Oncogenic and invasive potentials of human macrophage-stimulating protein receptor, the RON receptor tyrosine kinase
Ming-Hai Wang1,3,
Da Wang2 and
Yi-Qing Chen1
1 Laboratory of Chang-Jiang Scholar Endowment for Biomedical Sciences, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, Peoples Republic of China
2 Department of Medicine, University of Colorado School of Medicine, CU Cancer Center, and Denver Health Medical Center, Denver, CO 80204, USA
3 To whom correspondence should be addressed Email: ming-hai.wang{at}uchsc.edu
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Abstract
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The product of the RON (recepteur d'origine nantais) gene belongs to the MET proto-oncogene family, a distinct subfamily of receptor tyrosine kinases. The ligand of RON was identified as macrophage-stimulating protein (MSP), a member of the plasminogen-related growth factor family. RON is mainly expressed in cells of epithelial origin and is required for embryonic development. In vitro RON activation results in epithelial cell dissociation, migration and matrix invasion, suggesting that RON might be involved in the pathogenesis of certain epithelial cancers in vivo. Indeed, recent studies have shown that RON expression is significantly altered in several primary human cancers, including those of the breast and colon. Truncation of the RON protein has also been found in primary tumors from the gastrointestinal tract. These alterations lead to constitutive activation of RON that causes cell transformation in vitro, induces neoplasm formation in athymic nude mice, and promotes tumor metastasis into the lung. Studies employing transgenic models further demonstrated that over-expression of RON in lung epithelial cells results in multiple tumor formation with features of large cell undifferentiated carcinoma. The oncogenic activities of RON are mediated by RON-transduced signals that promote unbalanced cell growth and transformation leading to tumor development. Thus, abnormal accumulation and activation of RON could play a critical role in vivo in the progression of certain malignant human epithelial cancers.
Abbreviations: BAC, bronchioloalveolar carcinoma; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; Grb-2, growth factor receptor bound protein 2; HGF, hepatocyte growth factor; HGFL, hepatocyte growth factor-like; JNK; jun N-terminal kinase; JSRV, jaagsiekte sheep retrovirus; MAPK, mitogen activated protein kinase; MDCK, MadinDarby canine kidney; MSP, macrophage-stimulating protein; NF-
B, nuclear factor-
B; PI-3K, phosphotidylinositol-3 kinase; RON, recepteur d'origine nantais; Sea, sarcoma, erythroblastosis, and anemia; SF, scatter factor; Smad, Sma and mothers against decapentaplegic homolog; TGF, transforming growth factor; Tpr, translocated promoter region
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Introduction
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Initiation and progression of malignant tumors in the human are complicated processes manifested by a variety of factors including individual genetic background and environmental hazard. It is now widely accepted that tumor is a genetic disease with significant cellular disorganization and alteration (13). This conclusion is based on the cellular, molecular and genetic analyses, which demonstrated that the tumors are initiated and progressed from a series of genetic changes that are characterized by the activation of oncogenes and inactivation of tumor suppressor genes (13). Epigenetic changes, defined as modifications of the genome without changes of DNA sequence such as methylation of individual genes (4), together with protein alterations at the levels of phosphorylation, acetylation, amidation and deamidation (5), also contribute significantly to the cancer initiation and progression (4,5). Recently, a group of non-transforming genes, known as cancer progression genes, such as progression elevated gene-3, has been identified (6,7). These genes positively regulate cancer aggressiveness and angiogenesis (8). Thus, the genetic and epigenetic changes, together with other mechanisms, are the responsible factors that impact on cells leading to cancer initiation and progression (18).
Among oncogenes selectively activated in epithelial tumors are a group of cell surface proteins called receptor-type protein tyrosine kinases (RPTK) (9). RPTK consists of a large family of transmembrane proteins with unique structural properties and tyrosine kinase activity (9). Currently, 17 families of RPTK have been identified (10). They play an essential role in regulating cell growth, differentiation and survival (9,10). RPTK is also involved in cell transformation, tumor development and metastasis (11). The typical examples of RPTK are receptors for epidermal growth factor (EGF) (12), hepatocyte growth factor (HGF)/scatter factor (SF) (13) and nerve growth factor (14). They all share a similar structure consisting of a large extracellular domain, a transmembrane segment and an intracellular component with intrinsic tyrosine kinase (1214). Abnormal activation of RPTKs through gene mutation, deletion, chromosomal rearrangement or translocation has been widely documented in different types of human tumors (11). In the case of MET, the receptor for HGF/SF (15), the germ-line and somatic mutations are directly responsible for the initiation of hereditary and sporadic papillary renal cancers in vivo (16,17). Blocking RPTK activities by specific inhibitors, such as Herceptin, which inhibits EGF receptor, has been shown to significantly improve clinical conditions in patients with breast cancer (18).
