Receptor For Macrophage-stimulating Protein Research Articles

The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

Abstract 2242: KRAS mutations suppress the tumor immune microenvironment in colorectal cancer

Abstract Background: KRAS mutations (KRASmut) drive tumorigenesis through various signaling pathways that are associated with worse survival in colorectal cancer patients (CRC). Immune evasion is a hallmark of KRAS-driven cancer, but the role of KRASmut in the regulation of the immune response is unclear, particularly in the tumor microenvironment (TME), which often assists cancer cells to thrive and successfully escape immune surveillance. Here, we explore the immunobiological impacts of KRASmut in CRC patients. Methods: A total of 97 fresh frozen tumor samples were collected from each CRC patient with stage I-IV who underwent surgical treatment at the Department of Surgery, Faculty of Medicine Siriraj Hospital between October 2010 and March 2011. First, all patients were identified KRAS mutational status and compared overall survival (OS) with wild-type KRAS (KRASwt). Next, we analyzed the gene expression profiling of KRASmut which was performed using the Nanostring platform. Finally, we performed the biological network analysis focusing on immunological pathways associated with KRASmut using the Ingenuity Pathway Analysis (IPA). Results: We observed KRASmut in 41.24% (n=40) of the total cases. Mutations occurred primarily in exon 2 (total n=36; codon 12, n=28; codon 13, n=8) and rarely in exon 3 (n=4). Patients with KRASmut CRC in exon 2 and exon 3, showed significantly worse OS compared to KRASwt (HR 1.77, 95% CI 1.07-2.93; P<0.05). Comparing gene expression profiles between KRASmut and KRASwt, found that the survival outcome of patients with KRASmut was correlated with immunosuppressive TME by activation of genes encoded in TGFβ signaling pathway. In addition to activating TGFβ pathway, the gene expression profile of KRASmut also interrupted gene encoded pro-inflammatory cytokines, including TNFRSF12, IRF6, CCR2, CCL8, CCL11, CXCR3 and CXCL10. IPA analysis revealed that differentially expressed genes of KRASmut were significantly enriched in regulation of epithelial mesenchymal transition (EMT) and macrophage-stimulating protein receptor d'origine nantais (MSP-RON) signaling pathways (P value<0.001), which are strongly associated with maintaining the stability of the TME and contributing to immune escape in the immune TME. Conclusions: Our results suggested that KRASmut activated TGFβ and interrupted pro-inflammatory cytokine to suppress tumor immune microenvironment in CRC. Future studies and clinical trials in patients with CRC with KRASmut should take these transcriptional profiles into account. Citation Format: Pariyada Tanjak, Amphun Chaiboonchoe, Tharathorn Suwatthanarak, Onchira Acharayothin, Kullanist Thanormjit, Jantappapa Chanthercrob, Thanawat Suwatthanarak, Bundit Wannasuphaphol, Kemmapon Chumchuen, Bhoom Suktitipat, Somponnat Sampattavanich, Krittiya Korphaisarn, Ananya Pongpaibul, Naravat Poungvarin, Harald Grove, Woramin Riansuwan, Atthaphorn Trakarnsanga, Asada Methasate, Manop Pithukpakorn, Vitoon Chinswangwatanakul. KRAS mutations suppress the tumor immune microenvironment in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2242.

A combined FAK, c-MET, and MST1R three-protein panel risk-stratifies colorectal cancer patients.

