Recepteur D'origine Nantais Research Articles

Human immunodeficiency virus (HIV)-1 Tat downregulates the phosphorylation of recepteur d'origine nantais (RON) receptor tyrosine kinase induced by macrophage-stimulating protein

The aim of this study is to examine the effects of the human immunodeficiency virus (HIV)-1 Tat protein on the expression and phosphorylation of recepteur d'origine nantais (RON) receptor tyrosine kinase and the mechanisms involved. We first determined the expression levels of RON and macrophagestimulating protein (MSP) were determined in the peripheral blood of HIV-positive patients and control subjects. The 293T cells were transfected with the pDR2-RON plasmid to establish the 293T-RON cell line. The pcDNA3.1 (+)-Tat-His plasmid was constructed to obtain recombinant Tat protein. The 293TRON cells pretreated with MSP were co-cultured with the H9/HTLV-IIIB cells or were stimulated with the Tat protein; then, the change of RON expression was determined. This study revealed higher expression of RON and relatively lower expression of MSP in HIV-infected patients than in the uninfected patients. We demonstrated that HIV-1 Tat down-regulated the expression and phosphorylation of RON in 293T-RON cells. Flow cytometry analysis of 293T-RON cells revealed that RON was expressed in 293T-RON cells. The 293T-RON cells co-cultured with the H9/HTLV-IIIB cells RON expression did not change significantly, while stimulation with 500 ng/ml Tat considerably decreased RON expression in 293T-RON cells. These findings imply that Tat may play a role in modulating the function of RON, which is activated by MSP; this may provide a permissive environment for both HIV and other opportunistic microbes.

Human immunodeficiency virus (HIV)-1 Tat downregulates the phosphorylation of recepteur d'origine nantais (RON) receptor tyrosine kinase induced by macrophage-stimulating protein

The aim of this study is to examine the effects of the human immunodeficiency virus (HIV)-1 Tat protein on the expression and phosphorylation of recepteur d'origine nantais (RON) receptor tyrosine kinase and the mechanisms involved. We first determined the expression levels of RON and macrophagestimulating protein (MSP) were determined in the peripheral blood of HIV-positive patients and control subjects. The 293T cells were transfected with the pDR2-RON plasmid to establish the 293T-RON cell line. The pcDNA3.1 (+)-Tat-His plasmid was constructed to obtain recombinant Tat protein. The 293TRON cells pretreated with MSP were co-cultured with the H9/HTLV-IIIB cells or were stimulated with the Tat protein; then, the change of RON expression was determined. This study revealed higher expression of RON and relatively lower expression of MSP in HIV-infected patients than in the uninfected patients. We demonstrated that HIV-1 Tat down-regulated the expression and phosphorylation of RON in 293T-RON cells. Flow cytometry analysis of 293T-RON cells revealed that RON was expressed in 293T-RON cells. The 293T-RON cells co-cultured with the H9/HTLV-IIIB cells RON expression did not change significantly, while stimulation with 500 ng/ml Tat considerably decreased RON expression in 293T-RON cells. These findings imply that Tat may play a role in modulating the function of RON, which is activated by MSP; this may provide a permissive environment for both HIV and other opportunistic microbes.

Receptor tyrosine kinase pathway analysis sheds light on similarities between clear‐cell sarcoma and metastatic melanoma

To highlight possible similarities and differences in receptor tyrosine kinase (RTK) and downstream signalling activation profiles between clear-cell sarcomas (CCS) and metastatic melanomas (MM), frozen, and paired-matched fixed samples of six CCS with EWSR1 rearrangement (EWSR1+), five CCS without EWSR1 rearrangement (EWSR1-), and seven MM were investigated by means of biochemical, immunohistochemical, FISH, molecular analyses, and immunofluorescence confocal microscopy. Fixed samples of a further 10 CCS and 14 MM were investigated by means of sequencing for BRAF, NRAS, and KRAS mutations and FISH analyses for the gain of chromosomes 22 and 8. RTK analysis of all CCS/MM samples showed activation of short-form (sf) recepteur d'origine nantais (RON) RTK and of PDGFRB, MET, and HER3. Analysis of downstream signaling revealed consistent phosphorylation patterns of PI3K/AKT, RSK, and the mTOR targets S6 and 4EBP1. Analysis of frozen and fixed material from 21 CCS and 21 MM showed the presence of the V600E BRAF mutation in 2/12 EWSR1+ and 3/9 EWSR1- CCS and 9/21 MM and demonstrated a significant (P < 0.001) correlation between the gain of chromosomes 22 and 8 and EWSR1- CCS. Our results show that BRAF mutation can also be present in CCS and support the proposed aberration of chromosomes 22 and 8 as a possibly useful nonrandom hallmark of EWSR1- CCS. Besides, they broaden the spectrum of the similarities of RTK pathway activation between CCS and MM, thus suggesting that new drugs found to be active in melanoma and RON inhibitors could have a role in CCS treatment. © 2011 Wiley Periodicals, Inc.