This review summarizes our current knowledge of recepteur d'origine nantais (RON) in epithelial tumorigenesis. Our focus is on the oncogenic and invasive potentials of RON. Since its discovery in 1993 (19), our knowledge of the biochemical and biological aspects of RON has advanced significantly. Accumulated evidence from in vitro and in vivo studies have demonstrated that altered RON expression and activation might play a critical role in the development and progression of certain epithelial tumors in vivo.
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Biochemical properties of RON and macrophage- stimulating protein
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RON belongs to the MET proto-oncogene family (19,20), a distinct subfamily of RPTK (21). Only two members of the MET family, MET and RON, exist in humans as evident in human genomic sequence analysis (22). MET is the receptor for HGF/SF (15) and has a well-described role in cell transformation and tumor progression (23). RON is the receptor for macrophage-stimulating protein (MSP) (24,25), and its role in human epithelial tumorigenesis is currently under intensive investigation. Proteins highly homolog to human RON have been identified in other species including mouse (26,27), chicken (28,29), xenopus (30) and puffer fish (31). In avian erythroblastosis retrovirus S13 that causes sarcoma, erythroblastosis and anemia in young chicks (32), an oncoprotein called V-Sea (for sarcoma, erythroblastosis and anemia) was identified in the viral genome (33). V-Sea is a hybrid protein containing the chicken Sea kinase domain fused with viral envelope sequences (34,35). The data suggest that RON is evolutionally conserved in different species.
The cDNA encoding human RON was originally cloned from keratinocytes (19). The murine homolog of RON (27), STK (stem cell-derived tyrosine kinase), was isolated from bone marrow cells (26). The RON gene contains 20 exons and 19 introns and is located on chromosome 3p21 (19,36), a region frequently altered in certain cancers (37). The mouse RON gene, which contains similar structure as the human RON gene (36), has also been sequenced (38). Mature RON is a 180 kDa heterodimer composed of a 40 kDa
-chain and a 150 kDa transmembrane ß-chain with intrinsic tyrosine kinase activity (Figure 1) (19,24,25). Both chains are derived from proteolytic conversion of single-chain RON precursor and linked by a disulfide bond (Figure 1) (19,24,25). Mouse IgG monoclonal antibodies (clones ID1 and ID2) are two highly specific and well-characterized antibodies against human RON (CDw136) (39). They recognize the extracellular domain of RON in a natural form, and compete with MSP for receptor binding (39). These features make them the ideal reagents to study the functions of RON. By western and northern blot analyses, it is demonstrated that expression of RON is observed primarily in cells of epithelial origin including colon and breast (40). Certain immune cells such as tissue macrophages also express RON (4143). Fibroblasts do not express RON (25,40). Complete disruption of the RON gene (knockout) leads to the death of mouse embryos in the early stages (44), suggesting that RON is developmentally required.
The only ligand for RON was identified as MSP (24,25). MSP (45,46) is also known as HGF-like protein (47) or SF-2 (48). MSP is a serum protein originally discovered by Dr E.J.Leonard in NIH through its activities in mouse macrophages (49,50). Purification of MSP from human blood plasma and cloning of MSP cDNA from a hepatoma cell line revealed that mature MSP is 80 kDa heterodimer composed of 53 kDa
-chain and 30 kDa ß-chain linked by a disulfide bond (45,46). The
-chain contains four triple disulfide loop structures called kringle domains (51). The ß-chain contains a serine protease-like domain with substitution of three amino acids in the active site (45,46). Thus, MSP is devoid of enzymatic activities (52). Structurally, MSP belongs to the kringle protein family that includes plasminogen, HGF and others (53). The MSP gene is located on chromosome 3p21 (46,47), the same region where the RON gene resides (19,36). Live cells are the major sources of MSP (45,54), which is constantly produced and circulated in blood at optimal concentration as a biologically inactive pro-MSP (55,56). Recent studies have shown that MSP mRNA is also transcribed in cells from the kidney and lung (57,58). However, the significance of these findings remains to be determined. Like HGF/SF and plasminogen, proteolytic conversion of pro-MSP is required for MSP to bind to RON and to induce biological activities (52,56). It is established that the major receptor-binding site is located in MSP ß-chain (59,60). Unlike the RON gene, complete knockout of the mouse MSP gene is not lethal, and has no visible phenotypic changes (61). This suggests that MSP is not required for embryonic development and growth. It also implies that additional ligand(s) for RON might exist, which could compensate for the loss of MSP.