Focal adhesion kinase (FAK) is a key tyrosine kinase downstream of c-MET (or hepatocyte growth factor receptor, HGFR) and MST1R (macrophage-stimulating protein receptor or recepteur d'origine Nantais, RON) membrane receptors. The pathway plays an important role in cancer survival and invasion. In this study, we examined the protein expression of FAK, c-MET, and MST1R levels in a well-annotated cohort of 330 colorectal cancer patients. We found FAK to be overexpressed in colorectal adenocarcinomas (p = 0.0002), and FAK levels correlated positively with phospho-FAK levels (R2 = 0.81). In comparison, MST1R levels were not significantly different, and c-MET levels were slightly higher in the normal samples. We then developed a combined 3-protein panel of FAK, c-MET, and MST1R expression signatures that can robustly risk-stratify colorectal cancer across all stages into three clusters that differ in progression-free survival. The colorectal cancer subgroup with high FAK, low c-MET, and low MST1R protein levels showed the worst progression-free survival with particularly early progression of disease (p = 0.0053). Combined FAK, c-MET, and MST1R were independently prognostic for progression-free survival in stage II colorectal cancers in a multivariate model. The 3-protein panel provides a potentially clinically attractive method for risk-stratification and adjuvant therapy guidance, especially in stage II disease.

AMP-activated protein kinase regulates alternative pre-mRNA splicing by phosphorylation of SRSF1.

AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic processes and activating catabolic processes. Recent studies have demonstrated that metformin, which is an AMPK activator, modifies alternative precursor mRNA (pre-mRNA) splicing. However, no direct substrate of AMPK for alternative pre-mRNA splicing has been reported. In the present study, we identified the splicing factor serine/arginine-rich splicing factor 1 (SRSF1) as a novel AMPK substrate. AMPK directly phosphorylated SRSF1 at Ser133 in an RNA recognition motif. Ser133 phosphorylation suppressed the interaction between SRSF1 and specific RNA sequences without altering the subcellular localization of SRSF1. Moreover, AMPK regulated the SRSF1-mediated alternative pre-mRNA splicing of Ron, which is a macrophage-stimulating protein receptor, by suppressing its interaction with exon 12 of Ron pre-mRNA. The findings of this study revealed that the AMPK-dependent phosphorylation of SRSF1 at Ser133 inhibited the ability of SRSF1 to bind RNA and regulated alternative pre-mRNA splicing.

RRM but not the Asp/Glu domain of hnRNP C1/C2is required for splicing regulation of Ron exon 11 pre-mRNA

The Ron proto-oncogene is a human receptor for macrophage-stimulating protein (MSP). The exclusion of exon 11 in alternative splicing generates ΔRON protein that is constitutively activated. Heterogenous ribonucleaoprotein (hnRNP) C1/C2 is one of the most abundant proteins in cells. In this manuscript, we showed that both hnRNP C1 and C2 promoted exon 11 inclusion of Ron pre-mRNA and that hnRNP C1 and hnRNP C2 functioned independently but not cooperatively. Moreover, hnRNP C1 stimulated exon 11 splicing through intron 10 activation but not through intron 11 splicing. Furthermore, we showed that, whereas the RRM domain was required for hnRNP C1 function, the Asp/Glu domain was not. In conclusion, hnRNP C1/C2 promoted exon 11 splicing independently by stimulating intron 10 splicing through RRM but not through the Asp/Glu domain.

Global survey of the immunomodulatory potential of common drugs.

Small molecule drugs may complement antibody-based therapies in an immune-oncology setting, yet systematic methods for the identification and characterization of the immunomodulatory properties of these entities are lacking. We surveyed the immumomodulatory potential of 1,402 small chemical molecules as defined by their ability to alter the cell-cell interactions among peripheral mononuclear leukocytes ex vivo, using automated microscopy and population-wide single-cell image analysis. Surprisingly, some 10% of the agents tested affected these cell-cell interactions differentially. The results accurately recapitulated known immunomodulatory drug classes, and revealed several clinically approved drugs that unexpectedly harbor the ability to modulate the immune system, potentially contributing to their physiological mechanism of action. For instance, the kinase inhibitor crizotinib promoted T-cell interactions with monocytes as well as with cancer cells, through inhibition of MST1R/RON (macrophage-stimulating protein receptor) and subsequent upregulation of MHC (major histocompatibility complex) expression. The approach offers an attractive platform for the personalized identification and characterization of immunomodulatory therapeutics.