Splicing factor hnRNPH drives an oncogenic splicing switch in gliomas.

In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-α/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur d'Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16- or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness.

Knockdown of RON Inhibits AP-1 Activity and Induces Apoptosis and Cell Cycle Arrest Through the Modulation of Akt/FoxO Signaling in Human Colorectal Cancer Cells

Altered Recepteur d'Origine nantais (RON) expression transduces signals inducting invasive growth phenotype that includes cell proliferation, migration, matrix invasion, and protection of apoptosis in human cancer cells. The aims of the current study were to evaluate whether RON affects tumor cell behavior and cellular signaling pathways including activator protein-1 (AP-1) and Akt/forkhead box O (FoxO) in human colorectal cancer cells. To study the biological role of RON on tumor cell behavior and cellular signaling pathways in human colorectal cancer, we used small interfering RNA (siRNA) to knockdown RON gene expression in human colorectal cancer cell line, DKO-1. Knockdown of RON diminished migration, invasion, and proliferation of human colorectal cancer cells. Knockdown of RON decreased AP-1 transcriptional activity and expression of AP-1 target genes. Knockdown of RON activated cleaved caspase-3, -7, -9, and PARP, and down-regulated the expression of Mcl-1, survivin and XIAP, leading to induction of apoptosis. Knockdown of RON induced cell cycle arrest in the G2/M phase of cancer cells by an increase of p27 and a decrease of cyclin D3. Knockdown of RON inhibited the phosphorylation of Akt/FoxO signaling proteins such as Ser473 and Thr308 of Akt and FoxO1/3a. These results indicate that knockdown of RON inhibits AP-1 activity and induces apoptosis and cell cycle arrest through the modulation of Akt/FoxO signaling in human colorectal cancer cells.

Novel Function of Transcription Factor Nrf2 as an Inhibitor of RON Tyrosine Kinase Receptor-mediated Cancer Cell Invasion

Recepteur d' origine nantais (RON), a tyrosine kinase receptor, is aberrantly expressed in human tumors and promotes cancer cell invasion. RON receptor activation is also associated with resistance to tamoxifen treatment in breast cancer cells. Nrf2 is a positive regulator of cytoprotective genes. Using chromatin immunoprecipitation (ChIP) and site-directed mutagenesis studies of the RON promoter, we identified Nrf2 as a negative regulator of RON gene expression. High Nrf2 and low RON expression was observed in normal mammary tissue whereas high RON and low or undetectable expression of Nrf2 was observed in breast tumors. The Nrf2 inducer sulforaphane (SFN) as well as ectopic Nrf2 expression or knock-down of the Nrf2 negative regulator keap1, which stabilizes Nrf2, inhibited RON expression and invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" and RON kinase as a molecular target of SFN, which mediates the anti-tumor effects of SFN. These results are not limited to breast cancer cells since the Nrf2 inducer SFN stabilized Nrf2 and inhibited RON expression in carcinoma cells from various tumor types.

Small interfering RNA-directed targeting of RON alters invasive and oncogenic phenotypes of human hepatocellular carcinoma cells

The recepteur d'origine nantais (RON) receptor tyrosine kinase is highly expressed in various cancers including human hepatocellular carcinoma (HCC) and involved in tumor progression. The aims of the current study were to evaluate whether RON affects tumor cell behavior and oncogenic signaling cascades in HCC cells. We investigated the biologic role of RON on tumor cell behavior and oncogenic signaling cascades including Akt, c-Raf and extracellular signal-regulated kinase (ERK) by using the small interfering RNA (siRNA) in HCC cell lines, chang, HepG2 and Huh7. Knockdown of RON suppressed tumor cell migration and invasion in all tested HCC cell lines. The proportion of apoptotic cells induced by knockdown of RON was greater than that induced by transfection of the scramble siRNA in all tested HCC cell lines. Knockdown of RON resulted in cell cycle arrest in the G2/M phase of chang and Huh7 cells, and sub G1 phase of HepG2 cells. Knockdown of RON activated cleaved caspase-3 and PARP, and down-regulated the expression of Bcl-2, Bcl-xL and survivin, leading to induction of apoptosis in all tested cell lines. Knockdown of RON negatively regulates the progression of the cell cycle by decreasing cyclin D1 and D3, and increasing p21 and p27 in all tested cell lines. The phosphorylation of Akt, c-Raf and ERK1/2 signal proteins was significantly blocked by knockdown of RON in all tested cell lines. These results suggest that RON is associated with invasive and oncogenic phenotypes such as tumor cell migration, invasion, resistance to apoptosis and cell cycle arrest through the modulation of Akt, c-Raf and ERK signaling cascades in HCC cells.