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Abnormal accumulation and activation of RON and its variants in primary human cancers
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As described above, RON is mainly transcribed at relatively low levels in epithelial cells such as those from the skin, colon, breast, lung and kidney (40,62,63). Over-expression of RON was first demonstrated in primary breast cancer samples (48). Surveying RON expression in 94 breast tissue samples, it was found that RON expression is relatively low in normal breast epithelial cells and in benign lesions (adenoma and papillomas), but is highly expressed in 47% (35 out of 75 cases) of tumor specimens with different histotypic variants (48). The level of RON expression in malignant cells was increased from 2- to 20-fold as compared with benign epithelium. Elevated RON expression was strongly correlated to phosphorylation status and invasive activity of tumors (48), suggesting that increased RON expression plays a role in the progression of human breast carcinomas to invasive-metastatic phenotypes.
In the study of RON expression in epithelial cells of the digestive track, we found that RON is highly expressed and constitutively activated in a panel of human colorectal adenocarcinoma cell lines, but not in normal or SV-40 transformed colon epithelial cells (64), expression patterns similar to those observed in primary breast carcinomas (48). A recent study using immunohistochemical staining further demonstrated that RON is moderately expressed in normal colorectal mucosa, but increased significantly in the majority of primary human colorectal adenocarcinoma samples (29 of 49 cases) (65). The increase in expression ranged from 4- to 9-fold in comparison with the levels of RON expressed in normal colorectal mucosa (65). Accumulated RON is also constitutively active with autophosphorylation (64,65). However, no mutations in the kinase domain of the RON gene were found in colon carcinoma cell lines (64), indicating that the constitutive activation of RON is not caused by an abnormality in the kinase domain of RON. It probably resulted from the over-expression of the receptor protein leading to dimerization and activation (64,65). Other mechanisms, such as post-translational modifications in the RON kinase domain and deregulated cellular phosphorylation status, may also contribute to the constitutive activation of RON in colon cancer samples.
Altered RON expression is also accompanied by generation of biologically active RON variants through mRNA splicing in cancerous cells (6466), a mechanism responsible for generating protein diversity (67). Currently, three RON mRNA splicing products have been reported (Figure 1) (6466,68). The first RON variant, designated as RON
165 (also known as
-RON) with molecular mass of 165 kDa, was found in a stomach cancer cell line KATO III (68), and later in normal and malignant colon tissues samples (69). ROND165 has an in-frame deletion of 49 amino acids in the extracellular domain of the RON ß-chain, which resulted from the splicing out of exon 11 (66,68). This deletion prevents the proteolytic conversion of pro-RON
165 into the two-chain form and causes the protein to be retained in the cytoplasm (Figure 1) (66,68). RON
165 does not have cell-transforming activities but is capable of inducing cell motile activities in transfected cells (66,68).
The second RON variant, RON
160 (Figure 1), was cloned by us from colon cancer cell lines HT29 and SW837 (64,66). Our recent studies also found that RON
160 is present in two primary colorectal adenocarcinoma samples (65). RON
160 is a constitutively active variant (6466). It is synthesized from a transcript different from the full-length RON mRNA with several unique properties. (i) RON
160 has an in-frame deletion of 109 amino acids in the extracellular domain of the ß chain. These 109 amino acids are encoded by exons 5 and 6 of the RON gene (6466). (ii) RON
160 is first synthesized as a single-chain precursor and then converted into the two-chain form (6466). Unlike RON
165 (66,68), the deletion of 109 amino acids does not affect the proteolytic processing of pro-RON
160 (6466). (iii) The deletion of 109 amino acids containing three cysteines, results in unbalanced numbers of cysteine residues in the extracellular domain of the RON ß chain (6466). This results in abnormal dimerization causing the constitutive activation of the receptor variant (6466).