Analogs of the hepatocyte growth factor and macrophage-stimulating protein hinge regions act as Met and Ron dual inhibitors in pancreatic cancer cells

Pancreatic cancer is among the leading causes of cancer death in the USA, with limited effective treatment options. A major contributor toward the formation and persistence of pancreatic cancer is the dysregulation of the hepatocyte growth factor (HGF)/Met (HGF receptor) and the macrophage-stimulating protein (MSP)/Ron (MSP receptor) systems. These systems normally mediate a variety of cellular behaviors including proliferation, survival, and migration, but are often overactivated in pancreatic cancer and contribute toward cancer progression. Previous studies have shown that HGF must dimerize to activate Met. Small-molecule antagonists with homology to a 'hinge' region within the putative dimerization domain of HGF have been developed that bind to HGF and block dimerization, therefore inhibiting Met signaling. Because of the structural and sequence homology between MSP and HGF, we hypothesized that the inhibition of HGF by the hinge analogs may extend to MSP. The primary aim of this 'proof-of-concept' study was to determine whether hinge analogs could inhibit cellular responses to both HGF and MSP in pancreatic cancer cells. Our results showed that these compounds inhibited HGF and MSP activity. Hinge analog treatment resulted in decreased Met and Ron activation, and suppressed malignant cell behaviors including proliferation, migration, and invasion in pancreatic cancer cells in vitro. These results suggest that the hinge analogs represent a novel group of molecules that may offer a therapeutic approach for the treatment of pancreatic cancer and warrant further development and optimization.

MSP is a negative regulator of inflammation and lipogenesis in ex vivo models of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH), a metabolic disorder consisting of steatosis and inflammation, is considered the hepatic equivalent of metabolic syndrome and can result in irreversible liver damage. Macrophage-stimulating protein (MSP) is a hepatokine that potentially has a beneficial role in hepatic lipid and glucose metabolism via the activation of AMP-activated protein kinase (AMPK). In the current study, we investigated the regulatory role of MSP in the development of inflammation and lipid metabolism in various NASH models, both in vitro and ex vivo. We observed that MSP treatment activated the AMPK signaling pathway and inhibited lipopolysaccharide (LPS)- and palmitic acid (PA)-induced gene expression of pro-inflammatory cytokines in primary mouse hepatocytes. In addition, MSP treatment resulted in a significant reduction in PA-induced lipid accumulation and inhibited the gene expression of key lipogenic enzymes in HepG2 cells. Upon short hairpin RNA-induced knockdown of RON (the membrane-bound receptor for MSP), the anti-inflammatory and anti-lipogenic effects of MSP were markedly ablated. Finally, to mimic NASH ex vivo, we challenged bone marrow-derived macrophages with oxidized low-density lipoprotein (oxLDL) in combination with LPS. OxLDL+LPS exposure led to a marked inhibition of AMPK activity and a robust increase in inflammation. MSP treatment significantly reversed these effects by restoring AMPK activity and by suppressing pro-inflammatory cytokine gene expression and secretion under this condition. Taken together, these data suggest that MSP is an effective inhibitor of inflammation and lipid accumulation in the stressed liver, thereby indicating that MSP has a key regulatory role in NASH.

RON Nuclear Translocation under Hypoxia Potentiates Chemoresistance to DNA Double-Strand Break-Inducing Anticancer Drugs.

Tumor hypoxia is associated with radioresistance, chemoresistance, and metastasis, which eventually lead to cancer progression and a poor patient prognosis. RON [also known as macrophage-stimulating protein receptor (MST1R)] belongs to the c-MET [also known as hepatocyte growth factor receptor (HGFR)] receptor tyrosine kinase (RTK) superfamily. To identify the interaction partners of RON nuclear translocation in response to hypoxia, the nuclear extract of TSGH8301 bladder cancer cells was immunoprecipitated for tandem mass profiling analysis. Nuclear RON interacted with adenosine triphosphate (ATP)-dependent DNA helicase 2 (Ku70) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to activate nonhomologous end joining (NHEJ) DNA repair. The interaction was time dependent, extending 3 to 24 hours posthypoxia or until the components had been exposed to the chemotherapeutic drugs doxorubicin and epirubicin. Stable knockdown experiments in vitro suggest the importance of RON for the chemoresistance of cancer cells under hypoxia. In addition, the tyrosine kinase domain of nuclear RON is crucial for interaction with Ku70 under hypoxia. J82 cells transfected with RON showed a survival advantage in the presence of epirubicin and hypoxia. This suggests that nuclear RON activates NHEJ repair by interacting with Ku70/DNA-PKcs and inhibiting RON activity to increase cancer cell chemosensitivity. Mol Cancer Ther; 15(2); 276-86. ©2016 AACR.