Requirement for LMP1-induced RON receptor tyrosine kinase in Epstein-Barr virus–mediated B-cell proliferation

AbstractEBV, an oncogenic human herpesvirus, can transform primary B lymphocytes into immortalized lymphoblastoid cell lines (LCLs) through multiple regulatory mechanisms. However, the involvement of protein tyrosine kinases in the infinite proliferation of B cells is not clear. In this study, we performed kinase display assays to investigate this subject and identified a specific cellular target, Recepteur d'Origine Nantais (RON) tyrosine kinase, expressed in LCLs but not in primary B cells. Furthermore, we found that latent membrane protein 1 (LMP1), an important EBV oncogenic protein, enhanced RON expression through its C-terminal activation region-1 (CTAR1) by promoting NF-κB binding to the RON promoter. RON knockdown decreased the proliferation of LCLs, and transfection with RON compensated for the growth inhibition caused by knockdown of LMP1. Immunohistochemical analysis revealed a correlation between LMP1 and RON expression in biopsies from posttransplantation lymphoproliferative disorder (PTLD), suggesting that LMP1-induced RON expression not only is essential for the growth of LCLs but also may contribute to the pathogenesis of EBV-associated PTLD. Our study is the first to reveal the impact of RON on the proliferation of transformed B cells and to suggest that RON may be a novel therapeutic target for EBV-associated lymphoproliferative diseases.

MSP-induced RON activation upregulates uPAR expression and cell invasiveness via MAPK, AP-1 and NF- B signals in gastric cancer cells

Overexpression of recepteur d'Origine nantais (RON) and urokinase plasminogen activator receptor (uPAR) have been observed in human gastric cancers. However, the interaction between RON and uPAR in gastric cancer is unclear. The present study investigated the effect of macrophage-stimulating protein (MSP, the RON ligand) on uPAR expression and the underlying signal pathways in human gastric cancer AGS cells. uPAR messenger RNA expression was induced by MSP in a time- and concentration-dependent manner. MSP also induced uPAR promoter activity. The introduction of RON-specific small interfering RNA (siRNA) significantly affected the MSP-induced uPAR transcription. Deleted and site-directed mutagenesis studies demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the MSP-induced uPAR expression. Studies with expression vectors encoding mutated-type NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the MSP-induced uPAR expression. In addition, MSP induced the activation of extracellular signal-regulated kinase-1/2 (Erk-1/2), c-Jun amino terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Dominant-negative mutants (K97M and TAM67) and specific inhibitors of Erk-1/2 and JNK were able to suppress the MSP-induced uPAR expression. AGS cells pretreated with MSP showed a remarkably enhanced invasiveness, which was partially abrogated by siRNA-targeted RON and uPAR-neutralizing antibodies. The above results suggest that MSP induces uPAR expression via MAPK, AP-1 and NF-κB signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

Small interfering RNA targeting of Recepteur d'Origine Nantais induces apoptosis via modulation of nuclear factor-κB and Bcl-2 family in gastric cancer cells

The abnormal accumulation and activation of the receptor tyrosine kinase, Recepteur d'Origine Nantais (RON), has been implicated in tumorigenesis and metastasis in epithelial tumors including gastric cancer. This study examined whether the sequence-specific small interfering RNA (siRNA) suppression of the RON expression could induce apoptotic cell death, and investigated the involved molecular mechanisms. Sequence-specific siRNA effectively suppressed the RON expression at both the mRNA and protein levels. Silencing of the RON expression significantly inhibited gastric cancer cell proliferation and induced apoptosis in a time-dependent manner. The induction of apoptosis was confirmed by the ladder-patterned DNA fragmentation, the presence of cleaved and condensed nuclear chromatin and the increased number of annexin V-positive cells. RON-targeted siRNA effectively inhibited the constitutive nuclear factor-kappaB (NF-kappaB) activation as revealed by an altered electrophoretic mobility shift. In agreement with this, silencing of the RON expression resulted in a decrease in the nuclear level of the p65 subunit of NF-kappaB. The transfection of siRNA, which blocked the RON expression, also caused a change in the ratio of Bax/Bcl-2 in a manner that favored apoptosis. The siRNA silencing of RON induced cytochrome c release and the activation of caspase-8 and caspase-9. These results indicate that RON-targeted siRNA could be therapeutically efficacious by inducing cell apoptosis through the modulation of the NF-kappaB and Bcl-2 family in gastric cancer cells.