A third novel RON variant, named RON
155 (Figure 1), was recently cloned from two primary colorectal adenocarcinoma samples (65). RON
155 has a deletion of 158 amino acids in the extracellular domain of the ß-chain (65). These amino acids are encoded by exons 5, 6 and 11, respectively (65). Thus, RON
155 is the product of mRNA splicing with deletion of exons 5, 6 and 11 (65). RON
155 is also constitutively active with autophosphorlyation in tyrosine residues. It is a non-processed single-chain protein and retained in cytoplasm (65).
At present, it is unknown how these RON variants are generated. DNA analysis indicated that all three RON variants are not produced by an abnormality in the genomic sequences (6466). The roles of these RON variants in the progression of colorectal adenocarcinomas are also unknown. Nevertheless, the generation of RON
165, RON
160 and RON
155 provides a molecular mechanism for post-transcriptional activation of RON in certain colorectal adenocarcinomas.
The findings that RON is abnormally expressed, activated and generated in epithelial cancers suggest that cellular mechanisms that control RON expression are dysfunctional in primary colorectal and breast tumors. Also, systems that govern RON activation/inactivation are deregulated. It is speculated that RON may not play a role in initiating epithelial tumors, but is actively transcribed and functional in carcinoma cells (6466). Functional studies support these notions. RON activation induces migration and matrix invasion in SV-40 transformed colon epithelial cells and in certain colon carcinoma cells (6466). Thus, cell motile machinery is activated in colorectal carcinoma cells upon RON activation, which could facilitate tumor invasion and metastasis in vivo. Clearly, these results indicate that cellular disorganization and genetic changes during the progression of colon cancers result in abnormal RON accumulation and RON variant production, which might play an important role in regulating motile-invasive phenotypes in certain colorectal adenocarcinomas in vivo.
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Cell transforming and oncogenic activities of RON
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While there are transforming counterparts of MET, until recently, naturally occurring oncogenic forms of RON had not been identified. Previous work has studied the oncogenic potential of RON using molecular approaches, including the generation of chimeric translocated promoter region (Tpr)-RON, which mimics the oncogenic form of MET (Tpr-MET) (70). Unfortunately, Tpr-RON did not transform NIH3T3 cells (70). The inability of Tpr-RON to induce cell transformation is mainly due to the functional features of the kinase domain (70). Those features include the low catalytic efficiency of Tpr-RON and its weakness in activating MAP kinase signaling cascade (70). Thus, it appears that the Tpr-mediated RON dimerization is not an efficient way to induce RON activity. Other mechanisms capable of regulating kinase activity are essential to release the oncogenic potential of RON.
Experimental mutational studies confirmed that this is the case. By creating point mutations, such as D1232H/V or M1254T, in the kinase domain, RON is able to exert transforming activities in vitro, which lead to tumor growth and metastasis in nude mice (7173). These mutations mimic those naturally occurring in KIT and RET, which are associated with two human malignancies, mastocytosis and multiple endocrine neoplasia type 2B (74,75). It was revealed that D1232H or M1254T substitution in RON yields a dramatic increase in catalytic efficiency, indicating a direct correlation between kinase activity and oncogenic potential (71,72). Molecular modeling of the RON D1232H mutation suggests that this single amino acid substitution favors the transition of the kinase from the inactive to the active state (71,72).
The direct evidence that naturally expressed RON has oncogenic potentials came from the studies of two RON variants, RON
160 and RON
155 (65), as described above (6466). RON
p160 displayed cell-transforming activity as confirmed in focus formation and soft agar growth assays (65). Moreover, NIH3T3 cells expressing RON
160 formed tumors when inoculated into athymic nude mice and caused tumor metastasis in the lung (Figure 2AC) (65). Similarly, cells expressing another RON variant, RON
155, also induced tumor formation even though its efficiency is relatively low (65). To our knowledge, these are the first naturally occurring RON variants that have cell-transforming and tumor-producing activity. This finding is important for two reasons. First, RON
p160 is a constitutively active protein that has the ability to transduce signals that transform rodent fibroblasts (65). Thus, the oncogenic activities of RON
p160 could play an important role in the progression of certain colon cancers. Secondly, the generation of an altered form of the receptor tyrosine kinase by the RON gene may represent a unique mechanism for RON in the pathogenesis and progression of colorectal adenocarcinomas.