Abstract 2708: Development of allosteric regulators of the dimerization domains of the plasminogen-related growth factor family for the treatment of pancreatic cancer

Abstract The plasminogen-related family of growth factors includes the hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), both of which normally mediate a variety of cellular behaviors including proliferation, survival, and migration. A major contributor to the formation and persistence of various cancers is the dysregulation of these growth factor systems. Overexpression of the HGF receptor, c-Met, is common in many cancers including pancreatic cancer, and is associated with poor prognosis. Dysregulation of this system often leads to tumorigenesis and induction of various malignant phenotypes including increased proliferation, survival, migration, and invasion. In order to activate c-Met, HGF must first undergo an activation step in which it forms an HGF homodimer. Small molecule antagonists with homology to a putative “hinge” region within the HGF sequence have been developed that bind to HGF and allosterically block dimerization, therefore inhibiting c-Met signaling. Norleual [Nle-Tyr-Ile-ψ-(CH2-NH2)3-4-His-Pro-Phe] has previously been shown to inhibit HGF dimerization and HGF-dependent c-Met activation. We have assessed the ability of Norleual to block HGF dimerization and activation of c-Met within pancreatic cancer cells. Similar to the HGF/c-Met system, over-activation of the closely related MSP/Ron (MSP receptor) growth factor system has been identified as a critical contributor to pancreatic cancer progression. Due to overall high sequence homology between HGF and MSP, including the putative “hinge” region, we have hypothesized that MSP must also dimerize in order to efficiently activate the Ron receptor, and that this activation step can be targeted by “hinge” region homologs. Our results indicate that MSP naturally forms dimers and incubation with Norleual and similar “hinge” region antagonists inhibits dimerization. Further, we have demonstrated that MSP dimerization may be biologically significant and inhibition of dimerization suppresses MSP-dependent cell signaling and cell behaviors. The ultimate goal of this work is to develop a dual inhibitor capable of suppressing both the HGF and MSP growth factor systems, and further assess the therapeutic potential of these inhibitors for treatment of pancreatic cancer. Citation Format: Kevin J. Church, Leen Kawas, Joseph Harding, Malte Lang, Michelle McMicheal. Development of allosteric regulators of the dimerization domains of the plasminogen-related growth factor family for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2708. doi:10.1158/1538-7445.AM2014-2708

Abstract 956: Elucidating the mechanisms of acquired resistance in lung adenocarcinomas