Therapeutic effects of the Sp1 inhibitor mithramycin A in glioblastoma

Mithramycin A (MitA) is a chemotherapeutic compound which has been used in the therapy of several types of cancer. For experimental cancer it has been shown that MitA mediates the expression of genes involved in tumor progression such as genes involved in immunosurveillance, cell motility or cell death. MitA works synergistically with Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and with antiangiogenic agents. We were therefore interested in analyzing whether MitA might be a suitable agent for glioma therapy. We demonstrate herein that the cell death sensitizing effects of MitA are cell line specific, independent of the endogenous status of the tumor suppressor p53 as well as of the endogenous expression of X-linked inhibitor of apoptosis (XIAP) or basal sensitivity towards death ligand-induced cell death. In glioma cells, MitA reduced the secretion and activity of the migration-involved matrix metalloproteinases (MMP), diminished vascular endothelial growth factor (VEGF), and increased recepteur d'origine nantais (RON) kinase messenger RNA (mRNA), paralleled by a significant reduction of glioma cell migration. In contrast to other cancer types, in glioma cells MitA did not alter the expression of the immunorelevant genes major histocompatibility complex I class related (MIC)-A, MIC-B or UL16 binding proteins (ULBP). We conclude that, whereas MitA-mediated reduction of XIAP expression and sensitization to Apo2L/TRAIL are cell line specific, its antimigratory effects are more general and might be the result of altered expression of MMP, VEGF, and/or RON kinase. Therefore, MitA might be a potential agent to reduce glioma cell migration.

RON and Cisplatin Resistance in Ovarian Cancer Cell Lines

RON (recepteur d'origine nantais) tyrosine kinase receptor has revealed its tumorigenic potential in recent studies. RON was reported to be overexpressed in 55% of primary ovarian carcinoma samples and furthermore its activation increases cell motility and invasiveness. In this study, we investigated the correlation between RON expression and chemoresistance in ovarian cancer cells. In A2780 cells, a model featured by high chemosensitivity to cisplatin, stable overexpression of RON was able to reduce sensitivity to this agent, while incubation with a blocking anti-RON antibody (ID1) increased the cisplatin-induced growth inhibition effect. Moreover, we observed an increased RON expression both at the mRNA and protein level in A2780 cells made resistant to doxorubicin and paclitaxel (A2780ADR and TC 1, respectively), two cell lines exhibiting a collateral resistance to cisplatin. OVCAR-3 cells, showing high levels of RON expression, also displayed inherent cisplatin resistance. The morphology observed in these resistant cells is consistent with a scattering phenotype and a RON-activated state. RON expression levels were monitored upon hypoxia. A 2.5-fold increase of RON expression was noticed in response to hypoxia in OVCAR-3 cells, in parallel with a decrease of E-cadherin mRNA. Altogether these results suggest an involvement of RON in the acquisition of cisplatin resistance and highlight the importance of this factor as a promising target for combination with cisplatin-based chemotherapy in ovarian cancer.

Transactivation of RON receptor tyrosine kinase by interaction with PDGF receptor β during steady-state growth of human mesangial cells

Although it is well known that platelet-derived growth factor (PDGF) causes mesangial cell proliferation (presumably contributing to progression of glomerular disease), targeted inhibition of the PDGF receptor system has shown only limited efficacy against glomerular diseases. To examine whether this discrepancy is due to the involvement of other pathways, we used phosphorylated receptor tyrosine kinase arrays and found that RON (recepteur d'origine nantais) was phosphorylated while the PDGF receptor was dephosphorylated (thus inactive) in human mesangial cells (HMCs) at the time of cell cycle entry. Further, RON remained active during steady-state growth. Activation of RON was independent of its canonical ligand, macrophage-stimulating protein, but was mediated by transactivation from the PDGF-engaged PDGF receptor. Following stimulation with PDGF we found that the two receptors physically interacted. Knockdown of RON by siRNA increased the number of apoptotic cells without affecting the rate of DNA synthesis, suggesting that RON has anti-apoptotic functions. Immunohistochemical analysis found phosphorylated RON in glomerular lesions of patients with IgA nephropathy but not those with minimal change nephrotic syndrome, a disease not associated with mesangial proliferation. These results suggest that RON is involved in mesangial cell proliferation under both physiological and pathological conditions, and may be a relevant target for therapeutic intervention.