Studies from molecularly engineered (transgenic) animals also provide solid evidence indicating that over-expression of RON can lead to tumor formation in vivo (Figure 2D) (76,77). In studying the role of RON in the development and morphology of the lung (76,77), human wtRON was specifically expressed in the distal lung epithelial cells by using a lung-specific transcription element, the human surfactant protein C (SPC) promoter (78). Previous studies have shown that distal lung epithelial cells such as type II pneumocytes do not express endogenous RON (62). However, forced RON expression resulted in the formation of multiple lung adenomas and adenocarcinomas with unique cell morphology and growth pattern (Figure 2D) (76,77). Tumors residing in peripheral portions of the lung appeared as solid-alveolar adenomas/adenocarcinomas and progress slowly (76,77). Significant cellular atypia with a high mitotic index was observed in tumors at later stages. Some tumors showed distinct features of large cell undifferentiated carcinoma with the pallor of the cytoplasm present in the tumors as well as the tremendous pleomorphism of cellular size and nuclear morphology (76,77). Tumors displayed type II cell phenotype. The RON transgene is highly expressed in tumor cells with autophosphorylation (76,77). Immumohistochemical staining further demonstrated that Ras expression is significantly increased in tumors (77). Increased RON expression also led to genomic instability in tumor samples (77), suggesting that over-expression of wtRON is the driving force leading to the tumor initiation and progression in the lung distal epithelial cells in vivo.
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Motile and invasive functions of RON
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Differentiated epithelial cells maintain a unique structuralfunctional polarization and form tight junctions that mediate intracellular adhesion and paracellular contact (79). The loss of these restrictions is believed to be a critical step in epithelial tumorigenesis leading to invasive/metastatic potential (80,81). One of the distinct features of the MET proto-oncogene family is their ability to induce motile-invasive activities or phenotypes in epithelial cells (82), which are characterized by diminished polarization and tight junction (7981). The motile-invasive phenotypes, including cell spreading, dissociation, migration and matrix invasion, are important functions essential for epithelial cell development and homeostasis (83). These activities are also the hallmark of malignancy that distinguishes cancerous cells from benign tumors (8083). The earliest findings indicating that RON activation has the ability to regulate cellular motile activities were seen in tissue macrophages (49,50). Upon MSP stimulation, macrophages adhere to plastic surface within 20 min and undergo a dramatically morphological change known as cell spreading or shape change (49,50). Moreover, MSP induces rearrangement of the cytoskeleton and stimulates the formation of new cellsubstratum contacts (84,85), resulting in chemotactic migration (49,50). These findings provide an important clue suggesting that RON activation has the ability to initiate cell motile machinery leading to cell locomotion.
Up to now, RON has been found to induce cell spreading, dissociation, migration, matrix invasion and tubular formation in a variety of transformed or cancerous epithelial cells, including those from breast, colon, skin, liver, kidney and others (48,6264,86). Studies in vivo further demonstrated that altered RON expression increases the metastatic potentials of tumors (65,72,73). As shown in experimental lung metastasis experiments, NIH3T3 cells expressing RON mutants, such as M1254T or D1232V, displayed increased lung colonization activity (72). Similarly, cells harboring splicing RON variants RON
160 or RON
150 showed enhanced metastatic activities in athymic nude mice when cells were injected into the tail vein (65). These results provide convincing evidence suggesting that RON-mediated invasive growth is manifested not only in cell culture but also in living animals.
Migration of epithelial cells is controlled by cell adhesion to extracellular matrixes (ECM) (87). Cell/ECM interactions are mediated via integrin receptors (88). Accumulated evidence suggested that MSP promotes integrin-dependent epithelial cell adhesion and migration (89,90). First, the process of epithelial cells adhering to ECM was significantly accelerated upon RON activation (89,90). Secondly, adhesion of cells to ECM caused ligand-independent RON phosphorylation, which, through a paracrine fashion, further facilitates cell adhesion and spreading (89,90). Thirdly, RON-mediated epithelial cell migration and invasion are much more efficient on ECM-reconstituted membranes than on uncoated membranes (89,90). Thus, the cooperation between RON and integrins in modulating epithelial cell adhesion and migration is an obligatory step in MSP-induced cell motility and invasion.