Abstract In lung adenocarcinomas, targeted therapy with the EGFR tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and afatinib is associated with longer progression free survival (PFS) and higher radiographic response (RR) rates when compared to standard first-line chemotherapy. In ALK rearranged lung cancers, targeted therapy with crizotinib is associated with PFS of approximately 9,7 months and RR of 60.8%. However, despite the initial success of these agents, all patients progress with a median PFS of 7 to 16 months. Acquired resistance in EGFR mutant tumors is driven by the occurrence of a secondary EGFR mutation (T790M) in about 50% of the cases and by MET amplification in 5 to 10 % of the cases. Other mechanisms include HER2 amplification, PTEN loss, phenotypic change to small cell histology, rare mutations in BRAF and AXL activation. Resistance to crizotinib, on the other hand, is caused by secondary mutations in the ALK kinase domain, by ALK or cKIT amplification or by alterations in EGFR and KRAS. Here, we made use of next generation sequencing techniques to better understand the mechanisms that drive resistance in lung adenocarcinomas treated with erlotinib or crizotinib. For this purpose, we used transbronchial or CT-guided rebiopsies from patients that had either prolonged stable disease or partial response to therapy, and developed radiographic progression under TKI therapy. Samples were analyzed by FISH and sequenced on a benchtop Illumina platform (MiSeq) in order to evaluate the presence of known mechanisms of resistance. Samples that were negative for any of the reported mechanisms were analyzed by genome, exome or trascriptome sequencing. From the sequencing output of the pan-negative samples, filtering of mutation candidates included: absence of the mutation in the pre-treatment sample (when available), expression of the candidate gene in lung adenocarcinomas, absence of the mutation in primary lung adenocarcinomas, high impact of the mutation at protein level (Polyphen), mutant allelic fraction in the tumor higher than 10%, among other factors. After filtering, validation of mutation calls was performed by Sanger sequencing. Sequencing of the erlotinib resistant samples revealed mutations in members of a functionally wide spectrum of protein families including the proteoglycan family, the ATP-binding cassette (ABC) transporters family, an Fms-related tyrosine kinase receptor and a member of the transforming growth factor beta family of cytokines. On the other hand, crizotinib resistant samples showed mutations in a cell surface receptor for macrophage-stimulating protein with tyrosine kinase activity, in a C2H2 type zinc finger gene, a semaphorin, a mitogen-activated protein kinase and a member of the SWI/SNF family of proteins. Our results evidence the possible contribution of a wide range of cellular pathways in the process of acquired resistance to EGFR and ALK inhibitors in lung adenocarcinomas. Citation Format: Sandra Ortiz-Cuarán, Lynnette Fernandez-Cuesta, Marc Bos, Lukas Heukamp, Christine M. Lovly, Martin Peifer, Masyar Gardizi, Matthias Scheffler, Ilona Dahmen, Christian Müller, Katharina König, Kerstin Albus, Alexandra Florin, Sascha Ansén, Reinhard Buettner, Jürgen Wolf, William Pao, Roman K. Thomas. Elucidating the mechanisms of acquired resistance in lung adenocarcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 956. doi:10.1158/1538-7445.AM2014-956

Intron Retention in the Alternatively Spliced Region of RON Results from Weak 3’ Splice Site Recognition

The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3’ splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5’ of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo.

Entropy Model for Multiplex Drug-Target Interaction Endpoints of Drug Immunotoxicity

Entropy measures are universal parameters useful to codify biologically-relevant information in many systems. In our previous work, (Gonzalez-Diaz, H., et al. Chem. Res. Toxicol. 2003, 16, 1318-1327), we introduced the molecular structure information indices called 3D-Markovian electronic delocalization entropies (3D-MEDNEs) to study the quantitative structure-toxicity relationships (QSTR) of drugs. In a second part, (Cruz-Monteagudo, M. et al. Chem. Res. Toxicol., 2008, 21 (3), 619-632), we extended 3D-MEDNEs to numerically encode toxicologically-relevant information present in Mass Spectra of the serum proteome. These works demonstrated that the idea behind classic drug QSTR models can be extended to solve more general problems in toxicological chemical research. For instance, there are not many reports of multi-target QSTR (mt-QSTR) models useful to predict multiplexed endpoints of drugs in a high number of cytotoxicity assays. In this work, we train and validate for the first time a QSTR model that correctly classifies 8,806 out of 9,001 (Accuracy = 91.1%) multiplexing assay endpoints of 7903 drugs (including both training and validation series). Each endpoint corresponds to one out of 1443 assays, 32 molecular and cellular targets, 46 standard type measures, in two possible organisms (human and mouse). We have also determined experimentally, for the first time, the values of EC50 = 8.21 μg/mL and Cytotoxicity = 26.25 % for the antimicrobial / antiparasitic drug G1 on Balb/C mouse thymic macrophages using flow cytometry. In addition, we have used the new model to predict G1 endpoints in 1,283 assays finding a low average probability of p(1) = 0.50% in 152 cytotoxicity assays. Last, we have used the model to predict average probability of the interaction of G1 with different proteins in macrophages. Interestingly, the Macrophage colony-stimulating factor receptor, the Macrophage colony-stimulating factor 1 receptor, the Macrophage migration inhibitory factor, Macrophage scavenger receptor types I and II, and the Macrophage-stimulating protein receptor, have also very low average predicted probabilities of p(1) = 0.092, 0.038, 0.077, 0.026, 0.2, 0.106, respectively. Both experimental and theoretical results show a moderate thymic macrophage cytotoxicity of G1. The obtained results are significant because they complement the immunotoxicology studies of this important drug.