Recepteur d'Origine Nantais Tyrosine Kinase Is a Direct Target of Hypoxia-inducible Factor-1α-mediated Invasion of Breast Carcinoma Cells

Hypoxia-inducible factor-1alpha (HIF-1alpha) overexpression was shown to be associated with invasion and metastasis of tumors and tumor cell lines. The identification of molecular targets that contribute to HIF-1alpha-mediated invasion is under intensive investigation. We have analyzed the role of recepteur d'origine nantais (RON), a tyrosine kinase receptor for macrophage-stimulating protein (MSP) that plays a role in breast cancer cell invasion as one of the molecular targets of HIF-1alpha. Analysis of a panel of breast cancer cell lines indicated a correlation between HIF-1alpha and RON expression. Treatment of HIF-1alpha- and RON-positive breast cancer cells with HIF-1alpha inhibitor, echinomycin, led to the inhibition of HIF-1alpha activity and RON expression. We have identified HIF-1alpha binding site on the RON promoter. Chromatin immunoprecipitation analysis and site-directed mutagenesis of the RON promoter confirmed the binding of HIF-1alpha to RON promoter. HIF-1alpha inhibitor-, echinomycin-, or short hairpin RNA-mediated selective knockdown of HIF-1alpha or HIF-1alpha target RON tyrosine kinase abrogated RON gene expression, and the RON ligand macrophage-stimulating protein mediated invasion of breast cancer cells. Consequently, the data presented herein demonstrated RON as a novel molecular target of HIF-1alpha and suggest a potential therapeutic role for HIF-1alpha or RON tyrosine kinase inhibitors in the blockade of RON tyrosine kinase-mediated invasion of carcinoma cells.

Proteolytic cleavage and activation of pro-macrophage-stimulating protein by hepatocyte growth factor activator and human airway trypsin-like protease

Background and Objectives: Macrophage-stimulating protein (MSP) is a plasma protein homologous to hepatocyte growth factor (HGF). MSP circulates as inactive precursor (pro-MSP) in the blood and requires proteolytic activation to transduce signals via its specific cell surface receptor tyrosine kinase, RON (recepteur d’origine nantais). Serine proteinases on the cell surface and also in serum have been implicated in this activation. The aim of this study was to examine the effects of serum serine proteinase, HGF activator (HGFA) and cell surface serine proteinase of stratified squamous epithelium such as human airway trypsin-like proteinase (HAT) and its family protein, differentially expressed in squamous cell carcinoma gene 1 (DESC1), on the activation of pro-MSP.

RON receptor tyrosine kinase in human gliomas: expression, function, and identification of a novel soluble splice variant

Malignant gliomas are incurable because of their diffuse infiltration of the surrounding brain. The recepteur d'origine nantais (RON) receptor tyrosine kinase is highly expressed in several epithelial cancer types and mediates tumorigenic, pro-invasive as well as metastatic effects. Analyzing RON expression in human gliomas, we found that different splice variants with known oncogenic activity are expressed in glioblastomas (GBM). In addition, the RON ligand macrophage-stimulating protein (MSP) is secreted by cultured GBM cells. MSP showed no mitogenic effect on GBM cells but displayed significant chemotactic activity for several GBM cell lines. We identified a novel splice variant, RONDelta90, which is generated by a transcript missing exon 6. As a result of a frameshift, translation is terminated in exon 7, resulting in a truncated soluble protein. RONDelta90 transcripts are expressed in normal human brain as well as in low grade astrocytomas but only in approximately 50% of highly malignant astrocytomas. In addition, RONDelta90 is detectable in supernatants of GBM cell lines. We cloned the RONDelta90 cDNA, and purified the recombinant protein from transfected cells. RONDelta90 inhibited MSP-induced phosphorylation of cellular RON and also attenuated basal activation levels. In addition, RONDelta90 inhibited MSP-induced glioma cell migration as well as random motility. To conclude, RONDelta90 is a novel soluble receptor variant with antagonistic activity that may act as a physiological modulator of RON signaling. The expression of several oncogenic RON splice variants in malignant gliomas suggests that these could represent candidate targets for treatment with agents inhibiting RON activity.