Another important function of RON in vitro is its ability to act independently or cooperate with other growth factors to induce epithelial-mesenchymal transition (EMT) (91, Wang,D., Chen,Y.-Q., and Wang,M.-H., submitted), a distinct feature observed during embryonic development and in tumor progression towards metastasis (81,9295). EMT is a complicated event characterized by loss of epithelial characteristics and the acquisition of mesenchymal phenotypes (81,92). The typical EMT consists of the acquisition of a spindle-shaped morphology, delocalization of E-cadherin from cell junctions, elevated N-cadherin transcription and expression of mesenchymal cellular markers such as
-smooth muscle actin (81,92). Certain growth factors such as transforming growth factor (TGF)-ß or HGF/SF are known to induce EMT (9395). Although not all EMT exhibits the whole range of changes listed here, EMT is always associated with cell scattering, defined by the loss of intracellular junctions and acquisition of cell motility (81,92). Using molecularly engineered RON-expressing MadinDarby canine kidney (MDCK) (RE7) cells as a model, we found that over-expression and activation of RON cause RE7 cell scattering with clear spindle-shaped morphology (Wang,D., Chen,Y.-Q. and Wang,M.-H., submitted). Cells also migrate through the collagen IV-reconstituted basement membrane and penetrate into the Matrigel after MSP stimulation (Wang,D., Chen,Y.-Q. and Wang,M.-H., submitted). By immunohistochemical staining, it was found that RON activation results in redistribution of E-cadherin and re-expression of N-cadherin in spindle-shaped RE7 cells (Wang,D., Chen,Y.-Q. and Wang,M.-H., submitted). The data suggest that the RON activation directs a biochemical program that is indistinguishable from TGF-ß1 or other growth factor-induced EMT. Our recent studies further demonstrated that RON activation results in increased expression of the Smad 2 protein and directly causes its phosphorylation (Wang,D., Chen,Y.-Q. and Wang,M.-H., submitted). Smad 2 is a signal molecule responsible for TGF-ß-induced biological activities including EMT (96). The fact that RON activation mediates Smad 2 expression and phosphorylation suggests that RON-mediated EMT in epithelial cells might be channelled through TGF-ß/Smad signaling pathway. In supporting this notion, we found that even though MSP and TGF-ß are both capable of inducing EMT in MDCK cells, the complete epithelial-mesenchymal trans-differentiation, i.e. expressing specific mesenchymal cellular markers such as
-smooth muscle actin, requires the RON expression and activation (Wang,D., Chen, Y.-Q. and Wang,M.-H., submitted). In MDCK cells that do not express RON, neither TGF-ß nor MSP alone is capable of inducing the expression of
-smooth muscle actin. However, when RON is expressed, TGF-ß is able to induce
-smooth muscle actin expression (Wang,D., Chen,Y.-Q. and Wang, M.-H., submitted). These results suggest that RON expression is required for cell differentiation toward EMT with increased motile-invasive activities. As both MSP and TGF-ß are involved in regulating epithelial tumor motility, the signaling collaborations between Smad 2 and RON might be essential in regulating invasive and metastatic potentials of certain epithelial cancers.
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Mechanisms of RON activation that transduce oncogenic signals
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In vitro studies using various cell culture techniques have showed that under conditions mimicking physiological situations, RON can be activated through ligand-dependent or -independent mechanisms (24,90,91). Binding of MSP to RON leading to receptor homo-dimerization is the typical example for ligand-induced RON activation (24,27). Cell adhesion to ECM such as collagen IV also causes RON activation, which is ligand independent and relies on the action of integrins (90). As epithelial cells constantly interact with ECM in vivo, it is likely that RON homodimers pre-exist in normal epithelial tissues that naturally express RON. In support of this notion, a recent study demonstrated that RON at physiological levels forms heterodimers with MET upon HGF/SF stimulation (91). Transphosphorylation occurs directly in heterodimers (91). Interestingly, RON-MET heterodimers were also found in gastrointestinal tumor GTL-16 cells, which expresses high levels of RON and MET (91). These results clearly suggest that the cross-talk and transactivation exist not only in physiological but also in pathological situations. Thus, the ligand-independent RON activation could play a role during the progression of epithelial cancers.