Deletion of the epidermal growth factor receptor in renal proximal tubule epithelial cells delays recovery from acute kidney injury

To determine the role of epidermal growth factor receptor (EGFR) activation in renal functional and structural recovery from acute kidney injury (AKI), we generated mice with a specific EGFR deletion in the renal proximal tubule (EGFRptKO). Ischemia–reperfusion injury markedly activated EGFR in control littermate mice; however, this was inhibited in either the knockout or wild-type mice given erlotinib, a specific EGFR tyrosine kinase inhibitor. Blood urea nitrogen and serum creatinine increased to a comparable level in EGFRptKO and control mice 24 h after reperfusion, but the subsequent rate of renal function recovery was markedly slowed in the knockout mice. Twenty-four hours after reperfusion, both the knockout and the inhibitor-treated mice had a similar degree of histologic renal injury as control mice, but at day 6 there was minimal evidence of injury in the control mice while both EGFRptKO and erlotinib-treated mice still had persistent proximal tubule dilation, epithelial simplification, and cast formation. Additionally, renal cell proliferation was delayed due to decreased ERK and Akt signaling. Thus, our studies provide both genetic and pharmacologic evidence that proximal tubule EGFR activation plays an important role in the recovery phase after acute kidney injury.

Activation of macrophage-stimulating protein by human airway trypsin-like protease

Macrophage-stimulating protein (MSP) circulates as a proform protein and requires proteolytic processing for activation. Respiratory ciliated cells express the MSP receptor, recepteur d’origine nantais (RON), at the apical surface, which reportedly has an important role in ciliary function. Like RON, human airway trypsin-like protease (HAT) is also expressed at the apical surface of ciliated cells. Here we show that HAT cleaves proMSP at the physiological activation site, Arg483-Val484. MSP processed by HAT could induce chemotactic responses and morphological changes of peritoneal macrophages. In human respiratory epithelial cells, knock down of HAT expression reduced proMSP processing and RON autophosphorylation. We suggest that HAT is important for MSP-RON signaling in the respiratory tract. Structured summary of protein interactionsHATcleavesproMSP by protease assay (View interaction)

Comprehensive analysis of receptor tyrosine kinase activation in human melanomas reveals autocrine signaling through IGF-1R

Melanomas depend on autocrine signals for proliferation and survival; however, no systematic screen of known receptor tyrosine kinases (RTKs) has been performed to identify which autocrine signaling pathways are activated in melanoma. Here, we performed a comprehensive analysis of 42 RTKs in six individual human melanoma tumor specimens as well as 17 melanoma cell lines, some of which were derived from the tumor specimens. We identified five RTKs that were active in almost every one of the melanoma tissue specimens and cell lines, including two previously unreported receptors, insulin-like growth factor receptor 1 (IGF-1R) and macrophage-stimulating protein receptor (MSPR), in addition to three receptors (vascular endothelial growth factor receptor, fibroblast growth factor receptor, and hepatocyte growth factor receptor) known to be autocrine activated in melanoma. We show, by quantitative real time PCR, that all melanoma cell lines expressed genes for the RTK ligands such as HGF, IGF-1, and MSP. Addition of antibodies to either IGF-1 or HGF, but not to MSP, to the culture medium blocked melanoma cell proliferation, and even caused net loss of melanoma cells. Antibody addition deactivated IGF-1R and hepatocyte growth factor receptors, as well as mitogen-activated protein kinase signaling. Thus, IGF-1 is a new growth factor for autocrine driven proliferation of human melanoma in vitro. Our results suggest that IGF-1-IGF-1R autocrine pathway in melanoma is a possible target for therapy in human melanomas.