The RON receptor tyrosine kinase promotes MSP-independent cell spreading and survival in breast epithelial cells

The recepteur d’origne nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, the macrophage stimulating protein, when expressed in epithelial cells. In the current study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON is mediated in part by Src-family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.

HIV-1 Tat Mediates Degradation of RON Receptor Tyrosine Kinase, a Regulator of Inflammation

HIV encodes several proteins, including Tat, that have been demonstrated to modulate the expression of receptors critical for innate immunity, including MHC class I, mannose receptor, and beta(2)-microglobulin. We demonstrate that Tat targets the receptor tyrosine kinase recepteur d'origine nantais (RON), which negatively regulates inflammation and HIV transcription, for proteosome degradation. Tat decreases cell surface RON expression in HIV-infected monocytic cells, and Tat-mediated degradation of RON protein is blocked by inhibitors of proteosome activity. Tat specifically induced down-regulation of RON and not other cell surface receptors, such as the transferrin receptor, the receptor tyrosine kinase TrkA, or monocytic markers CD14 and ICAM-1. The Tat trans activation domain is required for RON degradation, and this down-regulation is dependent on the integrity of the kinase domain of RON receptor. We propose that Tat mediates degradation of RON through a ubiquitin-proteosome pathway, and suggest that by targeting signals that modulate inflammation, Tat creates a microenvironment that is optimal for HIV replication and progression of AIDS-associated diseases.

Curcumin Blocks RON Tyrosine Kinase–Mediated Invasion of Breast Carcinoma Cells

We have recently shown that macrophage-stimulating protein (MSP) promotes the invasion of recepteur d'origine nantais (RON), a tyrosine kinase receptor-positive MDA-MB-231, MDA-MB-468 breast cancer cells, and also identified the regulatory elements required for RON gene expression. In this report, we have analyzed the efficacy of a chemopreventive agent, curcumin, in blocking RON tyrosine kinase-mediated invasion of breast cancer cells. Reverse transcription-PCR and Western analysis indicated the down-regulation of the RON message and protein, respectively, in MDA-MB-231 and MDA-MB-468 cells. Significantly, curcumin-mediated inhibition of RON expression resulted in the blockade of RON ligand, MSP-induced invasion of breast cancer cells. We have identified two putative nuclear factor-kappaB p65 subunit binding sites on the RON promoter. Using chromatin immunoprecipitation analysis and site-directed mutagenesis of the RON promoter, we have confirmed the binding of p65 to the RON promoter. Our data show that curcumin reduces RON expression by affecting p65 protein expression and transcriptional activity. Treatment of MDA-MB-231 cells with pyrrolidine dithiocarbamate, an inhibitor of p65, or small interfering RNA knockdown of p65, blocked RON gene expression and MSP-mediated invasion of MDA-MB-231 cells. This is the first report showing the regulation of human RON gene expression by nuclear factor-kappaB and suggests a potential therapeutic role for curcumin in blocking RON tyrosine kinase-mediated invasion of carcinoma cells.

The Prognostic Significance of RON and MET Receptor Coexpression in Patients with Colorectal Cancer

Although Recepteur d'Origine Nantais (RON), a member of the MET receptor tyrosine kinase family, is overexpressed and constitutively active in some primary tumors and tumor cell lines, its expression pattern and clinical significance in colorectal cancer are not well documented. By using immunohistochemical staining, we examined RON and MET expression in 135 colorectal cancer specimens and investigated the association of the immunoreactivity of both receptors with colorectal cancer clinical parameters and prognosis. We found moderate to strong expression in 99 cases (73 percent) for RON and 97 cases (72 percent) for MET. Univariate analysis showed that increased immunoreactivity of RON or MET was associated with shorter patient survival and that moderate to strong coexpression of both receptors was associated with a significantly worse prognosis. Multivariate Cox analysis showed that the risk of tumor recurrence for patients with high-RON/high-MET expression was approximately 11 times greater than for patients with low-RON/low-MET expression (P = 0.001). In addition, RON and MET expression levels were positively correlated (P <or= 0.001; tau = 0.306). The crosstalk between RON and MET in colorectal cancer seems important. Evaluating the expression patterns of RON and MET was predictive of clinical outcome for patients with colorectal cancer.