Activated RON transduces a variety of signals that regulate different pathway cascades including Ras/mitogen activated protein kinase (MAPK) (97), phosphotidylinositol-3 kinase (PI-3K/Akt) (98), jun N-terminal kinase (JNK)/stress-activated protein kinase (64), ß-Catenin (100), Smad (Wang,D., Chen,Y.-Q. and Wang,M.-H., submitted) and nuclear factor-
B (NF-
B) (Figure 3) (100102). It is known that these pathways are essential for cell growth, migration, survival and differentiation (64,97102, Wang,D., Chen, Y.-Q. and Wang,M.-H., submitted). Currently, a number of signaling proteins have been shown to be regulated upon RON activation, including Sos (son of sevenless) (97), growth factor receptor bound protein 2 (Grb2) (97), Ras (97), PI-3K (98), MAPK/Erk 1/2 (71,72), JNK (64), ß-catenin (99), focal adhesion kinase (103), integrins (90), Smad 2/3 (Wang,D., Chen, Y.-Q. and Wang,M.-H., submitted) and NF-
B complex (100102). These proteins are the effector molecules responsible for RON-mediated cell replication, transformation, migration and matrix invasiveness (64,89,90,97103, Wang,D., Chen,Y.-Q. and Wang,M.-H., submitted).

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Fig. 3. Mechanisms of RON activation that transduce signals leading to increased cell migration and tumor formation. Activation of RON can be achieved by MSP stimulation (i), over-expression of the receptor (ii), generation of splicing variants (iii) and production of mutants with mutation in the kinase domain (iv). Tyrosine autophosphorylation creates specific-binding sites in the RON C-terminal tail that interact and recruit adaptor/docking proteins (containing Src homology 2 and other domains such as Sos and Grb2). The recruitment and assembly of signaling complexes provide a mechanism for RON to activate a variety of intracellular signaling pathways including Ras/MAPK, PI-3 kinase, JNK, and others. The consequences of the abnormal and persistent RON activation, dependent on individual cell conditions, are the acquisition of the motile/invasive phenotype or the unbalanced cell growth leading to tumor initiation and progression.
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The effect of RON on signaling proteins relies on the phosphorylation of several critical tyrosine residues in its kinase domain and in several tyrosine-containing motifs surrounding the kinase domain (97,98,104). Upon RON dimerization, the transition of the kinase from the inactive to the active state occurs rapidly with autophosphorylation in tyrosine residues such as Y1317 in the kinase domain or Y1353 and Y1360 in the C-terminal tail leading to a dramatic increase in catalytic efficiency (105,106). Mutational analysis revealed that the Y/F conversion of the Y1317 significantly impairs tumorigenic and metastatic properties of RON activated by M1254T substitution (105), indicating that Y1317 is essential for the M1254T mutant to exert oncogenic activities (105).
Phosphorylation also results in the formation of a distinctive multifunctional docking site in the RON C-terminal tail (104106). The docking site, also known as the bidentate motif, is composed of a conserved sequence encompassing two tyrosines (Y1353VQL-XXX-Y1360MNL-) (104). This motif is evolutionally conserved among the member of the MET family (104). Biochemical and biological studies demonstrated that the docking site is critical in recruiting intracellular components such as PI-3K, Grb2 and others (97,106). Substitution of Y1353 and Y1360 with other amino acids results in the significant impairment of the docking site in interacting with signaling proteins, leading to impaired cellular functions (106). In MET, the docking site has been shown to be essential for cell transformation (107). Studies from mutational analysis suggested that the docking site is also involved in RON-mediated cell transformation (7173). However, a recent study has shown that even though the docking site is required for the acquisition of the full oncogenic phenotype, certain RON mutants, such as RONM1254T, can exert cell transforming and metastatic activities without the docking site (105), indicating that increased kinase activity alone is sufficient to transduce the oncogenic signals of RON.
