experiments suggested reduced differentiation in TK(-/-) embryonic fibroblasts (MEFs) that was rescued by Ron reconstitution. Likewise, signal transducer and activator of transcription (STAT)-3 phosphorylation was diminished in TK(-/-) MEFs but was increased after Ron reconstitution. The adipogenic inhibitors, preadipocyte factor 1 and Sox9, were elevated in TK(-/-) MEFs and increased in both groups after STAT3 silencing. In total, these studies document a previously unknown function for the Ron receptor in mediating HFD-induced obesity and metabolic dysregulation.

Variant RONΔ160 of the RON receptor tyrosine kinase promotes the growth and invasion in vitro and in vivo in gastric cancer cell lines.

BackgroundRecepteur d’origine nantais (RON) is a receptor tyrosine kinase whose overexpression has been observed in human gastric cancers. This study aimed to determine whether overexpression of the variant RONΔ160 could induce tumorigenicity of gastric cancer cells in vitro or in vivo, and whether its specific small molecule inhibitor (Compound I) could inhibit the effect of RONΔ160.MethodsWe constructed human gastric cancer cell line MGC-803 that was stably transfected with a recombinant plasmid expressing RONΔ160, and the effect of RONΔ160 overexpression and macrophage-stimulating protein (MSP) activation on proliferation, migration and invasion abilities of MGC-803 cells were evaluated. Tumor-bearing mice with gastric cancer cells were used to analyze the effects of RONΔ160 overexpression and Compound I on implanted tumor growth.ResultsIn vitro, overexpression of RONΔ160 in MGC-803 cells resulted changes to their cell morphology, and promoted cell proliferation, migration and invasion. In addition, overexpression of RONΔ160 increased the proportion of cells in the S phase. The effect of RONΔ160 was significantly enhanced by induction of MSP inducing (p < 0.05). In vivo, RONΔ160 promoted the growth of MGC-803 cells in nude mice, including increased tumor size and weight, and lower tumor incubation period. The Compound I inhibited the tumorigenic abilities of RONΔ160 (p <0.05).ConclusionsThe results indicate that overexpression of the variant RONΔ160 altered the phenotype and tumorigenicity of MGC-803 cells. Its specific small molecule inhibitor could inhibit the effect of RONΔ160. Therefore, the variant RONΔ160 may become a potential therapeutic target for gastric cancer.

Identification of Regulatory-RNAs for Alternative Splicing of Ron Proto-Oncogene.

RON receptor tyrosine kinase is a proto-oncogene that induces cell migration and matrix invasion. RONΔ160 protein, which is produced by exclusion of exon 5 and 6, promotes cell migration, matrix invasion and protection from apoptosis. Alternative splicing regulation of exon 5 and 6 is not well understood. In this manuscript, we identified several new RNA regulatory elements for alternative splicing of Ron proto-oncogene. Firstly, we demonstrated that RNA sequences from EcoRI cleavage sites regulate alternative splicing of Ron exon 5 and 6. Secondly, we showed that the ~30 nt RNA at upstream end of exon 4 and the ~33 nt RNA at downstream end of exon 7 also modulate splicing of exon 5 and 6. Thirdly, our results indicate that the RNA sequences of the ends in exon 4 and 7 are required for the regulatory functions of the RNA from restriction enzyme cleavage sites. Our results provide a new insight for regulation of alternative splicing of Ron proto-oncogene.