Tyrosine kinase receptor RON and its ligand MSP in Merkel cell carcinoma

The RON gene, encoding the tyrosine-kinase receptor for macrophage-stimulating protein (MSP), is involved in a range of neoplastic processes. However, no aberration in RON or MSP has been identified in Merkel cell carcinoma (MCC). We investigated the RON signaling pathway in MCC. Fourteen cases of MCC were tested for the expression of RON and its ligand, MSP, using reverse transcription PCR (RT-PCR) and immunohistochemistry. The mutation of RON was also examined. RT-PCR identified transcription of both RON and MSP in all nine cases that were available for the examination. Immunohistochemistry showed the expression of RON (9/14 cases) and MSP (9/14 cases). Six cases out of 14 were positive for both RON and MSP. Normal Merkel cells were negative for RON and MSP expression. Missense mutation in the tyrosine kinase region of RON was detected in one of the 14 cases, and the case showed expression of RON. Transcription of RON and MSP was observed in MCC, and a considerable number of MCC cases expressed both RON and MSP, while Merkel cells do not express these molecules. The results suggest that RON signaling seems to play at least some role in the pathogenesis of MCC.

Abstract 5409: Ron silencing by shRNA inhibits growth, induces apoptosis and sensitizes tumor response to c-Met inhibitor treatment through down-regulating MAPK signaling in B-Raf mutant colon carcinomas

Abstract RON, the receptor for macrophage-stimulating protein, belongs to the MET receptor tyrosine kinase family and shares many common features with MET. Inappropriate activation of RON signaling plays an important roll in colon carcinoma cell proliferation, survival, migration and invasive growth. Ron and Met are overexpressed in patient colon tumors, where co-overexpression of both Ron and Met correlates with the worst patient survival compared to the overexpression of Ron or Met alone. BRaf-activating mutations occur in about 10% of colorectal carcinomas. Mutant BRaf has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase (MAPK) pathway, and BRaf mutant cancers are generally resistant to standard therapies. Here we report that knockdown of the Ron gene by Ron shRNA in BRafV600E mutant HT29 colon carcinoma cells induced HT29 tumor apoptosis and resulted in significant tumor growth inhibition (TGI) by 59%, which is comparable to that observed with the selective EGFR inhibitor-Erlotinib treatment (57% TGI), but more potent than that of the selective Met inhibitor-PF-04217903 treatment (38% TGI). When Ron shRNA knockdown and treatment with the selective Met inhibitor were combined, enhanced tumor growth inhibition (76%) was observed. Comparable experiments using the BRaf mutant Colo205 model yielded similar results. Western Blot analysis showed that combination of Ron shRNA and Met inhibition in HT29 cells decreased phosho-ERK, elevated cleaved caspase-3 and, surprisingly, increased total Ras protein levels. No significant changes in total and phospho-AKT levels were observed. In vitro, the stable HT29-shRon cells initially grew more slowly compared to HT29-vector cells, but the growth rate gradually increased, approaching that observed in normal cell culture conditions. Interestingly, when co-culturing stable HT29-shRon or HT29-vector cells with bone marrow derived mesenchymal stem cells, the selective Met inhibitor-PF-04217903 induced significant cell death only in the HT29-shRon cells under hypoxic conditions (1% oxygen). No cell death was observed with either HT29-shRon cells under normoxia or HT29-vector cells under hypoxia or normoxia conditions, indicating the collaborative roll of Ron and Met in colon cancer cell survival during hypoxia. In summary, inhibiting both Ron and Met pathways in BRaf mutant colon cancer cells down- regulated ERK activity, induced apoptosis and led to enhanced antitumor efficacy in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5409.

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.