As described above, the oncogenic potentials of RON are determined by the catalytic efficiency of the kinase activity (7173). Currently, three mechanisms, over-expression (74,75), mutation (7173) and truncation (6466), are involved in the abnormal up-regulation of the RON kinase activity (Figure 3). The over-expression model is documented in mouse lung epithelial cells, in which the accumulation of large amounts of wild-type RON results in constitutive activation of the kinase activity (74,75). The formation of tumors in RON over-expressing lung epithelial cells, and not in control littermate mice (74,75), indicates that increased RON kinase activity could initiate unbalanced cell growth leading to tumor formation. The mutation model is observed in several experimentally created RON mutants in which highly conserved residues in the RON kinase domain such as D1232V or M1254T are changed (7173). Substitution of these amino acid residues results in conformational changes in the kinase domain leading to dramatic increase of kinase activities (7173). Thus, oncogenic activities are easily acquired by such manipulation. The truncation model is observed in naturally occurring RON splicing variants such as RON
160 and RON
155 (6466). The deletion of the particular extracellular regions encoded by exons 5, 6 and 11 results in increased kinase activity and tumorigenic activities (6466). Detailed analysis of amino acid sequences showed that the deletion causes the unbalanced number of cysteine residues in the extracellular domains of the RON variants resulting in the abnormal formation of intermolecular disulfide bonds (64,66,68). The consequence of such abnormal bond formation is oligomerization of the altered receptor leading to the increased kinase activities, which are responsible for tumor formation in vivo.
The consequences of increased RON kinase activity are the significantly enhanced activation of Ras/MAPK and other signaling cascades, which are essential in cell transformation leading to tumor growth and formation (6466,7175). Results from analysis of lung tumors in RON transgenic mice also confirmed that enhanced Ras signaling pathways are directly involved in RON-mediated oncogenesis (77). Moreover, increased RON kinase activity led to different patterns of tyrosine-phosphorylated proteins in cells undergoing transformation (105), suggesting that the increased kinase activities are accompanied by the altered substrate specificities. RON activation also protected epithelial cells from apoptosis induced through different mechanisms (108110). Thus, it is believed that the RON kinase activity and the altered substrate specificity are the molecular basis accounting for the cellular mechanisms of oncogenic potentials of RON.
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Future direction
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The identification of oncogenic RON variants from primary colon cancer samples (65) and the discovery of featured lung tumors in RON transgenic mice (74,75) suggest that altered RON expression might be involved in the oncogenesis of certain epithelial tumors in vivo. Definitely, additional studies focusing on the cellular and molecular mechanisms of RON-mediated biological effects are needed. These studies will help us to uncover the significance of RON in epithelial tumor initiation and progression. Considering the available information, we believe that the following two areas need specific attention. The first is to determine the pathogenic roles of the three cloned RON variants in the development of certain colon adenocarcinomas in vivo. Targeted expression of RON variants in colon epithelial cells using a tissue-specific promoter in a transgenic mouse model will be worth trying. Furthermore, defining the relationships between the altered RON expression, including the variant forms of RON, and the observed colon cancer phenotypes is also worth pursuing. In this sense, using anti-sense strategies (111) or RNA interference approaches (112) to inactivate RON or its variants should provide important information about the pathological role of RON in colon cancer. The second is to explore the possible link between RON abnormalities and the progression of certain types of human lung cancer such as bronchioloalveolar carcinomas (BAC) (113). As described above, the pathological features of lung tumors in RON transgenic mice (76,77) resemble, to a certain degree, those of human BAC (113). Also, in ovine pulmonary adenomatosis, a model of sheep BAC induced by jaagsiekte sheep retrovirus (JSRV) (114), the interaction of RON with hyaluronidase 2, the cellular receptor for JSRV, in human bronchial epithelial cells was discovered (115). This suggests that RON might be involved in pathogenesis of JSRV oncogenesis. Moreover, a recent survey of RON expression in human BAC samples and cell lines indicated that RON expression is altered in the majority of BAS samples (our unpublished data). Thus, understanding the roles of RON in tumorigenesis in lung or colon cancers offers an opportunity to uncover the molecular mechanisms underlying epithelial cancer pathogenesis. It should also hold potentials for developing novel approaches to control oncogenesis of certain epithelial tumors in vivo.
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Acknowledgments
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We thank Dr James H.Fisher (University of Colorado School of Medicine) for discussion and encouragement. We are grateful to Ms J.Sharpnack (Denver Health Medical Center) for editing the manuscript. This work was supported by US NIH grants R01 AI43516 and CA91980 to M.H.W.
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Received January 20, 2003;
revised April 7, 2003;
accepted May 11, 2003.